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Wu X, Yang Z, Zou J, Gao H, Shao Z, Li C, Lei P. Protein kinases in neurodegenerative diseases: current understandings and implications for drug discovery. Signal Transduct Target Ther 2025; 10:146. [PMID: 40328798 PMCID: PMC12056177 DOI: 10.1038/s41392-025-02179-x] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/01/2024] [Revised: 01/03/2025] [Accepted: 02/12/2025] [Indexed: 05/08/2025] Open
Abstract
Neurodegenerative diseases (e.g., Alzheimer's, Parkinson's, Huntington's disease, and Amyotrophic Lateral Sclerosis) are major health threats for the aging population and their prevalences continue to rise with the increasing of life expectancy. Although progress has been made, there is still a lack of effective cures to date, and an in-depth understanding of the molecular and cellular mechanisms of these neurodegenerative diseases is imperative for drug development. Protein phosphorylation, regulated by protein kinases and protein phosphatases, participates in most cellular events, whereas aberrant phosphorylation manifests as a main cause of diseases. As evidenced by pharmacological and pathological studies, protein kinases are proven to be promising therapeutic targets for various diseases, such as cancers, central nervous system disorders, and cardiovascular diseases. The mechanisms of protein phosphatases in pathophysiology have been extensively reviewed, but a systematic summary of the role of protein kinases in the nervous system is lacking. Here, we focus on the involvement of protein kinases in neurodegenerative diseases, by summarizing the current knowledge on the major kinases and related regulatory signal transduction pathways implicated in diseases. We further discuss the role and complexity of kinase-kinase networks in the pathogenesis of neurodegenerative diseases, illustrate the advances of clinical applications of protein kinase inhibitors or novel kinase-targeted therapeutic strategies (such as antisense oligonucleotides and gene therapy) for effective prevention and early intervention.
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Affiliation(s)
- Xiaolei Wu
- Department of Neurology and State Key Laboratory of Biotherapy, National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University, Chengdu, Sichuan, China
| | - Zhangzhong Yang
- Department of Neurology and State Key Laboratory of Biotherapy, National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University, Chengdu, Sichuan, China
| | - Jinjun Zou
- Department of Neurology and State Key Laboratory of Biotherapy, National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University, Chengdu, Sichuan, China
| | - Huile Gao
- Key Laboratory of Drug Targeting and Drug Delivery Systems, West China School of Pharmacy, Sichuan University, Chengdu, China
| | - Zhenhua Shao
- Division of Nephrology and Kidney Research Institute, State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, Chengdu, Sichuan, China
| | - Chuanzhou Li
- Department of Medical Genetics, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
| | - Peng Lei
- Department of Neurology and State Key Laboratory of Biotherapy, National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University, Chengdu, Sichuan, China.
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2
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Rix B, Chauhan R, Masoumi Z, Grönroos E, Brain CE, Ogunbiyi OK, Swarbrick K, Swanton C, Bonnet D, Kurzawinski TR, Izatt L, McDonald NQ, Grey W. Kinome profiling reveals pathogenic variant specific protein signalling networks in MEN2 children with Medullary Thyroid Cancer. NPJ Precis Oncol 2025; 9:125. [PMID: 40316714 PMCID: PMC12048619 DOI: 10.1038/s41698-025-00919-4] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/22/2024] [Accepted: 04/22/2025] [Indexed: 05/04/2025] Open
Abstract
Multiple Endocrine Neoplasia Type 2 (MEN2) is an autosomal dominant disease caused by pathogenic variants in the receptor tyrosine kinase RET, with strong genotype-phenotype correlations. The development and progression of these tumours are not always predictable even within families with the same RET pathogenic variant, demonstrating a need for better understanding of the underlying molecular mechanisms. Precision molecular medicine is not widely used and the standard of care remains prophylactic thyroidectomy. This absence of curative approaches is exacerbated by the lack of novel therapeutic markers/targets. In this study, we investigated the functional kinome of 24 familial MEN2 patients. We identified MEN2 subtype and RET pathogenic variant-specific alterations in signalling pathways including mTOR, PKA, NF-κB and focal adhesions, which were validated in patient thyroid tissue. Overall, our study of MEN2 functional kinomes uncovers novel specific drivers of MEN2 disease and its pathogenic variant subtypes, identifying new potential therapeutic targets for MEN2.
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Affiliation(s)
- B Rix
- ProteoStem Lab, Centre for Blood Research, York Biomedical Research Institute, Department of Biology, University of York, York, UK
- Signalling and Structural Biology Laboratory, Francis Crick Institute, London, UK
| | - R Chauhan
- Signalling and Structural Biology Laboratory, Francis Crick Institute, London, UK
| | - Z Masoumi
- ProteoStem Lab, Centre for Blood Research, York Biomedical Research Institute, Department of Biology, University of York, York, UK
| | - E Grönroos
- Cancer evolution and genome instability laboratory, Francis Crick Institute, London, UK
| | - C E Brain
- Department of Endocrinology, Great Ormond Street Hospital, London, UK
| | - O K Ogunbiyi
- NIHR Great Ormond Street Hospital Biomedical Research Centre (BRC), London, UK
| | - K Swarbrick
- NIHR Great Ormond Street Hospital Biomedical Research Centre (BRC), London, UK
| | - C Swanton
- Cancer evolution and genome instability laboratory, Francis Crick Institute, London, UK
| | - D Bonnet
- Haematopoietic Stem Cell Laboratory, Francis Crick Institute, London, UK
| | - T R Kurzawinski
- Department of Endocrinology, Great Ormond Street Hospital, London, UK
| | - L Izatt
- Clinical Genetics Service, Guy's and St Thomas' NHS Foundation Trust, London, UK
- Department of Medical and Molecular Genetics, School of Basic and Medical Biosciences, King's College London, London, UK
| | - N Q McDonald
- Signalling and Structural Biology Laboratory, Francis Crick Institute, London, UK.
- Institute of Structural and Molecular Biology, School of Natural Sciences, Birkbeck College, University of London, London, UK.
| | - W Grey
- ProteoStem Lab, Centre for Blood Research, York Biomedical Research Institute, Department of Biology, University of York, York, UK.
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Mahin A, Gopalakrishnan AP, Ahmed M, Nisar M, John L, Shivamurthy PB, Ummar S, Varghese S, Modi PK, Pai VR, Prasad TSK, Raju R. Orchestrating Intracellular Calcium Signaling Cascades by Phosphosite-Centric Regulatory Network: A Comprehensive Analysis on Kinases CAMKK1 and CAMKK2. OMICS : A JOURNAL OF INTEGRATIVE BIOLOGY 2025; 29:139-153. [PMID: 40079160 DOI: 10.1089/omi.2024.0196] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 03/14/2025]
Abstract
Intracellular calcium signaling is a cornerstone in cell biology and a key molecular target for human health and disease. Calcium/calmodulin dependent protein kinase kinases, CAMKK1 and CAMKK2 are serine/threonine kinases that contribute to the regulation of intracellular calcium signals in response to diverse stimuli. CAMKK1 generally has stable dynamics, whereas CAMKK2 dysregulation triggers oncogenicity and neurological disorders. To differentiate the phosphosignaling hierarchy associated with predominant phosphosites of CAMKK1 and CAMKK2, we assembled and analyzed the global cellular phosphoproteome datasets. We found that predominant phosphosites in CAMKK1 and CAMKK2 are located outside the kinase domain, and their phosphomotifs are highly homologous. Further, we employed a coregulation analysis approach to these predominant phosphosites, to infer the co-occurrence patterns of phosphorylations within CAMKKs and the coregulation patterns of other protein phosphosites with CAMKK sites. We report herein that independent phosphorylations at CAMKK2 S100 and S511 increase their enzymatic activity in the presence of calcium/calmodulin. In addition, the study unveils kinase-substrate associations such as RPS6KB1 as a novel high-confidence upstream kinase of both CAMKK1 S74 and CAMKK2 S100. Further, CAMKK2 was identified as a primary orchestrator in mediating intracellular calcium signaling cascades compared to CAMKK1 based on coregulation patterns of phosphosites from proteins involved in the calcium signaling pathway. These molecular details shed promising insights into the pathophysiology of several diseases such as cancers and psychiatric disorders associated with kinase activity dysregulations of CAMKK2 and further open the avenue for novel PTM-directed therapeutic strategies to regulate CAMKK2.
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Affiliation(s)
- Althaf Mahin
- Centre for Integrative Omics Data Science (CIODS), Yenepoya (Deemed to be University), Mangalore, India
- Center for Systems Biology and Molecular Medicine (CSBMM) [an ICMR-Collaborating Centre of Excellence (ICMR-CCoE 2024)], Yenepoya Research Centre, Yenepoya (Deemed to be University), Mangalore, India
| | - Athira Perunelly Gopalakrishnan
- Centre for Integrative Omics Data Science (CIODS), Yenepoya (Deemed to be University), Mangalore, India
- Center for Systems Biology and Molecular Medicine (CSBMM) [an ICMR-Collaborating Centre of Excellence (ICMR-CCoE 2024)], Yenepoya Research Centre, Yenepoya (Deemed to be University), Mangalore, India
| | - Mukhtar Ahmed
- Department of Zoology, College of Science, King Saud University, Riyadh, Kingdom of Saudi Arabia
| | - Mahammed Nisar
- Centre for Integrative Omics Data Science (CIODS), Yenepoya (Deemed to be University), Mangalore, India
| | - Levin John
- Centre for Integrative Omics Data Science (CIODS), Yenepoya (Deemed to be University), Mangalore, India
| | | | - Samseera Ummar
- Centre for Integrative Omics Data Science (CIODS), Yenepoya (Deemed to be University), Mangalore, India
| | - Susmi Varghese
- Centre for Integrative Omics Data Science (CIODS), Yenepoya (Deemed to be University), Mangalore, India
| | - Prashant Kumar Modi
- Center for Systems Biology and Molecular Medicine (CSBMM) [an ICMR-Collaborating Centre of Excellence (ICMR-CCoE 2024)], Yenepoya Research Centre, Yenepoya (Deemed to be University), Mangalore, India
| | - Vinitha Ramanath Pai
- Department of Biochemistry, Yenepoya Medical College, Yenepoya (Deemed to be University), Mangaluru, India
| | - Thottethodi Subrahmanya Keshava Prasad
- Center for Systems Biology and Molecular Medicine (CSBMM) [an ICMR-Collaborating Centre of Excellence (ICMR-CCoE 2024)], Yenepoya Research Centre, Yenepoya (Deemed to be University), Mangalore, India
| | - Rajesh Raju
- Centre for Integrative Omics Data Science (CIODS), Yenepoya (Deemed to be University), Mangalore, India
- Center for Systems Biology and Molecular Medicine (CSBMM) [an ICMR-Collaborating Centre of Excellence (ICMR-CCoE 2024)], Yenepoya Research Centre, Yenepoya (Deemed to be University), Mangalore, India
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4
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Bremer HJ, Pflum MKH. Chemoproteomic Profiling of PKA Substrates with Kinase-catalyzed Crosslinking and Immunoprecipitation (K-CLIP). BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2025:2025.03.23.644825. [PMID: 40166339 PMCID: PMC11957104 DOI: 10.1101/2025.03.23.644825] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 04/02/2025]
Abstract
Phosphorylation is a highly regulated protein post-translational modification catalyzed by kinases. Kinases and phosphorylated proteins are key players in a myriad of cellular events, including cell signaling. When cell signaling networks are improperly regulated by kinases, various pathologies can arise, such as cancers and neurodegenerative disease. With critical roles in normal and disease biology, kinase-substrate interactions must be thoroughly characterized. Previously, the chemoproteomic method, kinase-catalyzed crosslinking and immunoprecipitation (K-CLIP), was developed to identify the kinases of a phosphoprotein substrate of interest. Here, K-CLIP was modified to profile the substrates of a kinase of interest. Specifically, the substrate profile of cAMP-dependent protein kinase (PKA) was studied with K-CLIP using a new ATP analog, ATP-alkyne aryl azide. Kinase-focused K-CLIP discovered SMC3 as a PKA substrate. With versatility for any kinase or phosphoprotein substrate of interest, K-CLIP will expand our understanding of kinase-mediated cell biology in healthy and diseased states.
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5
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Li T, Lin S, Zhu Y, Ye D, Rong X, Wang L. Basic biology and roles of CEBPD in cardiovascular disease. Cell Death Discov 2025; 11:102. [PMID: 40087290 PMCID: PMC11909146 DOI: 10.1038/s41420-025-02357-4] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/12/2024] [Revised: 01/25/2025] [Accepted: 02/12/2025] [Indexed: 03/17/2025] Open
Abstract
CCAAT/enhancer-binding protein delta (CEBPD), as an evolutionarily conserved protein in mammals, belongs to the CEBP transcription factor family, which modulates many biological processes. The diversity of CEBPD functions partly depends on the cell type and cellular context. Aberrant CEBPD expression and activity are associated with multiple organ diseases, including cardiovascular diseases. In this review, we describe the basic molecular biology of CEBPD to understand its expression regulation, modifications, and functions. Here, we summarize the recent advances in genetically modified animals with CEBPD. Finally, we discuss the contribution of CEBPD to cardiovascular diseases and highlight the strategies for developing novel therapies targeting CEBPD.
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Affiliation(s)
- Tongjun Li
- Guangdong Metabolic Diseases Research Center of Integrated Chinese and Western Medicine; Key Laboratory of Glucolipid Metabolic Disorder, Ministry of Education of China; Guangdong Key Laboratory of Metabolic Disease Prevention and Treatment of Traditional Chinese Medicine; Institute of Chinese Medicine, Guangdong Pharmaceutical University, Guangzhou, Guangdong Province, China
| | - Shaoling Lin
- Guangdong Metabolic Diseases Research Center of Integrated Chinese and Western Medicine; Key Laboratory of Glucolipid Metabolic Disorder, Ministry of Education of China; Guangdong Key Laboratory of Metabolic Disease Prevention and Treatment of Traditional Chinese Medicine; Institute of Chinese Medicine, Guangdong Pharmaceutical University, Guangzhou, Guangdong Province, China
| | - Yingyin Zhu
- Guangdong Metabolic Diseases Research Center of Integrated Chinese and Western Medicine; Key Laboratory of Glucolipid Metabolic Disorder, Ministry of Education of China; Guangdong Key Laboratory of Metabolic Disease Prevention and Treatment of Traditional Chinese Medicine; Institute of Chinese Medicine, Guangdong Pharmaceutical University, Guangzhou, Guangdong Province, China
| | - Dewei Ye
- Guangdong Metabolic Diseases Research Center of Integrated Chinese and Western Medicine; Key Laboratory of Glucolipid Metabolic Disorder, Ministry of Education of China; Guangdong Key Laboratory of Metabolic Disease Prevention and Treatment of Traditional Chinese Medicine; Institute of Chinese Medicine, Guangdong Pharmaceutical University, Guangzhou, Guangdong Province, China.
| | - Xianglu Rong
- Guangdong Metabolic Diseases Research Center of Integrated Chinese and Western Medicine; Key Laboratory of Glucolipid Metabolic Disorder, Ministry of Education of China; Guangdong Key Laboratory of Metabolic Disease Prevention and Treatment of Traditional Chinese Medicine; Institute of Chinese Medicine, Guangdong Pharmaceutical University, Guangzhou, Guangdong Province, China.
| | - Lexun Wang
- Guangdong Metabolic Diseases Research Center of Integrated Chinese and Western Medicine; Key Laboratory of Glucolipid Metabolic Disorder, Ministry of Education of China; Guangdong Key Laboratory of Metabolic Disease Prevention and Treatment of Traditional Chinese Medicine; Institute of Chinese Medicine, Guangdong Pharmaceutical University, Guangzhou, Guangdong Province, China.
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6
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Paul S, Biswas SR, Milner JP, Tomsick PL, Pickrell AM. Adaptor-Mediated Trafficking of Tank Binding Kinase 1 During Diverse Cellular Processes. Traffic 2025; 26:e70000. [PMID: 40047067 PMCID: PMC11883510 DOI: 10.1111/tra.70000] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/02/2024] [Revised: 02/11/2025] [Accepted: 02/14/2025] [Indexed: 03/09/2025]
Abstract
The serine/threonine kinase, Tank Binding Kinase 1 (TBK1), drives distinct cellular processes like innate immune signaling, selective autophagy, and mitosis. It is suggested that the translocation and activation of TBK1 at different subcellular locations within the cell, downstream of diverse stimuli, are driven by TBK1 adaptor proteins forming a complex directly or indirectly with TBK1. Various TBK1 adaptors and associated proteins like NAP1, TANK, SINTBAD, p62, optineurin (OPTN), TAX1BP1, STING, and NDP52 have been identified in facilitating TBK1 activation and recruitment with varying overlapping redundancy. This review focuses on what is known about these proteins, their interactions with TBK1, and the functional consequences of these associations. We shed light on underexplored areas of research on these TBK1 binding partners while emphasizing how future research is required to understand the function and flexibility of TBK1 signaling and crosstalk or regulation between different biological processes.
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Affiliation(s)
- Swagatika Paul
- Graduate Program in Biomedical and Veterinary SciencesVirginia‐Maryland College of Veterinary MedicineBlacksburgVirginiaUSA
| | - Sahitya Ranjan Biswas
- Translational Biology, Medicine, and Health Graduate ProgramVirginia Polytechnic Institute and State UniversityRoanokeVirginiaUSA
| | - Julia P. Milner
- School of NeuroscienceVirginia Polytechnic Institute and State UniversityBlacksburgVirginiaUSA
| | - Porter L. Tomsick
- School of NeuroscienceVirginia Polytechnic Institute and State UniversityBlacksburgVirginiaUSA
| | - Alicia M. Pickrell
- School of NeuroscienceVirginia Polytechnic Institute and State UniversityBlacksburgVirginiaUSA
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7
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Aboulache BL, Hoitsma NM, Luger K. Phosphorylation regulates the chromatin remodeler SMARCAD1 in nucleosome binding, ATP hydrolysis, and histone exchange. J Biol Chem 2024; 300:107893. [PMID: 39424143 PMCID: PMC11742319 DOI: 10.1016/j.jbc.2024.107893] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/12/2024] [Revised: 09/13/2024] [Accepted: 10/03/2024] [Indexed: 10/21/2024] Open
Abstract
Maintaining the dynamic structure of chromatin is critical for regulating the cellular processes that require access to the DNA template, such as DNA damage repair, transcription, and replication. Histone chaperones and ATP-dependent chromatin remodeling factors facilitate transitions in chromatin structure by assembling and positioning nucleosomes through a variety of enzymatic activities. SMARCAD1 is a unique chromatin remodeler that combines the ATP-dependent ability to exchange histones, with the chaperone-like activity of nucleosome deposition. We have shown previously that phosphorylated SMARCAD1 exhibits reduced binding to nucleosomes. However, it is unknown how phosphorylation affects SMARCAD1's ability to perform its various enzymatic activities. Here we use mutational analysis, activity assays, and mass spectrometry, to probe SMARCAD1 regulation and to investigate the role of its flexible N-terminal region. We show that phosphorylation affects SMARCAD1 binding to nucleosomes, DNA, and histones H2A-H2B, as well as ATP hydrolysis and histone exchange. Conversely, we report only a marginal effect of phosphorylation for histone H3-H4 binding and nucleosome assembly. In addition, the SMARCAD1 N-terminal region is revealed to be critical for nucleosome assembly and histone exchange. Together, this work examines the intricacies of how phosphorylation governs SMARCAD1 activity and provides insight into its complex regulation and diverse activities.
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Affiliation(s)
- Briana L Aboulache
- Department of Biochemistry, University of Colorado Boulder, Boulder, Colorado, USA; Howard Hughes Medical Institute, Chevy Chase, Maryland, USA
| | - Nicole M Hoitsma
- Department of Biochemistry, University of Colorado Boulder, Boulder, Colorado, USA; Howard Hughes Medical Institute, Chevy Chase, Maryland, USA
| | - Karolin Luger
- Department of Biochemistry, University of Colorado Boulder, Boulder, Colorado, USA; Howard Hughes Medical Institute, Chevy Chase, Maryland, USA.
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8
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Wang B, Zhang F, Wu X, Ji M. TBK1 is paradoxical in tumor development: a focus on the pathway mediating IFN-I expression. Front Immunol 2024; 15:1433321. [PMID: 39161768 PMCID: PMC11330819 DOI: 10.3389/fimmu.2024.1433321] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/15/2024] [Accepted: 07/22/2024] [Indexed: 08/21/2024] Open
Abstract
TANK-binding kinase 1 (TBK1) is a member of the IKK family and plays a crucial role in the activation of non-canonical NF-κB signaling and type I interferon responses. The aberrant activation of TBK1 contributes to the proliferation and survival of various types of tumor cells, particularly in specific mutational or tumorous contexts. Inhibitors targeting TBK1 are under development and application in both in vivo and in vitro settings, yet their clinical efficacy remains limited. Numerous literatures have shown that TBK1 can exhibit both tumor promoting and tumor inhibiting effects. TBK1 acts as a pivotal node within the innate immune pathway, mediating anti-tumor immunity through the activation of innate immune responses. Facilitating interferon-I (IFN-I) production represents a critical mechanism through which TBK1 bridges these processes. IFN has been shown to exert both beneficial and detrimental effects on tumor progression. Hence, the paradoxical role of TBK1 in tumor development may necessitate acknowledgment in light of its downstream IFN-I signaling cascade. In this paper, we review the signaling pathways mediated by TBK1 in various tumor contexts and summarize the dual roles of TBK1 and the TBK1-IFN pathways in both promoting and inhibiting tumor progression. Additionally, we highlight the significance of the TBK1-IFN pathway in clinical therapy, particularly in the context of immune response. We anticipate further advancements in the development of TBK1 inhibitors as part of novel cancer treatment strategies.
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Affiliation(s)
| | | | | | - Mei Ji
- Department of Oncology, The Third Affiliated Hospital of Soochow University, Changzhou, China
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Liao Q, Yang Y, Li Y, Zhang J, Fan K, Guo Y, Chen J, Chen Y, Zhu P, Huang L, Liu Z. Targeting TANK-binding kinase 1 attenuates painful diabetic neuropathy via inhibiting microglia pyroptosis. Cell Commun Signal 2024; 22:368. [PMID: 39030571 PMCID: PMC11264750 DOI: 10.1186/s12964-024-01723-6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/23/2024] [Accepted: 06/22/2024] [Indexed: 07/21/2024] Open
Abstract
BACKGROUND Painful diabetic neuropathy (PDN) is closely linked to inflammation, which has been demonstrated to be associated with pyroptosis. Emerging evidence has implicated TANK-binding kinase 1 (TBK1) in various inflammatory diseases. However, it remains unknown whether activated TBK1 causes hyperalgesia via pyroptosis. METHODS PDN mice model of type 1 or type 2 diabetic was induced by C57BL/6J or BKS-DB mice with Lepr gene mutation. For type 2 diabetes PDN model, TBK1-siRNA, Caspase-1 inhibitor Ac-YVAD-cmk or TBK1 inhibitor amlexanox (AMX) were delivered by intrathecal injection or intragastric administration. The pain threshold and plantar skin blood perfusion were evaluated through animal experiments. The assessments of spinal cord, dorsal root ganglion, sciatic nerve, plantar skin and serum included western blotting, immunofluorescence, ELISA, and transmission electron microscopy. RESULTS In the PDN mouse model, we found that TBK1 was significantly activated in the spinal dorsal horn (SDH) and mainly located in microglia, and intrathecal injection of chemically modified TBK1-siRNA could improve hyperalgesia. Herein, we described the mechanism that TBK1 could activate the noncanonical nuclear factor κB (NF-κB) pathway, mediate the activation of NLRP3 inflammasome, trigger microglia pyroptosis, and ultimately induce PDN, which could be reversed following TBK1-siRNA injection. We also found that systemic administration of AMX, a TBK1 inhibitor, could effectively improve peripheral nerve injury. These results revealed the key role of TBK1 in PDN and that TBK1 inhibitor AMX could be a potential strategy for treating PDN. CONCLUSIONS Our findings revealed a novel causal role of TBK1 in pathogenesis of PDN, which raises the possibility of applying amlexanox to selectively target TBK1 as a potential therapeutic strategy for PDN.
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Affiliation(s)
- Qinming Liao
- Department of Neurosurgery, The Third Affiliated Hospital of Southern Medical University, Guangzhou, 510630, Guangdong, China
| | - Yimei Yang
- Department of Neurosurgery, Guangdong Provincial Hospital of Chinese Medicine, Guangdong Provincial Academy of Chinese Medical Sciences, Guangzhou, 510030, Guangdong, China
- Department of Neurosurgery, The Third Affiliated Hospital of Southern Medical University, Guangzhou, 510630, Guangdong, China
| | - Yilu Li
- Department of Neurosurgery, The Third Affiliated Hospital of Southern Medical University, Guangzhou, 510630, Guangdong, China
| | - Jun Zhang
- Department of Neurosurgery, Dalang Hospital, Dongguan, 523775, Guangdong, China
| | - Keke Fan
- Department of Anesthesiology, Shenzhen Children's Hospital, Shenzhen, 518000, Guangdong, China
| | - Yihao Guo
- Department of Anesthesiology, Zhujiang Hospital of Southern Medical University, Guangzhou, 510220, Guangdong, China
| | - Jun Chen
- Department of Neurosurgery, The Third Affiliated Hospital of Southern Medical University, Guangzhou, 510630, Guangdong, China
| | - Yinhao Chen
- Department of Neurosurgery, The Third Affiliated Hospital of Southern Medical University, Guangzhou, 510630, Guangdong, China
| | - Pian Zhu
- Department of Anesthesiology, Zhujiang Hospital of Southern Medical University, Guangzhou, 510220, Guangdong, China
- Department of Anesthesiology, Zhongshan Hospital Affiliated to Fudan University, Shanghai, 200032, China
| | - Lijin Huang
- Department of Neurosurgery, Guangdong Provincial Hospital of Chinese Medicine, Guangdong Provincial Academy of Chinese Medical Sciences, Guangzhou, 510030, Guangdong, China.
- Department of Neurosurgery, The Third Affiliated Hospital of Southern Medical University, Guangzhou, 510630, Guangdong, China.
| | - Zhongjie Liu
- Department of Anesthesiology, Shenzhen Children's Hospital, Shenzhen, 518000, Guangdong, China.
- Department of Anesthesiology, Zhujiang Hospital of Southern Medical University, Guangzhou, 510220, Guangdong, China.
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10
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Paul S, Sarraf SA, Nam KH, Zavar L, DeFoor N, Biswas SR, Fritsch LE, Yaron TM, Johnson JL, Huntsman EM, Cantley LC, Ordureau A, Pickrell AM. NAK-associated protein 1/NAP1 activates TBK1 to ensure accurate mitosis and cytokinesis. J Cell Biol 2024; 223:e202303082. [PMID: 38059900 PMCID: PMC10702366 DOI: 10.1083/jcb.202303082] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/20/2023] [Revised: 10/03/2023] [Accepted: 11/10/2023] [Indexed: 12/08/2023] Open
Abstract
Subcellular location and activation of Tank Binding Kinase 1 (TBK1) govern precise progression through mitosis. Either loss of activated TBK1 or its sequestration from the centrosomes causes errors in mitosis and growth defects. Yet, what regulates its recruitment and activation on the centrosomes is unknown. We identified that NAK-associated protein 1 (NAP1) is essential for mitosis, binding to and activating TBK1, which both localize to centrosomes. Loss of NAP1 causes several mitotic and cytokinetic defects due to inactivation of TBK1. Our quantitative phosphoproteomics identified numerous TBK1 substrates that are not only confined to the centrosomes but are also associated with microtubules. Substrate motifs analysis indicates that TBK1 acts upstream of other essential cell cycle kinases like Aurora and PAK kinases. We also identified NAP1 as a TBK1 substrate phosphorylating NAP1 at S318 to promote its degradation by the ubiquitin proteasomal system. These data uncover an important distinct function for the NAP1-TBK1 complex during cell division.
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Affiliation(s)
- Swagatika Paul
- Graduate Program in Biomedical and Veterinary Sciences, Virginia-Maryland College of Veterinary Medicine, Blacksburg, VA, USA
| | - Shireen A. Sarraf
- Biochemistry Section, National Institutes of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA
| | - Ki Hong Nam
- Cell Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA
| | - Leila Zavar
- School of Neuroscience, Virginia Polytechnic Institute and State University, Blacksburg, VA, USA
| | - Nicole DeFoor
- School of Neuroscience, Virginia Polytechnic Institute and State University, Blacksburg, VA, USA
| | - Sahitya Ranjan Biswas
- Translational Biology, Medicine, and Health Graduate Program, Virginia Polytechnic Institute and State University, Roanoke, VA, USA
| | - Lauren E. Fritsch
- Translational Biology, Medicine, and Health Graduate Program, Virginia Polytechnic Institute and State University, Roanoke, VA, USA
| | - Tomer M. Yaron
- Meyer Cancer Center, Weill Cornell Medicine, New York, NY, USA
- Englander Institute for Precision Medicine, Institute for Computational Biomedicine, Weill Cornell Medicine, New York, NY, USA
| | | | - Emily M. Huntsman
- Meyer Cancer Center, Weill Cornell Medicine, New York, NY, USA
- Englander Institute for Precision Medicine, Institute for Computational Biomedicine, Weill Cornell Medicine, New York, NY, USA
| | - Lewis C. Cantley
- Meyer Cancer Center, Weill Cornell Medicine, New York, NY, USA
- Department of Cell Biology, Harvard Medical School, Boston, MA, USA
- Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA
| | - Alban Ordureau
- Cell Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA
| | - Alicia M. Pickrell
- School of Neuroscience, Virginia Polytechnic Institute and State University, Blacksburg, VA, USA
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11
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Yang X, Liu Z. Role of TBK1 Inhibition in Targeted Therapy of Cancer. Mini Rev Med Chem 2024; 24:1031-1045. [PMID: 38314681 DOI: 10.2174/0113895575271977231115062803] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/13/2023] [Revised: 09/16/2023] [Accepted: 09/16/2023] [Indexed: 02/06/2024]
Abstract
TANK-binding kinase 1 (TBK1) is a serine/threonine protein that plays a crucial role in various biological processes like immunity, autophagy, cell survival, and proliferation. The level and kinase activity of the TBK1 protein is regulated through post-translational modifications (PTMs). TBK1 mainly mediates the activation of IRF3/7 and NF-κB signaling pathways while also participating in the regulation of cellular activities such as autophagy, mitochondrial metabolism, and cell proliferation. TBK1 regulates immune, metabolic, inflammatory, and tumor occurrence and development within the body through these cellular activities. TBK1 kinase has emerged as a promising therapeutic target for tumor immunity. However, its molecular mechanism of action remains largely unknown. The identification of selective TBK1 small molecule inhibitors can serve as valuable tools for investigating the biological function of TBK1 protein and also as potential drug candidates for tumor immunotherapy. The current research progress indicates that some TBK1 inhibitors (compounds 15,16 and 21) exhibit certain antitumor effects in vitro culture systems. Here, we summarize the mechanism of action of TBK1 in tumors in recent years and the progress of small molecule inhibitors of TBK1.
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Affiliation(s)
- Xueqing Yang
- School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai University), Ministry of Education, Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Yantai University, Yantai, China
| | - Zongliang Liu
- School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai University), Ministry of Education, Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Yantai University, Yantai, China
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12
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Gong J, Gao X, Ge S, Li H, Wang R, Zhao L. The Role of cGAS-STING Signalling in Metabolic Diseases: from Signalling Networks to Targeted Intervention. Int J Biol Sci 2024; 20:152-174. [PMID: 38164186 PMCID: PMC10750282 DOI: 10.7150/ijbs.84890] [Citation(s) in RCA: 12] [Impact Index Per Article: 12.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/02/2023] [Accepted: 10/17/2023] [Indexed: 01/03/2024] Open
Abstract
The cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) is a crucial innate defence mechanism against viral infection in the innate immune system, as it principally induces the production of type I interferons. Immune responses and metabolic control are inextricably linked, and chronic low-grade inflammation promotes the development of metabolic diseases. The cGAS-STING pathway activated by double-stranded DNA (dsDNA), cyclic dinucleotides (CDNs), endoplasmic reticulum stress (ER stress), mitochondrial stress, and energy imbalance in metabolic cells and immune cells triggers proinflammatory responses and metabolic disorders. Abnormal overactivation of the pathway is closely associated with metabolic diseases such as obesity, nonalcoholic fatty liver disease (NAFLD), insulin resistance and cardiovascular diseases (CVDs). The interaction of cGAS-STING with other pathways, such as the nuclear factor-kappa B (NF-κB), Jun N-terminal kinase (JNK), AMP-activated protein kinase (AMPK), mammalian target of rapamycin (mTOR), autophagy, pyroptosis and insulin signalling pathways, is considered an important mechanism by which cGAS-STING regulates inflammation and metabolism. This review focuses on the link between immune responses related to the cGAS-STING pathway and metabolic diseases and cGAS-STING interaction with other pathways for mediating signal input and affecting output. Moreover, potential inhibitors of the cGAS-STING pathway and therapeutic prospects against metabolic diseases are discussed. This review provides a comprehensive perspective on the involvement of STING in immune-related metabolic diseases.
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Affiliation(s)
- Jiahui Gong
- College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China
| | - Xilong Gao
- Key Laboratory of Functional Dairy, Department of Nutrition and Health, China Agricultural University, Beijing 100193, China
| | - Shaoyang Ge
- Hebei Engineering Research Center of Animal Product, Sanhe 065200, China
| | - Hongliang Li
- Inner Mongolia Mengniu Dairy (Group) Co., Ltd., Hohhot 011517, China
| | - Ran Wang
- Key Laboratory of Functional Dairy, Department of Nutrition and Health, China Agricultural University, Beijing 100193, China
- Research Center for Probiotics, China Agricultural University, Sanhe 065200, China
| | - Liang Zhao
- College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China
- Key Laboratory of Functional Dairy, Department of Nutrition and Health, China Agricultural University, Beijing 100193, China
- Food Laboratory of Zhongyuan, Luohe 462300, China
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13
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Tian X, Zhang Z, Ding M. TXLNA enhances TBK1 phosphorylation by suppressing PPM1B recruitment. BIOCHIMICA ET BIOPHYSICA ACTA. MOLECULAR CELL RESEARCH 2023; 1870:119550. [PMID: 37506885 DOI: 10.1016/j.bbamcr.2023.119550] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/06/2023] [Revised: 07/19/2023] [Accepted: 07/19/2023] [Indexed: 07/30/2023]
Abstract
In recent years, there has been a notable increase in cancer incidence and mortality, and immune abnormalities have been closely linked to malignancy development. TANK-binding kinase 1 (TBK1) is a non-classical IκB kinase that regulates interferon and NF-κB signaling pathways and plays a crucial role in innate immunity. Recent studies have shown high expression levels of TBK1 and increased activity in various tumor cells, suggesting its involvement in the development and progression of multiple cancers. Targeting TBK1 for tumor therapy may be a possibility. However, little is known about the abnormal activation and dynamic regulation of TBK1 in cancer. First, we utilized the BioID biotinylation technique combined with TMT-based quantitative proteomics to analyze the TBK1 interacting proteins. Our results revealed that TXLNA interacts with TBK1 and binds to the α-helical scaffold of TBK1. The expression of TXLNA could affect the S172 phosphorylation of TBK1. PPM1B is a phosphatase that can dephosphorylate TBK1 S172, so we used the APEX2 proximity labeling technique combined with TMT-based quantitative proteomics to explore the interacting proteins of PPM1B and search for the regulatory pathway of TXLNA on TBK1 phosphorylation. We found that PPM1B interacts with TXLNA. Based on these results, we further found that TXLNA impairs the binding of PPM1B to TBK1, inhibiting the dephosphorylation of TBK1 and contributing to the abnormal enhancement of TBK1 activity in cancer cells. This study sheds light on the potential mechanism of aberrant activation and dynamic regulation of TBK1 in tumors and provides a potential target for tumor therapy.
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Affiliation(s)
- Xiao Tian
- School of Life Science and Technology, China Pharmaceutical University, Nanjing 211199, China
| | - Zhiyuan Zhang
- School of Life Science and Technology, China Pharmaceutical University, Nanjing 211199, China
| | - Ming Ding
- School of Life Science and Technology, China Pharmaceutical University, Nanjing 211199, China.
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14
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Song S, Tang H, Ran T, Fang F, Tong L, Chen H, Xie H, Lu X. Application of deep generative model for design of Pyrrolo[2,3-d] pyrimidine derivatives as new selective TANK binding kinase 1 (TBK1) inhibitors. Eur J Med Chem 2023; 247:115034. [PMID: 36603506 DOI: 10.1016/j.ejmech.2022.115034] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/31/2022] [Revised: 12/08/2022] [Accepted: 12/17/2022] [Indexed: 12/24/2022]
Abstract
The deep conditional transformer neural network SyntaLinker was applied to identify compounds with pyrrolo[2,3-d]pyrimidine scaffold as potent selective TBK1 inhibitor. Further medicinal chemistry optimization campaign led to the discovery of the most potent compound 7l, which exhibited strong enzymatic inhibitory activity against TBK1 with an IC50 value of 22.4 nM 7l had a superior inhibitory activity in human monocytic THP1-Blue cells reporter gene assay than MRT67307. Furthermore, 7l significantly inhibited TBK1 downstream target genes cxcl10 and ifnβ expression in THP1 and RAW264.7 cells induced by poly (I:C) and lipopolysaccharide, respectively. This study suggested that combination of deep conditional transformer neural network SyntaLinker and transfer learning could be a powerful tool for scaffold hopping in drug discovery.
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Affiliation(s)
- Shukai Song
- School of Pharmacy, Jinan University, #855 Xingye Avenue, Guangzhou, 510632, China
| | - Haotian Tang
- Division of Antitumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, #555 Zuchongzhi Road, Shanghai, 201203, China; University of Chinese Academy of Sciences, No.19(A) Yuquan Road, Shijingshan District, Beijing, 100049, China
| | - Ting Ran
- Division of Drug and Vaccine Research, Guangzhou Laboratory, Guangzhou, 510530, China
| | - Feng Fang
- Division of Antitumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, #555 Zuchongzhi Road, Shanghai, 201203, China
| | - Linjiang Tong
- Division of Antitumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, #555 Zuchongzhi Road, Shanghai, 201203, China
| | - Hongming Chen
- Division of Drug and Vaccine Research, Guangzhou Laboratory, Guangzhou, 510530, China.
| | - Hua Xie
- Division of Antitumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, #555 Zuchongzhi Road, Shanghai, 201203, China; Zhongshan Institute for Drug Discovery, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Cuiheng New District, Zhongshan City, China.
| | - Xiaoyun Lu
- School of Pharmacy, Jinan University, #855 Xingye Avenue, Guangzhou, 510632, China.
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15
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Vacchini A, Maffioli E, Di Silvestre D, Cancellieri C, Milanesi S, Nonnis S, Badanai S, Mauri P, Negri A, Locati M, Tedeschi G, Borroni EM. Phosphoproteomic mapping of CCR5 and ACKR2 signaling properties. Front Mol Biosci 2022; 9:1060555. [PMID: 36483536 PMCID: PMC9723398 DOI: 10.3389/fmolb.2022.1060555] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/03/2022] [Accepted: 10/31/2022] [Indexed: 07/25/2024] Open
Abstract
ACKR2 is an atypical chemokine receptor which is structurally uncoupled from G proteins and is unable to activate signaling pathways used by conventional chemokine receptors to promote cell migration. Nonetheless, ACKR2 regulates inflammatory and immune responses by shaping chemokine gradients in tissues via scavenging inflammatory chemokines. To investigate the signaling pathways downstream to ACKR2, a quantitative SILAC-based phosphoproteomic analysis coupled with a systems biology approach with network analysis, was carried out on a HEK293 cell model expressing either ACKR2 or its conventional counterpart CCR5. The model was stimulated with the common agonist CCL3L1 for short (3 min) and long (30 min) durations. As expected, many of the identified proteins are known to participate in conventional signal transduction pathways and in the regulation of cytoskeleton dynamics. However, our analyses revealed unique phosphorylation and network signatures, suggesting roles for ACKR2 other than its scavenger activity. In conclusion, the mapping of phosphorylation events at a holistic level indicated that conventional and atypical chemokine receptors differ in signaling properties. This provides an unprecedented level of detail in chemokine receptor signaling and identifying potential targets for the regulation of ACKR2 and CCR5 function.
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Affiliation(s)
- Alessandro Vacchini
- IRCCS Humanitas Research Hospital, Milan, Italy
- Department of Medical Biotechnologies and Translational Medicine, University of Milan, Segrate, Italy
| | - Elisa Maffioli
- Department of Veterinary Medicine and Animal Science, University of Milan, Lodi, Italy
| | - Dario Di Silvestre
- Institute of Technologies in Biomedicine, National Research Council (ITB-CNR), Milan, Italy
| | | | - Samantha Milanesi
- IRCCS Humanitas Research Hospital, Milan, Italy
- Department of Medical Biotechnologies and Translational Medicine, University of Milan, Segrate, Italy
| | - Simona Nonnis
- Department of Veterinary Medicine and Animal Science, University of Milan, Lodi, Italy
| | | | | | - Armando Negri
- Department of Veterinary Medicine and Animal Science, University of Milan, Lodi, Italy
| | - Massimo Locati
- IRCCS Humanitas Research Hospital, Milan, Italy
- Department of Medical Biotechnologies and Translational Medicine, University of Milan, Segrate, Italy
| | - Gabriella Tedeschi
- Department of Veterinary Medicine and Animal Science, University of Milan, Lodi, Italy
- CIMAINA, Milan, Italy
| | - Elena Monica Borroni
- IRCCS Humanitas Research Hospital, Milan, Italy
- Department of Medical Biotechnologies and Translational Medicine, University of Milan, Segrate, Italy
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16
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Zhao B, Ni Y, Zhang H, Zhao Y, Li L. Endothelial deletion of TBK1 contributes to BRB dysfunction via CXCR4 phosphorylation suppression. Cell Death Dis 2022; 8:429. [PMID: 36307391 PMCID: PMC9616849 DOI: 10.1038/s41420-022-01222-y] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/19/2022] [Revised: 10/11/2022] [Accepted: 10/13/2022] [Indexed: 11/18/2022]
Abstract
Blood-retinal barrier (BRB) dysfunction has been recognized as an early pathological feature in common eye diseases that cause blindness. The breakdown of endothelial cell-to-cell junctions is the main reason for BRB dysfunction, yet our understanding of junctional modulation remains limited. Here, we demonstrated that endothelial-specific deletion of TBK1 (Tbk1ΔEC) disrupted retinal vascular development, and induced vascular leakage. LC-MS/MS proteomic analysis was used to identify candidate substrates of TBK1. We found that TBK1 interacted with CXCR4, and the phosphorylation level of CXCR4-Serine 355 (Ser355) was decreased in Tbk1ΔEC retina samples. Furthermore, TBK1-mediated phosphorylation of CXCR4 at Ser355 played an indispensable role in maintaining endothelial junctions. Interestingly, we also detected an increased expression of TBK1 in diabetic retinopathy samples, which suggested an association between TBK1 and the disease. Taken together, these results provided insight into the mechanisms involved in the regulation of endothelial cell-to-cell junctions via TBK1-dependent CXCR4 phosphorylation.
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17
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Verzella D, Cornice J, Arboretto P, Vecchiotti D, Di Vito Nolfi M, Capece D, Zazzeroni F, Franzoso G. The NF-κB Pharmacopeia: Novel Strategies to Subdue an Intractable Target. Biomedicines 2022; 10:2233. [PMID: 36140335 PMCID: PMC9496094 DOI: 10.3390/biomedicines10092233] [Citation(s) in RCA: 17] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2022] [Revised: 08/31/2022] [Accepted: 09/03/2022] [Indexed: 11/19/2022] Open
Abstract
NF-κB transcription factors are major drivers of tumor initiation and progression. NF-κB signaling is constitutively activated by genetic alterations or environmental signals in many human cancers, where it contributes to almost all hallmarks of malignancy, including sustained proliferation, cell death resistance, tumor-promoting inflammation, metabolic reprogramming, tissue invasion, angiogenesis, and metastasis. As such, the NF-κB pathway is an attractive therapeutic target in a broad range of human cancers, as well as in numerous non-malignant diseases. Currently, however, there is no clinically useful NF-κB inhibitor to treat oncological patients, owing to the preclusive, on-target toxicities of systemic NF-κB blockade. In this review, we discuss the principal and most promising strategies being developed to circumvent the inherent limitations of conventional IκB kinase (IKK)/NF-κB-targeting drugs, focusing on new molecules that target upstream regulators or downstream effectors of oncogenic NF-κB signaling, as well as agents targeting individual NF-κB subunits.
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Affiliation(s)
- Daniela Verzella
- Department of Biotechnological and Applied Clinical Sciences (DISCAB), University of L’Aquila, 67100 L’Aquila, Italy
- Department of Immunology and Inflammation, Imperial College London, London W12 0NN, UK
| | - Jessica Cornice
- Department of Immunology and Inflammation, Imperial College London, London W12 0NN, UK
| | - Paola Arboretto
- Department of Immunology and Inflammation, Imperial College London, London W12 0NN, UK
| | - Davide Vecchiotti
- Department of Biotechnological and Applied Clinical Sciences (DISCAB), University of L’Aquila, 67100 L’Aquila, Italy
| | - Mauro Di Vito Nolfi
- Department of Biotechnological and Applied Clinical Sciences (DISCAB), University of L’Aquila, 67100 L’Aquila, Italy
| | - Daria Capece
- Department of Biotechnological and Applied Clinical Sciences (DISCAB), University of L’Aquila, 67100 L’Aquila, Italy
- Department of Immunology and Inflammation, Imperial College London, London W12 0NN, UK
| | - Francesca Zazzeroni
- Department of Biotechnological and Applied Clinical Sciences (DISCAB), University of L’Aquila, 67100 L’Aquila, Italy
| | - Guido Franzoso
- Department of Immunology and Inflammation, Imperial College London, London W12 0NN, UK
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18
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Khatoon F, Kumar V, Anjum F, Shafie A, Adnan M, Hassan MI. Frustration analysis of TBK1 missense mutations reported in ALS/FTD and cancer patients. 3 Biotech 2022; 12:174. [PMID: 35845111 PMCID: PMC9283588 DOI: 10.1007/s13205-022-03240-0] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/27/2022] [Accepted: 06/23/2022] [Indexed: 11/29/2022] Open
Abstract
Tank-binding kinase 1 (TBK1) is a multifunctional kinase having essential roles in cellular processes, autophagy/mitophagy, and selective clearance of damaged proteins. More than 90 mutations in the TBK1 gene are linked with multiple cancer types, amyotrophic lateral sclerosis (ALS), and frontotemporal dementia (FTD). Some of these missense mutations disrupt the abilities of TBK1 to dimerize, associate with the mitophagy receptor optineurin (OPTN), autoactivate, or catalyze phosphorylation. Some mutations may cause severe dysregulation of the pathway, while others induce a limited disruption. Here, we have studied those mutations reported in cancer, ALS and FTD, and subsequently investigated the effect of missense mutations on the structure and function of TBK1 for localized residual frustration change. Out of 33 ALS/FTD causing mutations and 28 oncogenic mutations, 10 mutations and 12 oncogenic mutations showed significant change in the residual frustration. The local frustration plays an important role in the conformation of protein structure in active and inactive kinases. Our analysis reports the change in residual frustration state, conformational change and effect on active and inactive TBK1 function due to ALS/FTD causing and oncogenic missense mutations. Supplementary Information The online version contains supplementary material available at 10.1007/s13205-022-03240-0.
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19
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Therapeutic targeting of TANK-binding kinase signaling towards anticancer drug development: Challenges and opportunities. Int J Biol Macromol 2022; 207:1022-1037. [PMID: 35358582 DOI: 10.1016/j.ijbiomac.2022.03.157] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/01/2022] [Revised: 03/23/2022] [Accepted: 03/24/2022] [Indexed: 12/15/2022]
Abstract
TANK-binding kinase 1 (TBK1) plays a fundamental role in regulating the cellular responses and controlling several signaling cascades. It regulates inflammatory, interferon, NF-κB, autophagy, and Akt pathways. Post-translational modifications (PTM) of TBK1 control its action and subsequent cellular signaling. The dysregulation of the TBK1 pathway is correlated to many pathophysiological conditions, including cancer, that implicates the promising therapeutic advantage for targeting TBK1. The present study summarizes current updates on the molecular mechanisms and cancer-inducing roles of TBK1. Designed inhibitors of TBK1 are considered a potential therapeutic agent for several diseases, including cancer. Data from pre-clinical tumor models recommend that the targeting of TBK1 could be an attractive strategy for anti-tumor therapy. This review further highlighted the therapeutic potential of potent and selective TBK1 inhibitors, including Amlexanox, Compound II, BX795, MRT67307, SR8185 AZ13102909, CYT387, GSK8612, BAY985, and Domainex. These inhibitors may be implicated to facilitate therapeutic management of cancer and TBK1-associated diseases in the future.
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20
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Runde AP, Mack R, S J PB, Zhang J. The role of TBK1 in cancer pathogenesis and anticancer immunity. JOURNAL OF EXPERIMENTAL & CLINICAL CANCER RESEARCH : CR 2022; 41:135. [PMID: 35395857 PMCID: PMC8994244 DOI: 10.1186/s13046-022-02352-y] [Citation(s) in RCA: 74] [Impact Index Per Article: 24.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 02/12/2022] [Accepted: 03/29/2022] [Indexed: 02/07/2023]
Abstract
The TANK-binding kinase 1 (TBK1) is a serine/threonine kinase belonging to the non-canonical inhibitor of nuclear factor-κB (IκB) kinase (IKK) family. TBK1 can be activated by pathogen-associated molecular patterns (PAMPs), inflammatory cytokines, and oncogenic kinases, including activated K-RAS/N-RAS mutants. TBK1 primarily mediates IRF3/7 activation and NF-κB signaling to regulate inflammatory cytokine production and the activation of innate immunity. TBK1 is also involved in the regulation of several other cellular activities, including autophagy, mitochondrial metabolism, and cellular proliferation. Although TBK1 mutations have not been reported in human cancers, aberrant TBK1 activation has been implicated in the oncogenesis of several types of cancer, including leukemia and solid tumors with KRAS-activating mutations. As such, TBK1 has been proposed to be a feasible target for pharmacological treatment of these types of cancer. Studies suggest that TBK1 inhibition suppresses cancer development not only by directly suppressing the proliferation and survival of cancer cells but also by activating antitumor T-cell immunity. Several small molecule inhibitors of TBK1 have been identified and interrogated. However, to this point, only momelotinib (MMB)/CYT387 has been evaluated as a cancer therapy in clinical trials, while amlexanox (AMX) has been evaluated clinically for treatment of type II diabetes, nonalcoholic fatty liver disease, and obesity. In this review, we summarize advances in research into TBK1 signaling pathways and regulation, as well as recent studies on TBK1 in cancer pathogenesis. We also discuss the potential molecular mechanisms of targeting TBK1 for cancer treatment. We hope that our effort can help to stimulate the development of novel strategies for targeting TBK1 signaling in future approaches to cancer therapy.
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Affiliation(s)
- Austin P Runde
- Department of Cancer Biology, Oncology Institute, Cardinal Bernardin Cancer Center, Loyola University Medical Center, Maywood, IL, 60153, USA
| | - Ryan Mack
- Department of Cancer Biology, Oncology Institute, Cardinal Bernardin Cancer Center, Loyola University Medical Center, Maywood, IL, 60153, USA
| | - Peter Breslin S J
- Department of Cancer Biology, Oncology Institute, Cardinal Bernardin Cancer Center, Loyola University Medical Center, Maywood, IL, 60153, USA.,Departments of Molecular/Cellular Physiology and Biology, Loyola University Medical Center and Loyola University Chicago, Chicago, IL, 60660, USA
| | - Jiwang Zhang
- Department of Cancer Biology, Oncology Institute, Cardinal Bernardin Cancer Center, Loyola University Medical Center, Maywood, IL, 60153, USA. .,Departments of Pathology and Radiation Oncology, Loyola University Medical Center, Maywood, IL, 60153, USA.
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21
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E3 Ubiquitin Ligase Regulators of Notch Receptor Endocytosis: From Flies to Humans. Biomolecules 2022; 12:biom12020224. [PMID: 35204725 PMCID: PMC8961608 DOI: 10.3390/biom12020224] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/03/2021] [Revised: 01/18/2022] [Accepted: 01/21/2022] [Indexed: 02/04/2023] Open
Abstract
Notch is a developmental receptor, conserved in the evolution of the metazoa, which regulates cell fate proliferation and survival in numerous developmental contexts, and also regulates tissue renewal and repair in adult organisms. Notch is activated by proteolytic removal of its extracellular domain and the subsequent release of its intracellular domain, which then acts in the nucleus as part of a transcription factor complex. Numerous regulatory mechanisms exist to tune the amplitude, duration and spatial patterning of this core signalling mechanism. In Drosophila, Deltex (Dx) and Suppressor of dx (Su(dx)) are E3 ubiquitin ligases which interact with the Notch intracellular domain to regulate its endocytic trafficking, with impacts on both ligand-dependent and ligand-independent signal activation. Homologues of Dx and Su(dx) have been shown to also interact with one or more of the four mammalian Notch proteins and other target substrates. Studies have shown similarities, specialisations and diversifications of the roles of these Notch regulators. This review collates together current research on vertebrate Dx and Su(dx)-related proteins, provides an overview of their various roles, and discusses their contributions to cell fate regulation and disease.
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22
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Zaman A, Wu X, Lemoff A, Yadavalli S, Lee J, Wang C, Cooper J, McMillan EA, Yeaman C, Mirzaei H, White MA, Bivona TG. Exocyst protein subnetworks integrate Hippo and mTOR signaling to promote virus detection and cancer. Cell Rep 2021; 36:109491. [PMID: 34348154 DOI: 10.1016/j.celrep.2021.109491] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/02/2020] [Revised: 05/20/2021] [Accepted: 07/14/2021] [Indexed: 11/25/2022] Open
Abstract
The exocyst is an evolutionarily conserved protein complex that regulates vesicular trafficking and scaffolds signal transduction. Key upstream components of the exocyst include monomeric RAL GTPases, which help mount cell-autonomous responses to trophic and immunogenic signals. Here, we present a quantitative proteomics-based characterization of dynamic and signal-dependent exocyst protein interactomes. Under viral infection, an Exo84 exocyst subcomplex assembles the immune kinase Protein Kinase R (PKR) together with the Hippo kinase Macrophage Stimulating 1 (MST1). PKR phosphorylates MST1 to activate Hippo signaling and inactivate Yes Associated Protein 1 (YAP1). By contrast, a Sec5 exocyst subcomplex recruits another immune kinase, TANK binding kinase 1 (TBK1), which interacted with and activated mammalian target of rapamycin (mTOR). RALB was necessary and sufficient for induction of Hippo and mTOR signaling through parallel exocyst subcomplex engagement, supporting the cellular response to virus infection and oncogenic signaling. This study highlights RALB-exocyst signaling subcomplexes as mechanisms for the integrated engagement of Hippo and mTOR signaling in cells challenged by viral pathogens or oncogenic signaling.
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Affiliation(s)
- Aubhishek Zaman
- Department of Medicine, University of California, San Francisco, 600 16th Street, San Francisco, CA 94158, USA; UCSF Helen Diller Comprehensive Cancer Center, University of California, San Francisco, 600 16th Street, San Francisco, CA 94158, USA; Department of Cell Biology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390, USA.
| | - Xiaofeng Wu
- Department of Physiology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390, USA
| | - Andrew Lemoff
- Department of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390, USA
| | - Sivaramakrishna Yadavalli
- Department of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390, USA
| | - Jeon Lee
- Department of Cell Biology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390, USA; Bioinformatics Core Facility, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390, USA
| | - Chensu Wang
- Department of Cell Biology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390, USA
| | - Jonathan Cooper
- Department of Cell Biology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390, USA
| | - Elizabeth A McMillan
- Department of Cell Biology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390, USA
| | - Charles Yeaman
- Department of Anatomy and Cell Biology, University of Iowa, 51 Newton Road, Iowa City, IA 52242, USA
| | - Hamid Mirzaei
- Department of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390, USA
| | - Michael A White
- Department of Cell Biology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390, USA
| | - Trever G Bivona
- Department of Medicine, University of California, San Francisco, 600 16th Street, San Francisco, CA 94158, USA; UCSF Helen Diller Comprehensive Cancer Center, University of California, San Francisco, 600 16th Street, San Francisco, CA 94158, USA.
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23
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Rehman R, Tar L, Olamide AJ, Li Z, Kassubek J, Böckers T, Weishaupt J, Ludolph A, Wiesner D, Roselli F. Acute TBK1/IKK-ε Inhibition Enhances the Generation of Disease-Associated Microglia-Like Phenotype Upon Cortical Stab-Wound Injury. Front Aging Neurosci 2021; 13:684171. [PMID: 34326766 PMCID: PMC8313992 DOI: 10.3389/fnagi.2021.684171] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/22/2021] [Accepted: 06/21/2021] [Indexed: 12/11/2022] Open
Abstract
Traumatic brain injury has a poorer prognosis in elderly patients, possibly because of the enhanced inflammatory response characteristic of advanced age, known as “inflammaging.” Recently, reduced activation of the TANK-Binding-Kinase 1 (Tbk1) pathway has been linked to age-associated neurodegeneration and neuroinflammation. Here we investigated how the blockade of Tbk1 and of the closely related IKK-ε by the small molecule Amlexanox could modify the microglial and immune response to cortical stab-wound injury in mice. We demonstrated that Tbk1/IKK-ε inhibition resulted in a massive expansion of microglial cells characterized by the TMEM119+/CD11c+ phenotype, expressing high levels of CD68 and CD317, and with the upregulation of Cst7a, Prgn and Ccl4 and the decrease in the expression levels of Tmem119 itself and P2yr12, thus a profile close to Disease-Associated Microglia (DAM, a subset of reactive microglia abundant in Alzheimer’s Disease and other neurodegenerative conditions). Furthermore, Tbk1/IKK-ε inhibition increased the infiltration of CD3+ lymphocytes, CD169+ macrophages and CD11c+/CD169+ cells. The enhanced immune response was associated with increased expression of Il-33, Ifn-g, Il-17, and Il-19. This upsurge in the response to the stab wound was associated with the expanded astroglial scars and increased deposition of chondroitin-sulfate proteoglycans at 7 days post injury. Thus, Tbk1/IKK-ε blockade results in a massive expansion of microglial cells with a phenotype resembling DAM and with the substantial enhancement of neuroinflammatory responses. In this context, the induction of DAM is associated with a detrimental outcome such as larger injury-related glial scars. Thus, the Tbk1/IKK-ε pathway is critical to repress neuroinflammation upon stab-wound injury and Tbk1/IKK-ε inhibitors may provide an innovative approach to investigate the consequences of DAM induction.
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Affiliation(s)
- Rida Rehman
- Department of Neurology, Ulm University, Ulm, Germany
| | - Lilla Tar
- Department of Neurology, Ulm University, Ulm, Germany.,German Center for Neurodegenerative Diseases (DZNE)-Ulm, Ulm, Germany
| | - Adeyemi Jubril Olamide
- Department of Neurology, Ulm University, Ulm, Germany.,Master in Translational and Molecular Neuroscience, Ulm University, Ulm, Germany
| | - Zhenghui Li
- Department of Neurology, Ulm University, Ulm, Germany.,Department of Neurosurgery, Kaifeng Central Hospital, Kaifeng, China
| | - Jan Kassubek
- Department of Neurology, Ulm University, Ulm, Germany
| | - Tobias Böckers
- Institute of Anatomy and Cell Biology, Ulm University, Ulm, Germany.,Neurozentrum Ulm, Ulm, Germany
| | - Jochen Weishaupt
- Department of Neurology, Ulm University, Ulm, Germany.,Neurozentrum Ulm, Ulm, Germany
| | - Albert Ludolph
- Department of Neurology, Ulm University, Ulm, Germany.,German Center for Neurodegenerative Diseases (DZNE)-Ulm, Ulm, Germany.,Neurozentrum Ulm, Ulm, Germany
| | - Diana Wiesner
- Department of Neurology, Ulm University, Ulm, Germany.,German Center for Neurodegenerative Diseases (DZNE)-Ulm, Ulm, Germany.,Neurozentrum Ulm, Ulm, Germany
| | - Francesco Roselli
- Department of Neurology, Ulm University, Ulm, Germany.,German Center for Neurodegenerative Diseases (DZNE)-Ulm, Ulm, Germany.,Neurozentrum Ulm, Ulm, Germany
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24
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Umair M, Khan S, Mohammad T, Shafie A, Anjum F, Islam A, Hassan MI. Impact of single amino acid substitution on the structure and function of TANK-binding kinase-1. J Cell Biochem 2021; 122:1475-1490. [PMID: 34237165 DOI: 10.1002/jcb.30070] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/29/2021] [Revised: 05/26/2021] [Accepted: 05/31/2021] [Indexed: 12/14/2022]
Abstract
Tank-binding kinase 1 (TBK1) is a serine/threonine protein kinase involved in various signaling pathways and subsequently regulates cell proliferation, apoptosis, autophagy, antiviral and antitumor immunity. Dysfunction of TBK1 can cause many complex diseases, including autoimmunity, neurodegeneration, and cancer. This dysfunction of TBK1 may result from single amino acid substitutions and subsequent structural alterations. This study analyzed the effect of substituting amino acids on TBK1 structure, function, and subsequent disease using advanced computational methods and various tools. In the initial assessment, a total of 467 mutations were found to be deleterious. After that, in detailed structural and sequential analyses, 13 mutations were found to be pathogenic. Finally, based on the functional importance, two variants (K38D and S172A) of the TBK1 kinase domain were selected and studied in detail by utilizing all-atom molecular dynamics (MD) simulation for 200 ns. MD simulation, including correlation matrix and principal component analysis, helps to get deeper insights into the TBK1 structure at the atomic level. We observed a substantial change in variants' conformation, which may be possible for structural alteration and subsequent TBK1 dysfunction. However, substitution S172A shows a significant conformational change in TBK1 structure as compared to K38D. Thus, this study provides a structural basis to understand the effect of mutations on the kinase domain of TBK1 and its function associated with disease progression.
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Affiliation(s)
- Mohd Umair
- Department of Computer Science, Jamia Millia Islamia, Jamia Nagar, New Delhi, India
| | - Shama Khan
- Drug Discovery and Development Centre (H3D), University of Cape Town, Rondebosch, South Africa
| | - Taj Mohammad
- Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, Jamia Nagar, New Delhi, India
| | - Alaa Shafie
- Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, Taif University, Taif, Saudi Arabia
| | - Farah Anjum
- Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, Taif University, Taif, Saudi Arabia
| | - Asimul Islam
- Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, Jamia Nagar, New Delhi, India
| | - Md Imtaiyaz Hassan
- Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, Jamia Nagar, New Delhi, India
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25
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Niinae T, Imami K, Sugiyama N, Ishihama Y. Identification of Endogenous Kinase Substrates by Proximity Labeling Combined with Kinase Perturbation and Phosphorylation Motifs. Mol Cell Proteomics 2021; 20:100119. [PMID: 34186244 PMCID: PMC8325102 DOI: 10.1016/j.mcpro.2021.100119] [Citation(s) in RCA: 23] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/05/2021] [Revised: 06/08/2021] [Accepted: 06/22/2021] [Indexed: 02/08/2023] Open
Abstract
Mass-spectrometry-based phosphoproteomics can identify more than 10,000 phosphorylated sites in a single experiment. But, despite the fact that enormous phosphosite information has been accumulated in public repositories, protein kinase–substrate relationships remain largely unknown. Here, we describe a method to identify endogenous substrates of kinases by using a combination of a proximity-dependent biotin identification method, called BioID, with two other independent methods, kinase-perturbed phosphoproteomics and phosphorylation motif matching. For proof of concept, this approach was applied to casein kinase 2 (CK2) and protein kinase A (PKA), and we identified 24 and 35 putative substrates, respectively. We also show that known cancer-associated missense mutations near phosphosites of substrates affect phosphorylation by CK2 or PKA and thus might alter downstream signaling in cancer cells bearing these mutations. This approach extends our ability to probe physiological kinase–substrate networks by providing new methodology for large-scale identification of endogenous substrates of kinases.
Identification of novel kinase interactors by BioID. Applying two orthogonal filters, kinase perturbation and phosphorylation motif. Identification of novel CK2 and PKA substrates. A universal method for the identification of endogenous substrates for all kinases.
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Affiliation(s)
- Tomoya Niinae
- Department of Molecular & Cellular BioAnalysis, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto, Japan
| | - Koshi Imami
- Department of Molecular & Cellular BioAnalysis, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto, Japan; PRESTO, Japan Science and Technology Agency (JST), Chiyoda-ku, Tokyo, Japan
| | - Naoyuki Sugiyama
- Department of Molecular & Cellular BioAnalysis, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto, Japan
| | - Yasushi Ishihama
- Department of Molecular & Cellular BioAnalysis, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto, Japan; Laboratory of Clinical and Analytical Chemistry, National Institute of Biomedical Innovation, Health and Nutrition, Ibaraki, Osaka, Japan.
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26
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Alam M, Hasan GM, Hassan MI. A review on the role of TANK-binding kinase 1 signaling in cancer. Int J Biol Macromol 2021; 183:2364-2375. [PMID: 34111484 DOI: 10.1016/j.ijbiomac.2021.06.022] [Citation(s) in RCA: 31] [Impact Index Per Article: 7.8] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/28/2021] [Revised: 05/19/2021] [Accepted: 06/03/2021] [Indexed: 02/06/2023]
Abstract
TANK-binding kinase 1 (TBK1) regulates various biological processes including, NF-κB signaling, immune response, autophagy, cell division, Ras-mediated oncogenesis, and AKT pro-survival signaling. Enhanced TBK1 activity is associated with autoimmune diseases and cancer, suggesting its role in therapeutic targeting of interferonopathies. In addition, dysregulation of TBK1 activity promotes several inflammatory disorders and oncogenesis. Structural and biochemical study reports provide the molecular process of TBK1 activation and recap the substrate selection about TBK1. This review summarizes recent findings on the molecular mechanisms by which TBK1 is involved in cancer signaling. The IKK-ε and TBK1 are together associated with inflammatory diseases by inducing type I IFNs. Furthermore, TBK1 signaling regulates radiation-induced epithelial-mesenchymal transition by controlling phosphorylation of GSK-3β and expression of Zinc finger E-box-binding homeobox 1, suggesting, TBK1 could be targeted for radiotherapy-induced metastasis therapy. Despite a considerable increase in the list of TBK1 inhibitors, only a few has potential to control cancer. Among them, a compound BX795 is considered a potent and selective inhibitor of TBK1. We discussed the therapeutic potential of small-molecule inhibitors of TBK1, particularly those with high selectivity, which will enable further exploration in the therapeutic management of cancer and inflammatory diseases.
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Affiliation(s)
- Manzar Alam
- Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi 110025, India
| | - Gulam Mustafa Hasan
- Department of Biochemistry, College of Medicine, Prince Sattam Bin Abdulaziz University, PO Box 173, Al-Kharj 11942, Saudi Arabia
| | - Md Imtaiyaz Hassan
- Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi 110025, India.
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27
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Xiang S, Song S, Tang H, Smaill JB, Wang A, Xie H, Lu X. TANK-binding kinase 1 (TBK1): An emerging therapeutic target for drug discovery. Drug Discov Today 2021; 26:2445-2455. [PMID: 34051368 DOI: 10.1016/j.drudis.2021.05.016] [Citation(s) in RCA: 16] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/13/2020] [Revised: 04/20/2021] [Accepted: 05/22/2021] [Indexed: 12/16/2022]
Abstract
Dysregulation of TANK-binding kinase 1 (TBK1) homeostasis leads to the occurrence and progression of many diseases, such as inflammation, autoimmune diseases, metabolic diseases, and cancer. Therefore, there is a need to develop TBK1 inhibitors as therapeutic agents. In this review, we highlight the diverse biological functions of TBK1 and summarize the promising small-molecule inhibitors of TBK1 that have the potential to be developed as therapeutic candidates.
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Affiliation(s)
- Shuang Xiang
- Jinan University, 601 Huangpu Avenue West, Guangzhou 510632, China
| | - Shukai Song
- Jinan University, 601 Huangpu Avenue West, Guangzhou 510632, China
| | - Haotian Tang
- Division of Antitumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, China
| | - Jeff B Smaill
- Auckland Cancer Society Research Centre, School of Medical Sciences, The University of Auckland, Private Bag 92019, Auckland 1142, New Zealand
| | - Aiqun Wang
- Department of Anesthesiology, Guangzhou Red Cross Hospital Affiliated to Jinan University, Guangzhou 510220, China.
| | - Hua Xie
- Division of Antitumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, China.
| | - Xiaoyun Lu
- Jinan University, 601 Huangpu Avenue West, Guangzhou 510632, China.
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28
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Antonia RJ, Hagan RS, Baldwin AS. Expanding the View of IKK: New Substrates and New Biology. Trends Cell Biol 2021; 31:166-178. [PMID: 33422358 DOI: 10.1016/j.tcb.2020.12.003] [Citation(s) in RCA: 66] [Impact Index Per Article: 16.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/04/2020] [Revised: 11/16/2020] [Accepted: 12/07/2020] [Indexed: 01/07/2023]
Abstract
The inhibitor of kappa B kinase (IKK) family consists of IKKα, IKKβ, and the IKK-related kinases TBK1 and IKKε. These kinases are considered master regulators of inflammation and innate immunity via their control of the transcription factors NF-κB, IRF3, and IRF7. Novel phosphorylated substrates have been attributed to these kinases, a subset of which is not directly related to either inflammation or innate immunity. These findings have greatly expanded the perspectives on the biological activities of these kinases. In this review we highlight some of the novel substrates for this kinase family and discuss the biological implications of these phosphorylation events.
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Affiliation(s)
- Ricardo J Antonia
- Department of Surgery, Division of Surgical Oncology, and The Hellen Diller Comprehensive Cancer Center, The University of California San Francisco, San Francisco, CA, USA
| | - Robert S Hagan
- Division of Pulmonary Diseases and Critical Care Medicine, Department of Medicine, University of North Carolina, Chapel Hill, NC 27599, USA; Marsico Lung Institute, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
| | - Albert S Baldwin
- Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
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29
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Paul S, Pickrell AM. Hidden phenotypes of PINK1/Parkin knockout mice. Biochim Biophys Acta Gen Subj 2021; 1865:129871. [PMID: 33571581 DOI: 10.1016/j.bbagen.2021.129871] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2020] [Revised: 01/26/2021] [Accepted: 02/05/2021] [Indexed: 12/26/2022]
Abstract
PINK1, a serine/threonine ubiquitin kinase, and Parkin, an E3 ubiquitin ligase, work in coordination to target damaged mitochondria to the lysosome in a process called mitophagy. This review will cover what we have learned from PINK1 and Parkin knockout (KO) mice. Systemic PINK1 and Parkin KO mouse models haven't faithfully recapitulated early onset forms of Parkinson's disease found in humans with recessive mutations in these genes. However, the utilization of these mouse models has given us insight into how PINK1 and Parkin contribute to mitochondrial quality control and function in different tissues beyond the brain such as in heart and adipose tissue. Although PINK1 and Parkin KO mice have been generated over a decade ago, these models are still being used today to creatively elucidate cell-type specific functions. Recently, these mouse models have uncovered that these proteins contribute to innate immunity and cancer phenotypes.
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Affiliation(s)
- Swagatika Paul
- Graduate Studies in Biomedical and Veterinary Sciences, Virginia-Maryland College of Veterinary Medicine, Blacksburg, VA 24601, USA; School of Neuroscience, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA
| | - Alicia M Pickrell
- School of Neuroscience, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA.
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30
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Zheng H, Zhang J, Ma J, Jia Q. Engineering Magnetic Guanidyl-Functionalized Supramolecular Organic Framework for Efficient Enrichment of Global Phosphopeptides. ACS APPLIED MATERIALS & INTERFACES 2020; 12:57468-57476. [PMID: 33295748 DOI: 10.1021/acsami.0c18803] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/12/2023]
Abstract
Comprehensive mass spectrometry-based proteomics analysis is currently available but remains challenging, especially for post-translational modifications of phosphorylated proteins. Herein, multifunctional magnetic pillar[5]arene supramolecular organic frameworks were fabricated and immobilized with arginine (mP5SOF-Arg) for highly effective enrichment of global phosphopeptides. The specific phosphate-P5/phosphate-guanidine affinities and large surface area with regular porosity contribute to the high enrichment capacity. By coupling with mass spectrometry, high detection sensitivity (0.1 fmol), excellent selectivity (1:5000 molar ratios of β-casein/cytochrome c), and high recyclability (seven cycles) were achieved for phosphopeptide analysis. mP5SOF-Arg can efficiently enrich phosphopeptides from practical samples, including defatted milk, egg yolk, and human saliva. Notably, a total of 450 phosphopeptides were explored for highly selective identification from A594 cells and 1445 phosphopeptides were identified from mouse liver tissue samples. mP5SOF-Arg exhibited great potential to serve as the basis for peptidomic research to identify phosphopeptides and provided insight for biomarker discovery.
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Affiliation(s)
- Haijiao Zheng
- College of Chemistry, Jilin University, Changchun 130012, China
| | - Jingchun Zhang
- China-Japan Union Hospital, Jilin University, Changchun 130033, China
| | - Jiutong Ma
- College of Chemistry, Jilin University, Changchun 130012, China
| | - Qiong Jia
- College of Chemistry, Jilin University, Changchun 130012, China
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31
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Revach OY, Liu S, Jenkins RW. Targeting TANK-binding kinase 1 (TBK1) in cancer. Expert Opin Ther Targets 2020; 24:1065-1078. [PMID: 32962465 PMCID: PMC7644630 DOI: 10.1080/14728222.2020.1826929] [Citation(s) in RCA: 33] [Impact Index Per Article: 6.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/03/2020] [Accepted: 09/18/2020] [Indexed: 12/16/2022]
Abstract
INTRODUCTION TANK-binding kinase 1 (TBK1) is a Ser/Thr kinase with a central role in coordinating the cellular response to invading pathogens and regulating key inflammatory signaling cascades. While intact TBK1 signaling is required for successful anti-viral signaling, dysregulated TBK1 signaling has been linked to a variety of pathophysiologic conditions, including cancer. Several lines of evidence support a role for TBK1 in cancer pathogenesis, but the specific roles and regulation of TBK1 remain incompletely understood. A key challenge is the diversity of cellular processes that are regulated by TBK1, including inflammation, cell cycle, autophagy, energy homeostasis, and cell death. Nevertheless, evidence from pre-clinical cancer models suggests that targeting TBK1 may be an effective strategy for anti-cancer therapy in specific settings. AREAS COVERED This review provides an overview of the roles and regulation of TBK1 with a focus on cancer pathogenesis and drug targeting of TBK1 as an anti-cancer strategy. Relevant literature was derived from a PubMed search encompassing studies from 1999 to 2020. EXPERT OPINION TBK1 is emerging as a potential target for anti-cancer therapy. Inhibition of TBK1 alone may be insufficient to restrain the growth of most cancers; hence, combination strategies will likely be necessary. Improved understanding of tumor-intrinsic and tumor-extrinsic TBK1 signaling will inform novel therapeutic strategies.
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Affiliation(s)
- Or-yam Revach
- Massachusetts General Hospital Cancer Center, Harvard Medical School, Boston, MA, USA
- Laboratory of Systems Pharmacology, Harvard Medical School, Boston, MA, USA
| | - Shuming Liu
- Laboratory of Systems Pharmacology, Harvard Medical School, Boston, MA, USA
| | - Russell W. Jenkins
- Massachusetts General Hospital Cancer Center, Harvard Medical School, Boston, MA, USA
- Laboratory of Systems Pharmacology, Harvard Medical School, Boston, MA, USA
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32
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De R, Mazumder S, Bandyopadhyay U. Mediators of mitophagy that regulate mitochondrial quality control play crucial role in diverse pathophysiology. Cell Biol Toxicol 2020; 37:333-366. [PMID: 33067701 DOI: 10.1007/s10565-020-09561-1] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/23/2020] [Accepted: 10/05/2020] [Indexed: 02/06/2023]
Abstract
Mitochondria are double membrane-bound cellular work-horses constantly functioning to regulate vital aspects of cellular metabolism, bioenergetics, proliferation and death. Biogenesis, homeostasis and regulated turnover of mitochondria are stringently regulated to meet the bioenergetic requirements. Diverse external and internal stimuli including oxidative stress, diseases, xenobiotics and even age profoundly affect mitochondrial integrity. Damaged mitochondria need immediate segregation and selective culling to maintain physiological homeostasis. Mitophagy is a specialised form of macroautophagy that constantly checks mitochondrial quality followed by elimination of rogue mitochondria by lysosomal targeting through multiple pathways tightly regulated and activated in context-specific manners. Mitophagy is implicated in diverse oxidative stress-associated metabolic, proliferating and degenerative disorders owing to the centrality of mitopathology in diseases as well as the common mandate to eliminate damaged mitochondria for restoring physiological homeostasis. With improved health care and growing demand for precision medicine, specifically targeting the keystone factors in pathogenesis, more exploratory studies are focused on mitochondrial quality control as underlying guardian of cellular pathophysiology. In this context, mitophagy emerged as a promising area to focus biomedical research for identifying novel therapeutic targets against diseases linked with physiological redox perturbation. The present review provides a comprehensive account of the recent developments on mitophagy along with precise discussion on its impact on major diseases and possibilities of therapeutic modulation.
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Affiliation(s)
- Rudranil De
- Amity Institute of Biotechnology, Amity University, Kolkata, Plot No: 36, 37 & 38, Major Arterial Road, Action Area II, Kadampukur Village, Newtown, Kolkata, West Bengal, 700135, India
| | - Somnath Mazumder
- Division of Infectious Diseases and Immunology, CSIR-Indian Institute of Chemical Biology, 4 Raja S.C. Mullick Road, Kolkata, West Bengal, 700032, India
- Department of Zoology, Raja Peary Mohan College, 1 Acharya Dhruba Pal Road, Uttarpara, West Bengal, 712258, India
| | - Uday Bandyopadhyay
- Division of Infectious Diseases and Immunology, CSIR-Indian Institute of Chemical Biology, 4 Raja S.C. Mullick Road, Kolkata, West Bengal, 700032, India.
- Division of Molecular Medicine, Bose Institute, P-1/12, CIT Rd, Scheme VIIM, Kankurgachi, Kolkata, West Bengal, 700054, India.
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33
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Sarraf SA, Sideris DP, Giagtzoglou N, Ni L, Kankel MW, Sen A, Bochicchio LE, Huang CH, Nussenzweig SC, Worley SH, Morton PD, Artavanis-Tsakonas S, Youle RJ, Pickrell AM. PINK1/Parkin Influences Cell Cycle by Sequestering TBK1 at Damaged Mitochondria, Inhibiting Mitosis. Cell Rep 2020; 29:225-235.e5. [PMID: 31577952 PMCID: PMC6880866 DOI: 10.1016/j.celrep.2019.08.085] [Citation(s) in RCA: 66] [Impact Index Per Article: 13.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/26/2018] [Revised: 05/13/2019] [Accepted: 08/27/2019] [Indexed: 12/21/2022] Open
Abstract
PINK1 and Parkin are established mediators of mitophagy, the selective removal of damaged mitochondria by autophagy. PINK1 and Parkin have been proposed to act as tumor suppressors, as loss-of-function mutations are correlated with enhanced tumorigenesis. However, it is unclear how PINK1 and Parkin act in coordination during mitophagy to influence the cell cycle. Here we show that PINK1 and Parkin genetically interact with proteins involved in cell cycle regulation, and loss of PINK1 and Parkin accelerates cell growth. PINK1- and Parkin-mediated activation of TBK1 at the mitochondria during mitophagy leads to a block in mitosis due to the sequestration of TBK1 from its physiological role at centrosomes during mitosis. Our study supports a diverse role for the far-reaching, regulatory effects of mitochondrial quality control in cellular homeostasis and demonstrates that the PINK1/Parkin pathway genetically interacts with the cell cycle, providing a framework for understanding the molecular basis linking PINK1 and Parkin to mitosis. Sarraf et al. use mouse and fly genetics to discover that PINK1 and Parkin influence cell cycle progression. Mitophagy and mitosis independently activate TBK1 at damaged mitochondria and centrosomes, respectively, influencing whether the cell will address mitochondrial quality control or progress with proliferation.
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Affiliation(s)
- Shireen A Sarraf
- Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke, NIH, Bethesda, MD 20892, USA
| | - Dionisia P Sideris
- Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke, NIH, Bethesda, MD 20892, USA
| | | | - Lina Ni
- School of Neuroscience, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA
| | - Mark W Kankel
- Neuromuscular & Movement Disorders, Biogen, Inc., Cambridge, MA 02142, USA
| | - Anindya Sen
- Pathway Discovery Laboratory, Biogen, Inc., Cambridge, MA 02142, USA
| | - Lauren E Bochicchio
- Translational Biology, Medicine, and Health Graduate Program, Virginia Polytechnic Institute and State University, Roanoke, VA 24016, USA
| | - Chiu-Hui Huang
- Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke, NIH, Bethesda, MD 20892, USA
| | - Samuel C Nussenzweig
- Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke, NIH, Bethesda, MD 20892, USA
| | - Stuart H Worley
- School of Neuroscience, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA
| | - Paul D Morton
- Department of Biomedical Sciences and Pathobiology, College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA
| | - Spyros Artavanis-Tsakonas
- Pathway Discovery Laboratory, Biogen, Inc., Cambridge, MA 02142, USA; Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA
| | - Richard J Youle
- Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke, NIH, Bethesda, MD 20892, USA
| | - Alicia M Pickrell
- Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke, NIH, Bethesda, MD 20892, USA; School of Neuroscience, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA.
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Abstract
Fodrin and its erythroid cell-specific isoform spectrin are actin-associated fibrous proteins that play crucial roles in the maintenance of structural integrity in mammalian cells, which is necessary for proper cell function. Normal cell morphology is altered in diseases such as various cancers and certain neuronal disorders. Fodrin and spectrin are two-chain (αβ) molecules that are encoded by paralogous genes and share many features but also demonstrate certain differences. Fodrin (in humans, typically a heterodimer of the products of the SPTAN1 and SPTBN1 genes) is expressed in nearly all cell types and is especially abundant in neuronal tissues, whereas spectrin (in humans, a heterodimer of the products of the SPTA1 and SPTB1 genes) is expressed almost exclusively in erythrocytes. To fulfill a role in such a variety of different cell types, it was anticipated that fodrin would need to be a more versatile scaffold than spectrin. Indeed, as summarized here, domains unique to fodrin and its regulation by Ca2+, calmodulin, and a variety of posttranslational modifications (PTMs) endow fodrin with additional specific functions. However, how fodrin structural variations and misregulated PTMs may contribute to the etiology of various cancers and neurodegenerative diseases needs to be further investigated.
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Phosphorylation of BCKDK of BCAA catabolism at Y246 by Src promotes metastasis of colorectal cancer. Oncogene 2020; 39:3980-3996. [PMID: 32238881 PMCID: PMC7220852 DOI: 10.1038/s41388-020-1262-z] [Citation(s) in RCA: 49] [Impact Index Per Article: 9.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/24/2019] [Revised: 03/04/2020] [Accepted: 03/06/2020] [Indexed: 12/18/2022]
Abstract
Branched-chain α-keto acid dehydrogenase kinase (BCKDK), the key enzyme of branched-chain amino acids (BCAAs) metabolism, has been reported to promote colorectal cancer (CRC) tumorigenesis by upregulating the MEK-ERK signaling pathway. However, the profile of BCKDK in metastatic colorectal cancer (mCRC) remains unknown. Here, we report a novel role of BCKDK in mCRC. BCKDK is upregulated in CRC tissues. Increased BCKDK expression was associated with metastasis and poor clinical prognosis in CRC patients. Knockdown of BCKDK decreased CRC cell migration and invasion ex vivo, and lung metastasis in vivo. BCKDK promoted the epithelial mesenchymal transition (EMT) program, by decreasing the expression of E-cadherin, epithelial marker, and increasing the expression of N-cadherin and Vimentin, which are mesenchymal markers. Moreover, BCKDK-knockdown experiments in combination with phosphoproteomics analysis revealed the potent role of BCKDK in modulating multiple signal transduction pathways, including EMT and metastasis. Src phosphorylated BCKDK at the tyrosine 246 (Y246) site in vitro and ex vivo. Knockdown and knockout of Src downregulated the phosphorylation of BCKDK. Importantly, phosphorylation of BCKDK by Src enhanced the activity and stability of BCKDK, thereby promoting the migration, invasion, and EMT of CRC cells. In summary, the identification of BCKDK as a novel prometastatic factor in human CRC will be beneficial for further diagnostic biomarker studies and suggests novel targeting opportunities.
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36
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Marcinkowski M, Pilžys T, Garbicz D, Steciuk J, Zugaj D, Mielecki D, Sarnowski TJ, Grzesiuk E. Human and Arabidopsis alpha-ketoglutarate-dependent dioxygenase homolog proteins-New players in important regulatory processes. IUBMB Life 2020; 72:1126-1144. [PMID: 32207231 DOI: 10.1002/iub.2276] [Citation(s) in RCA: 27] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/03/2020] [Revised: 02/20/2020] [Accepted: 03/08/2020] [Indexed: 12/31/2022]
Abstract
The family of AlkB homolog (ALKBH) proteins, the homologs of Escherichia coli AlkB 2-oxoglutarate (2OG), and Fe(II)-dependent dioxygenase are involved in a number of important regulatory processes in eukaryotic cells including repair of alkylation lesions in DNA, RNA, and nucleoprotein complexes. There are nine human and thirteen Arabidopsis thaliana ALKBH proteins described, which exhibit diversified functions. Among them, human ALKBH5 and FaT mass and Obesity-associated (FTO) protein and Arabidopsis ALKBH9B and ALKBH10B have been recognized as N6 methyladenine (N6 meA) demethylases, the most abundant posttranscriptional modification in mRNA. The FTO protein is reported to be associated with obesity and type 2 diabetes, and involved in multiple other processes, while ALKBH5 is induced by hypoxia. Arabidopsis ALKBH9B is an N6 meA demethylase influencing plant susceptibility to viral infections via m6 A/A ratio control in viral RNA. ALKBH10B has been discovered to be a functional Arabidopsis homolog of FTO; thus, it is also an RNA N6 meA demethylase involved in plant flowering and several other regulatory processes including control of metabolism. High-throughput mass spectrometry showed multiple sites of human ALKBH phosphorylation. In the case of FTO, the type of modified residue decides about the further processing of the protein. This modification may result in subsequent protein ubiquitination and proteolysis, or in the blocking of these processes. However, the impact of phosphorylation on the other ALKBH function and their downstream pathways remains nearly unexplored in both human and Arabidopsis. Therefore, the investigation of evolutionarily conserved functions of ALKBH proteins and their regulatory impact on important cellular processes is clearly called for.
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Affiliation(s)
- Michał Marcinkowski
- Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland
| | - Tomaš Pilžys
- Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland
| | - Damian Garbicz
- Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland
| | - Jaroslaw Steciuk
- Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland
| | - Dorota Zugaj
- Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland
| | - Damian Mielecki
- Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland
| | - Tomasz J Sarnowski
- Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland
| | - Elżbieta Grzesiuk
- Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland
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Santana-Codina N, Chandhoke AS, Yu Q, Małachowska B, Kuljanin M, Gikandi A, Stańczak M, Gableske S, Jedrychowski MP, Scott DA, Aguirre AJ, Fendler W, Gray NS, Mancias JD. Defining and Targeting Adaptations to Oncogenic KRASG12C Inhibition Using Quantitative Temporal Proteomics. Cell Rep 2020; 30:4584-4599.e4. [DOI: 10.1016/j.celrep.2020.03.021] [Citation(s) in RCA: 48] [Impact Index Per Article: 9.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/16/2019] [Revised: 02/04/2020] [Accepted: 03/07/2020] [Indexed: 02/07/2023] Open
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The kinases IKBKE and TBK1 regulate MYC-dependent survival pathways through YB-1 in AML and are targets for therapy. Blood Adv 2019; 2:3428-3442. [PMID: 30504235 DOI: 10.1182/bloodadvances.2018016733] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/27/2018] [Accepted: 10/24/2018] [Indexed: 12/27/2022] Open
Abstract
To identify novel therapeutic targets in acute myeloid leukemia (AML), we examined kinase expression patterns in primary AML samples. We found that the serine/threonine kinase IKBKE, a noncanonical IkB kinase, is expressed at higher levels in myeloid leukemia cells compared with normal hematopoietic cells. Inhibiting IKBKE, or its close homolog TANK-binding kinase 1 (TBK1), by either short hairpin RNA knockdown or pharmacological compounds, induces apoptosis and reduces the viability of AML cells. Using gene expression profiling and gene set enrichment analysis, we found that IKBKE/TBK1-sensitive AML cells typically possess an MYC oncogenic signature. Consistent with this finding, the MYC oncoprotein was significantly downregulated upon IKBKE/TBK1 inhibition. Using proteomic analysis, we found that the oncogenic gene regulator YB-1 was activated by IKBKE/TBK1 through phosphorylation, and that YB-1 binds to the MYC promoter to enhance MYC gene transcription. Momelotinib (CYT387), a pharmacological inhibitor of IKBKE/TBK1, inhibits MYC expression, reduces viability and clonogenicity of primary AML cells, and demonstrates efficacy in a murine model of AML. Together, these data identify IKBKE/TBK1 as a promising therapeutic target in AML.
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39
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Antonia RJ, Castillo J, Herring LE, Serafin DS, Liu P, Graves LM, Baldwin AS, Hagan RS. TBK1 Limits mTORC1 by Promoting Phosphorylation of Raptor Ser877. Sci Rep 2019; 9:13470. [PMID: 31530866 PMCID: PMC6748941 DOI: 10.1038/s41598-019-49707-8] [Citation(s) in RCA: 31] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/04/2018] [Accepted: 08/29/2019] [Indexed: 01/02/2023] Open
Abstract
While best known for its role in the innate immune system, the TANK-binding kinase 1 (TBK1) is now known to play a role in modulating cellular growth and autophagy. One of the major ways that TBK1 accomplishes this task is by modulating the mechanistic Target of Rapamycin (mTOR), a master regulator that when activated promotes cell growth and inhibits autophagy. However, whether TBK1 promotes or inhibits mTOR activity is highly cell type and context dependent. To further understand the mechanism whereby TBK1 regulates mTOR, we tested the hypothesis that TBK1 phosphorylates a key component of the mTOR complex 1 (mTORC1), Raptor. Using kinase assays coupled with mass spectrometry, we mapped the position of the TBK1 dependent phosphorylation sites on Raptor in vitro. Among the sites identified in vitro, we found that TBK1 promotes Raptor Ser877 phosphorylation in cells both basally and in response to pathogen-associated molecules known to induce TBK1 activity. The levels of Raptor Ser877 phosphorylation were inversely correlated with the levels of mTOR activity. Expression of a mutant Raptor that could not be phosphorylated at Ser877 led to an increase in mTORC1 activity. We conclude that TBK1 limits mTORC1 activity by promoting Raptor Ser877 phosphorylation.
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Affiliation(s)
- Ricardo J Antonia
- Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.,The Helen Diller Family Comprehensive Cancer Center, The University of California San Francisco, San Francisco, California, USA
| | - Johnny Castillo
- Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
| | - Laura E Herring
- UNC Proteomics Core Facility, Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, USA
| | - D Stephen Serafin
- Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.,Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, USA
| | - Pengda Liu
- Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.,Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, USA
| | - Lee M Graves
- UNC Proteomics Core Facility, Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, USA
| | - Albert S Baldwin
- Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
| | - Robert S Hagan
- Division of Pulmonary Diseases and Critical Care Medicine, Department of Medicine, University of North Carolina, Chapel Hill, NC, 27599, USA. .,Marsico Lung Institute, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA.
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40
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Saul VV, Seibert M, Krüger M, Jeratsch S, Kracht M, Schmitz ML. ULK1/2 Restricts the Formation of Inducible SINT-Speckles, Membraneless Organelles Controlling the Threshold of TBK1 Activation. iScience 2019; 19:527-544. [PMID: 31442668 PMCID: PMC6710720 DOI: 10.1016/j.isci.2019.08.001] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/25/2019] [Revised: 07/08/2019] [Accepted: 08/01/2019] [Indexed: 12/13/2022] Open
Abstract
Membraneless organelles (MLOs) are liquid-like subcellular compartments providing spatiotemporal control to biological processes. This study reveals that cellular stress leads to the incorporation of the adaptor protein SINTBAD (TBKBP1) into membraneless, cytosolic speckles. Determination of the interactome identified >100 proteins forming constitutive and stress-inducible members of an MLO that we termed SINT-speckles. SINT-speckles partially colocalize with activated TBK1, and deletion of SINTBAD and the SINT-speckle component AZI2 leads to impaired TBK1 phosphorylation. Dynamic formation of SINT-speckles is positively controlled by the acetyltransferase KAT2A (GCN5) and antagonized by heat shock protein-mediated chaperone activity. SINT-speckle formation is also inhibited by the autophagy-initiating kinases ULK1/2, and knockdown of these kinases prevented focal TBK1 phosphorylation in a pathway-specific manner. The phlebovirus-encoded non-structural protein S enhances ULK1-mediated TBK1 phosphorylation and shows a stress-induced translocation to SINT-speckles, raising the possibility that viruses can also target this signaling hub to manipulate host cell functions.
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Affiliation(s)
- Vera Vivian Saul
- Institute of Biochemistry, Medical Faculty, Friedrichstrasse 24, Justus-Liebig-University, D-35392 Giessen, Germany, Member of the German Center for Lung Research
| | - Markus Seibert
- Institute of Biochemistry, Medical Faculty, Friedrichstrasse 24, Justus-Liebig-University, D-35392 Giessen, Germany, Member of the German Center for Lung Research
| | - Marcus Krüger
- Institute for Genetics, Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), 50931 Cologne, Germany; Center for Molecular Medicine (CMMC), University of Cologne, 50931 Cologne, Germany
| | - Sylvia Jeratsch
- Max Planck Institute for Heart and Lung Research, 61231 Bad Nauheim, Germany
| | - Michael Kracht
- Rudolf-Buchheim-Institute of Pharmacology, Justus-Liebig-University, D-35392 Giessen, Germany, Member of the German Center for Lung Research
| | - Michael Lienhard Schmitz
- Institute of Biochemistry, Medical Faculty, Friedrichstrasse 24, Justus-Liebig-University, D-35392 Giessen, Germany, Member of the German Center for Lung Research.
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Wang X, Teng F, Lu J, Mu D, Zhang J, Yu J. Expression and prognostic role of IKBKE and TBK1 in stage I non-small cell lung cancer. Cancer Manag Res 2019; 11:6593-6602. [PMID: 31406474 PMCID: PMC6642623 DOI: 10.2147/cmar.s204924] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/11/2019] [Accepted: 05/27/2019] [Indexed: 12/14/2022] Open
Abstract
BACKGROUND The inhibitors of nuclear factor kappa-B kinase subunit epsilon (IKBKE) and TANK-binding kinase 1 (TBK1) are important members of the nonclassical IKK family that share the kinase domain. They are important oncogenes for activation of several signaling pathways in several tumors. This study aims to explore the expression of IKBKE and TBK1 and their prognostic role in stage I non-small cell lung cancer (NSCLC). PATIENTS AND METHODS A total of 142 surgically resected stage I NSCLC patients were enrolled and immunohistochemistry of IKBKE and TBK1 was performed. RESULTS IKBKE and TBK1 were expressed in 121 (85.2%) and 114 (80.3%) of stage I NSCLC patients respectively. IKBKE expression was significantly associated with TBK1 expression (P=0.004). Furthermore, multivariate regression analyses showed there was a significant relationship between patients with risk factors, the recurrence pattern of metastasis and IKBKE+/TBK1+ co-expression (P=0.032 and P=0.022, respectively). In Kaplan-Meier survival curve analyses, the IKBKE+/TBK1+ co-expression subgroup was significantly associated with poor overall survival (P=0.014). CONCLUSIONS This is the first study to investigate the relationship between IKBKE and TBK1 expression and clinicopathologic characteristics in stage I NSCLC patients. IKBKE+/TBK1+ co-expression was significantly obvious in patients with risk factors and with recurrence pattern of distant metastasis. Furthermore, IKBKE+/TBK1+ is also an effective prognostic predictor for poor overall survival.
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Affiliation(s)
- Xin Wang
- Department of Oncology, Renmin Hospital of Wuhan University, Wuhan, Hubei430060, People’s Republic of China
- Department of Radiation Oncology, Shandong Cancer Hospital Affiliated to Shandong University, Shandong Academy of Medical Sciences, Jinan, Shandong250117, People’s Republic of China
| | - Feifei Teng
- Department of Radiation Oncology, Shandong Cancer Hospital Affiliated to Shandong University, Shandong Academy of Medical Sciences, Jinan, Shandong250117, People’s Republic of China
| | - Jie Lu
- Department of Neurosurgery, Shandong Province Qianfoshan Hospital of Shandong University, Jinan, Shandong250014, People’s Republic of China
| | - Dianbin Mu
- Department of Pathology, Shandong Cancer Hospital Affiliated to Shandong University, Shandong Academy of Medical Sciences, Jinan, Shandong250117, People’s Republic of China
| | - Jianbo Zhang
- Department of Pathology, Shandong Cancer Hospital Affiliated to Shandong University, Shandong Academy of Medical Sciences, Jinan, Shandong250117, People’s Republic of China
| | - Jinming Yu
- Department of Oncology, Renmin Hospital of Wuhan University, Wuhan, Hubei430060, People’s Republic of China
- Department of Radiation Oncology, Shandong Cancer Hospital Affiliated to Shandong University, Shandong Academy of Medical Sciences, Jinan, Shandong250117, People’s Republic of China
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Qu H, Liu L, Liu Z, Qin H, Liao Z, Xia P, Yang Y, Li B, Gao F, Cai J. Blocking TBK1 alleviated radiation-induced pulmonary fibrosis and epithelial-mesenchymal transition through Akt-Erk inactivation. Exp Mol Med 2019; 51:1-17. [PMID: 30988282 PMCID: PMC6465273 DOI: 10.1038/s12276-019-0240-4] [Citation(s) in RCA: 27] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/16/2018] [Revised: 11/25/2018] [Accepted: 12/11/2018] [Indexed: 12/23/2022] Open
Abstract
As a common serious complication of thoracic radiotherapy, radiation-induced pulmonary fibrosis (RIPF) severely limits radiation therapy approaches. Epithelial–mesenchymal transition (EMT) is a direct contributor to the fibroblast pool during fibrogenesis, and prevention of EMT is considered an effective strategy to inhibit tissue fibrosis. Our previous study revealed that TANK-binding kinase 1 (TBK1) regulates EMT in lung cancer cells. In the present study, we aimed to investigate the therapeutic potential of targeting TBK1 to prevent RIPF and EMT progression. We found radiation-induced EMT and pulmonary fibrosis in normal alveolar epithelial cells and lung tissues. TBK1 knockdown or inhibition significantly reversed EMT in vivo and in vitro and attenuated pulmonary fibrosis and collagen deposition. Moreover, we observed that TBK1 was elevated in a time- and dose-dependent manner by radiation. Meanwhile, radiation also induced time- and dose-dependent activation of AKT and ERK, each of whose inhibitors suppressed radiation-induced EMT. Intriguingly, silencing of TBK1 with shRNA also blocked the radiation-induced activation of AKT and ERK signaling. The ERK inhibitor did not obviously affect the expression of TBK1 or phosphorylated AKT, while AKT inhibition suppressed activation of ERK without changing the expression of TBK1. Finally, we found that a TBK1 inhibitor inhibited inflammatory cytokine expression in a RIPF model and Amlexanox protected normal cells and mice from ionizing radiation. In conclusion, our results indicate that the TBK1–AKT–ERK signaling pathway regulates radiation-induced EMT in normal alveolar epithelial cells, suggesting that TBK1 is a potential target for pulmonary fibrosis prevention during cancer radiotherapy. The risk of scarred tissues and respiratory distress during radiation treatment of lung cancer could be reduced by targeting an enzyme that alters healthy cells. Lung cancer radiotherapy often causes pulmonary fibrosis, excessive growth of fibrous tissues in the lungs, involving the transition of normal epithelial cells into an invasive form of multipotent stem cells. The development of pulmonary fibrosis limits the clinical application of radiotherapy. Hongjin Qu and co-workers at the Second Military University in Shanghai, China, previously demonstrated that the TANK-binding kinase 1 (TBK1) enzyme regulates this transition. Now, the team have shown that levels of TBK1 itself increased during radiation treatment, together with two proteins that would normally suppress alterations in healthy cells. Inhibiting TBK1, both in cell cultures and mouse models, reversed the cell transitions and prevented pulmonary fibrosis.
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Affiliation(s)
- Hongjin Qu
- Department of Radiation Medicine, Faculty of Naval Medicine, Second Military Medical University, 800, Xiangyin Road, 200433, Shanghai, P. R. China
| | - Lei Liu
- Department of Radiation Medicine, Faculty of Naval Medicine, Second Military Medical University, 800, Xiangyin Road, 200433, Shanghai, P. R. China
| | - Zhe Liu
- Department of Radiation Medicine, Faculty of Naval Medicine, Second Military Medical University, 800, Xiangyin Road, 200433, Shanghai, P. R. China
| | - Hongran Qin
- Department of Radiation Medicine, Faculty of Naval Medicine, Second Military Medical University, 800, Xiangyin Road, 200433, Shanghai, P. R. China
| | - Zebin Liao
- Department of Radiation Medicine, Faculty of Naval Medicine, Second Military Medical University, 800, Xiangyin Road, 200433, Shanghai, P. R. China
| | - Penglin Xia
- Department of Radiation Medicine, Faculty of Naval Medicine, Second Military Medical University, 800, Xiangyin Road, 200433, Shanghai, P. R. China
| | - Yanyong Yang
- Department of Radiation Medicine, Faculty of Naval Medicine, Second Military Medical University, 800, Xiangyin Road, 200433, Shanghai, P. R. China
| | - Bailong Li
- Department of Radiation Medicine, Faculty of Naval Medicine, Second Military Medical University, 800, Xiangyin Road, 200433, Shanghai, P. R. China.
| | - Fu Gao
- Department of Radiation Medicine, Faculty of Naval Medicine, Second Military Medical University, 800, Xiangyin Road, 200433, Shanghai, P. R. China.
| | - Jianming Cai
- Department of Radiation Medicine, Faculty of Naval Medicine, Second Military Medical University, 800, Xiangyin Road, 200433, Shanghai, P. R. China.
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Cruz VH, Arner EN, Du W, Bremauntz AE, Brekken RA. Axl-mediated activation of TBK1 drives epithelial plasticity in pancreatic cancer. JCI Insight 2019; 5:126117. [PMID: 30938713 PMCID: PMC6538328 DOI: 10.1172/jci.insight.126117] [Citation(s) in RCA: 24] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2018] [Accepted: 03/27/2019] [Indexed: 01/11/2023] Open
Abstract
Pancreatic ductal adenocarcinoma (PDA) is characterized by an activating mutation in KRAS. Direct inhibition of KRAS through pharmacological means remains a challenge; however, targeting key KRAS effectors has therapeutic potential. We investigated the contribution of TANK-binding kinase 1 (TBK1), a critical downstream effector of mutant active KRAS, to PDA progression. We report that TBK1 supports the growth and metastasis of KRAS-mutant PDA by driving an epithelial plasticity program in tumor cells that enhances invasive and metastatic capacity. Further, we identify that the receptor tyrosine kinase Axl induces TBK1 activity in a Ras-RalB-dependent manner. These findings demonstrate that TBK1 is central to an Axl-driven epithelial-mesenchymal transition in KRAS-mutant PDA and suggest that interruption of the Axl-TBK1 signaling cascade above or below KRAS has potential therapeutic efficacy in this recalcitrant disease.
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Affiliation(s)
- Victoria H. Cruz
- Division of Surgical Oncology, Department of Surgery, and the Hamon Center for Therapeutic Oncology Research
| | - Emily N. Arner
- Division of Surgical Oncology, Department of Surgery, and the Hamon Center for Therapeutic Oncology Research
| | - Wenting Du
- Division of Surgical Oncology, Department of Surgery, and the Hamon Center for Therapeutic Oncology Research
| | | | - Rolf A. Brekken
- Division of Surgical Oncology, Department of Surgery, and the Hamon Center for Therapeutic Oncology Research
- Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas, USA
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Needham EJ, Parker BL, Burykin T, James DE, Humphrey SJ. Illuminating the dark phosphoproteome. Sci Signal 2019; 12:12/565/eaau8645. [PMID: 30670635 DOI: 10.1126/scisignal.aau8645] [Citation(s) in RCA: 214] [Impact Index Per Article: 35.7] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
Protein phosphorylation is a major regulator of protein function and biological outcomes. This was first recognized through functional biochemical experiments, and in the past decade, major technological advances in mass spectrometry have enabled the study of protein phosphorylation on a global scale. This rapidly growing field of phosphoproteomics has revealed that more than 100,000 distinct phosphorylation events occur in human cells, which likely affect the function of every protein. Phosphoproteomics has improved the understanding of the function of even the most well-characterized protein kinases by revealing new downstream substrates and biology. However, current biochemical and bioinformatic approaches have only identified kinases for less than 5% of the phosphoproteome, and functional assignments of phosphosites are almost negligible. Notably, our understanding of the relationship between kinases and their substrates follows a power law distribution, with almost 90% of phosphorylation sites currently assigned to the top 20% of kinases. In addition, more than 150 kinases do not have a single known substrate. Despite a small group of kinases dominating biomedical research, the number of substrates assigned to a kinase does not correlate with disease relevance as determined by pathogenic human mutation prevalence and mouse model phenotypes. Improving our understanding of the substrates targeted by all kinases and functionally annotating the phosphoproteome will be broadly beneficial. Advances in phosphoproteomics technologies, combined with functional screening approaches, should make it feasible to illuminate the connectivity and functionality of the entire phosphoproteome, providing enormous opportunities for discovering new biology, therapeutic targets, and possibly diagnostics.
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Affiliation(s)
- Elise J Needham
- School of Life and Environmental Sciences, University of Sydney, Sydney, NSW 2006, Australia.,Charles Perkins Centre, University of Sydney, Sydney, NSW 2006, Australia
| | - Benjamin L Parker
- School of Life and Environmental Sciences, University of Sydney, Sydney, NSW 2006, Australia.,Charles Perkins Centre, University of Sydney, Sydney, NSW 2006, Australia
| | - Timur Burykin
- Charles Perkins Centre, University of Sydney, Sydney, NSW 2006, Australia
| | - David E James
- School of Life and Environmental Sciences, University of Sydney, Sydney, NSW 2006, Australia. .,Charles Perkins Centre, University of Sydney, Sydney, NSW 2006, Australia.,Sydney Medical School, University of Sydney, Sydney, NSW 2006, Australia
| | - Sean J Humphrey
- School of Life and Environmental Sciences, University of Sydney, Sydney, NSW 2006, Australia. .,Charles Perkins Centre, University of Sydney, Sydney, NSW 2006, Australia
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Ctortecka C, Palve V, Kuenzi BM, Fang B, Sumi NJ, Izumi V, Novakova S, Kinose F, Remsing Rix LL, Haura EB, Koomen JM, Rix U. Functional Proteomics and Deep Network Interrogation Reveal a Complex Mechanism of Action of Midostaurin in Lung Cancer Cells. Mol Cell Proteomics 2018; 17:2434-2447. [PMID: 30217950 PMCID: PMC6283294 DOI: 10.1074/mcp.ra118.000713] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2018] [Revised: 08/16/2018] [Indexed: 12/21/2022] Open
Abstract
Lung cancer is associated with high prevalence and mortality, and despite significant successes with targeted drugs in genomically defined subsets of lung cancer and immunotherapy, the majority of patients currently does not benefit from these therapies. Through a targeted drug screen, we found the recently approved multi-kinase inhibitor midostaurin to have potent activity in several lung cancer cells independent of its intended target, PKC, or a specific genomic marker. To determine the underlying mechanism of action we applied a layered functional proteomics approach and a new data integration method. Using chemical proteomics, we identified multiple midostaurin kinase targets in these cells. Network-based integration of these targets with quantitative tyrosine and global phosphoproteomics data using protein-protein interactions from the STRING database suggested multiple targets are relevant for the mode of action of midostaurin. Subsequent functional validation using RNA interference and selective small molecule probes showed that simultaneous inhibition of TBK1, PDPK1 and AURKA was required to elicit midostaurin's cellular effects. Immunoblot analysis of downstream signaling nodes showed that combined inhibition of these targets altered PI3K/AKT and cell cycle signaling pathways that in part converged on PLK1. Furthermore, rational combination of midostaurin with the potent PLK1 inhibitor BI2536 elicited strong synergy. Our results demonstrate that combination of complementary functional proteomics approaches and subsequent network-based data integration can reveal novel insight into the complex mode of action of multi-kinase inhibitors, actionable targets for drug discovery and cancer vulnerabilities. Finally, we illustrate how this knowledge can be used for the rational design of synergistic drug combinations with high potential for clinical translation.
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Affiliation(s)
- Claudia Ctortecka
- Department of Drug Discovery, H. Lee Moffitt Cancer Center & Research Institute, Tampa, Florida 33612
| | - Vinayak Palve
- Department of Drug Discovery, H. Lee Moffitt Cancer Center & Research Institute, Tampa, Florida 33612
| | - Brent M Kuenzi
- Department of Drug Discovery, H. Lee Moffitt Cancer Center & Research Institute, Tampa, Florida 33612; Cancer Biology PhD Program, University of South Florida, Tampa, Florida 33620
| | - Bin Fang
- Proteomics and Metabolomics Core, H. Lee Moffitt Cancer Center & Research Institute, Tampa, Florida 33612
| | - Natalia J Sumi
- Department of Drug Discovery, H. Lee Moffitt Cancer Center & Research Institute, Tampa, Florida 33612; Cancer Biology PhD Program, University of South Florida, Tampa, Florida 33620
| | - Victoria Izumi
- Proteomics and Metabolomics Core, H. Lee Moffitt Cancer Center & Research Institute, Tampa, Florida 33612
| | - Silvia Novakova
- Department of Drug Discovery, H. Lee Moffitt Cancer Center & Research Institute, Tampa, Florida 33612
| | - Fumi Kinose
- Department of Thoracic Oncology, H. Lee Moffitt Cancer Center & Research Institute, Tampa, Florida 33612
| | - Lily L Remsing Rix
- Department of Drug Discovery, H. Lee Moffitt Cancer Center & Research Institute, Tampa, Florida 33612
| | - Eric B Haura
- Department of Thoracic Oncology, H. Lee Moffitt Cancer Center & Research Institute, Tampa, Florida 33612
| | - John Matthew Koomen
- Department of Molecular Oncology, H. Lee Moffitt Cancer Center & Research Institute, Tampa, Florida 33612
| | - Uwe Rix
- Department of Drug Discovery, H. Lee Moffitt Cancer Center & Research Institute, Tampa, Florida 33612.
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Roles for the IKK-Related Kinases TBK1 and IKKε in Cancer. Cells 2018; 7:cells7090139. [PMID: 30223576 PMCID: PMC6162516 DOI: 10.3390/cells7090139] [Citation(s) in RCA: 57] [Impact Index Per Article: 8.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/29/2018] [Revised: 09/11/2018] [Accepted: 09/13/2018] [Indexed: 01/21/2023] Open
Abstract
While primarily studied for their roles in innate immune response, the IκB kinase (IKK)-related kinases TANK-binding kinase 1 (TBK1) and IKKε also promote the oncogenic phenotype in a variety of cancers. Additionally, several substrates of these kinases control proliferation, autophagy, cell survival, and cancer immune responses. Here we review the involvement of TBK1 and IKKε in controlling different cancers and in regulating responses to cancer immunotherapy.
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Weil R, Laplantine E, Curic S, Génin P. Role of Optineurin in the Mitochondrial Dysfunction: Potential Implications in Neurodegenerative Diseases and Cancer. Front Immunol 2018; 9:1243. [PMID: 29971063 PMCID: PMC6018216 DOI: 10.3389/fimmu.2018.01243] [Citation(s) in RCA: 56] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/15/2018] [Accepted: 05/17/2018] [Indexed: 12/11/2022] Open
Abstract
Optineurin (Optn) is a 577 aa protein encoded by the Optn gene. Mutations of Optn are associated with normal tension glaucoma and amyotrophic lateral sclerosis, and its gene has also been linked to the development of Paget’s disease of bone and Crohn’s disease. Optn is involved in diverse cellular functions, including NF-κB regulation, membrane trafficking, exocytosis, vesicle transport, reorganization of actin and microtubules, cell cycle control, and autophagy. Besides its role in xenophagy and autophagy of aggregates, Optn has been identified as a primary autophagy receptor, among the five adaptors that translocate to mitochondria during mitophagy. Mitophagy is a selective macroautophagy process during which irreparable mitochondria are degraded, preventing accumulation of defective mitochondria and limiting the release of reactive oxygen species and proapoptotic factors. Mitochondrial quality control via mitophagy is central to the health of cells. One of the important surveillance pathways of mitochondrial health is the recently defined signal transduction pathway involving the mitochondrial PTEN-induced putative kinase 1 (PINK1) protein and the cytosolic RING-between-RING ubiquitin ligase Parkin. Both of these proteins, when mutated, have been identified in certain forms of Parkinson’s disease. By targeting ubiquitinated mitochondria to autophagosomes through its association with autophagy related proteins, Optn is responsible for a critical step in mitophagy. This review reports recent discoveries on the role of Optn in mitophagy and provides insight into its link with neurodegenerative diseases. We will also discuss the involvement of Optn in other pathologies in which mitophagy dysfunctions are involved including cancer.
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Affiliation(s)
- Robert Weil
- Laboratory of Signaling and Pathogenesis, Institut Pasteur, CNRS UMR3691, Paris, France
| | - Emmanuel Laplantine
- Laboratory of Signaling and Pathogenesis, Institut Pasteur, CNRS UMR3691, Paris, France
| | - Shannel Curic
- Laboratory of Signaling and Pathogenesis, Institut Pasteur, CNRS UMR3691, Paris, France
| | - Pierre Génin
- Laboratory of Signaling and Pathogenesis, Institut Pasteur, CNRS UMR3691, Paris, France
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Abstract
Translation is a key step in the regulation of gene expression and one of the most energy-consuming processes in the cell. In response to various stimuli, multiple signaling pathways converge on the translational machinery to regulate its function. To date, the roles of phosphoinositide 3-kinase (PI3K)/AKT and the mitogen-activated protein kinase (MAPK) pathways in the regulation of translation are among the best understood. Both pathways engage the mechanistic target of rapamycin (mTOR) to regulate a variety of components of the translational machinery. While these pathways regulate protein synthesis in homeostasis, their dysregulation results in aberrant translation leading to human diseases, including diabetes, neurological disorders, and cancer. Here we review the roles of the PI3K/AKT and MAPK pathways in the regulation of mRNA translation. We also highlight additional signaling mechanisms that have recently emerged as regulators of the translational apparatus.
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Lombardi LM, Zaghlula M, Sztainberg Y, Baker SA, Klisch TJ, Tang AA, Huang EJ, Zoghbi HY. An RNA interference screen identifies druggable regulators of MeCP2 stability. Sci Transl Med 2018; 9:9/404/eaaf7588. [PMID: 28835516 DOI: 10.1126/scitranslmed.aaf7588] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/26/2016] [Revised: 04/14/2016] [Accepted: 06/13/2017] [Indexed: 12/14/2022]
Abstract
Alterations in gene dosage due to copy number variation are associated with autism spectrum disorder, intellectual disability (ID), and other psychiatric disorders. The nervous system is so acutely sensitive to the dose of methyl-CpG-binding protein 2 (MeCP2) that even a twofold change in MeCP2 protein-either increased or decreased-results in distinct disorders with overlapping features including ID, autistic behavior, and severe motor dysfunction. Rett syndrome is caused by loss-of-function mutations in MECP2, whereas duplications spanning the MECP2 locus result in MECP2 duplication syndrome (MDS), which accounts for ~1% of X-linked ID. Despite evidence from mouse models that restoring MeCP2 can reverse the course of disease, there are currently no U.S. Food and Drug Administration-approved therapies available to clinically modulate MeCP2 abundance. We used a forward genetic screen against all known human kinases and phosphatases to identify druggable regulators of MeCP2 stability. Two putative modulators of MeCP2, HIPK2 (homeodomain-interacting protein kinase 2) and PP2A (protein phosphatase 2A), were validated as stabilizers of MeCP2 in vivo. Further, pharmacological inhibition of PP2A in vivo reduced MeCP2 in the nervous system and rescued both overexpression and motor abnormalities in a mouse model of MDS. Our findings reveal potential therapeutic targets for treating disorders of altered MECP2 dosage.
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Affiliation(s)
- Laura M Lombardi
- Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA.,Jan and Dan Duncan Neurological Research Institute at Texas Children's Hospital, Houston, TX 77030, USA.,Howard Hughes Medical Institute, Baylor College of Medicine, Houston, TX 77030, USA
| | - Manar Zaghlula
- Jan and Dan Duncan Neurological Research Institute at Texas Children's Hospital, Houston, TX 77030, USA.,Graduate Program in Translational Biology and Molecular Medicine, Baylor College of Medicine, Houston, TX 77030, USA
| | - Yehezkel Sztainberg
- Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA.,Jan and Dan Duncan Neurological Research Institute at Texas Children's Hospital, Houston, TX 77030, USA
| | - Steven A Baker
- Department of Pathology, Stanford University School of Medicine, Palo Alto, CA 94305, USA
| | - Tiemo J Klisch
- Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA.,Jan and Dan Duncan Neurological Research Institute at Texas Children's Hospital, Houston, TX 77030, USA
| | - Amy A Tang
- Department of Pathology, University of California, San Francisco, San Francisco, CA 94158, USA
| | - Eric J Huang
- Department of Pathology, University of California, San Francisco, San Francisco, CA 94158, USA
| | - Huda Y Zoghbi
- Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA. .,Jan and Dan Duncan Neurological Research Institute at Texas Children's Hospital, Houston, TX 77030, USA.,Howard Hughes Medical Institute, Baylor College of Medicine, Houston, TX 77030, USA.,Graduate Program in Translational Biology and Molecular Medicine, Baylor College of Medicine, Houston, TX 77030, USA.,Department of Neuroscience, Baylor College of Medicine, Houston, TX 77030, USA.,Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030, USA
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50
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Zhong M, Jiang Q, Jin R. USP4 expression independently predicts favorable survival in lung adenocarcinoma. IUBMB Life 2018; 70:670-677. [PMID: 29667299 DOI: 10.1002/iub.1755] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/18/2018] [Accepted: 03/29/2018] [Indexed: 01/29/2023]
Abstract
Ubiquitin specific protease 4 (USP4) is a member of the USPs family, which catalyzes the cleavage of ubiquitin from a series of protein substrates, thereby modulating a number of cellular signaling pathways. In this study, we aimed to explore the expression profile of USP4 in lung adenocarcinoma (LUAD) using large patient cohorts in the Cancer Genome Atlas and the International Cancer Genome Consortium and to investigate its prognostic value and the possible mechanisms of its dysregulation. Results showed that USP4 was significantly downregulated in LUAD tissues (N = 514) compared with the normal controls (N = 59). The high USP4 expression group had significantly better overall survival (OS) and recurrence-free survival (RFS). Multivariate analysis showed that preserved USP4 expression was an independent prognostic factor of favorable OS (HR: 0.574, 95%CI: 0.427-0.771, P < 0.001) and RFS (HR: 0.625, 95%CI: 0.444-0.880, P = 0.007) in LUAD. In comparison, although USP4 was downregulated in lung squamous cell carcinoma, its expression had no prognostic value in term of OS and RFS. By examining USP4 DNA copy number alterations (CNAs) (N = 511) and DNA methylation (N = 453) in LUAD, we found that DNA shallow deletion was frequent (-1, N = 239, 46.8%) and was associated with significantly decreased USP4 expression compared with the copy-neutral (0) cases. The methylation status of some CpG sites in USP4 DNA was negatively correlated with USP4 expression. Based on these findings, we infer that USP4 expression might be a favorable biomarker in terms of OS and RFS in LUAD patients. DNA shallow deletion and hypermethylation might be two important mechanisms of decreased USP4 in these patients. © 2018 IUBMB Life, 70(7):670-677, 2018.
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Affiliation(s)
- Ming Zhong
- Department of Respiration, China-Japan Union Hospital of Jilin University, Changchun, Jilin, China
| | - Qi Jiang
- Department of Nephrology, China-Japan Union Hospital of Jilin University, Changchun, Jilin, China
| | - Ronghui Jin
- Department of Respiration, China-Japan Union Hospital of Jilin University, Changchun, Jilin, China
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