|
1
|
Folahan JT, Barabutis N. NEK kinases in cell cycle regulation, DNA damage response, and cancer progression. Tissue Cell 2025; 94:102811. [PMID: 40037068 PMCID: PMC11912005 DOI: 10.1016/j.tice.2025.102811] [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: 11/20/2024] [Revised: 02/16/2025] [Accepted: 02/21/2025] [Indexed: 03/06/2025]
Abstract
The NIMA-related kinase (NEK) family of serine/threonine kinases is essential for the regulation of cell cycle progression, mitotic spindle assembly, and genomic stability. In this review, we explore the structural and functional diversity of NEK kinases, highlighting their roles in both canonical and non-canonical cellular processes. We examine recent preclinical findings on NEK inhibition, showcasing promising results for NEK-targeted therapies, particularly in cancer types characterized by high NEK expression. We discussed the therapeutic potential of targeting NEKs as modulators of cell cycle and DDR pathways, with a focus on identifying strategies to exploit NEK activity for enhanced treatment efficacy. Future research directions are proposed to further elucidate NEK-mediated mechanisms and to develop selective inhibitors that target NEK-related pathways.
Collapse
Affiliation(s)
- Joy T Folahan
- School of Basic Pharmaceutical and Toxicological Sciences, College of Pharmacy, University of Louisiana Monroe, Monroe, LA 71201, USA
| | - Nektarios Barabutis
- School of Basic Pharmaceutical and Toxicological Sciences, College of Pharmacy, University of Louisiana Monroe, Monroe, LA 71201, USA.
| |
Collapse
|
|
2
|
Baskin A, Soudah N, Gilad N, Halevi N, Darlyuk-Saadon I, Schoffman H, Engelberg D. All intrinsically active Erk1/2 mutants autophosphorylate threonine207/188, a plausible regulator of the TEY motif phosphorylation. J Biol Chem 2025; 301:108509. [PMID: 40222547 DOI: 10.1016/j.jbc.2025.108509] [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: 10/23/2024] [Revised: 03/19/2025] [Accepted: 04/08/2025] [Indexed: 04/15/2025] Open
Abstract
The extracellular-activated kinases 1 & 2 (Erk1/2) are catalytically active when dually phosphorylated on a TEY motif located at the activation loop. In human patients with cardiac hypertrophy, Erk1/2 are phosphorylated on yet another activation loop's residue, T207/188. Intrinsically active variants of Erk1/2, mutated at R84/65, are also (auto)phosphorylated on T207/188. It is not known whether T207/188 phosphorylation is restricted to these cases, nor how it affects Erks' activity. We report that T207/188 phosphorylation is not rare, as we found that: 1) All known auto-activated Erk1/2 variants are phosphorylated on T207/188. 2) It occurs in various cell lines and mouse tissues. 3) It is extremely high in patients with skeletal muscle atrophies or myopathies. We propose that T207/188 controls the permissiveness of the TEY motif for phosphorylation because T207/188-mutated Erk1/2 and the yeast Erk/Mpk1 were efficiently dually phosphorylated when expressed in HEK293 or yeast cells, respectively. The T207/188-mutated Mpk1 was not TEY-phosphorylated in cells knocked out for MEKs, suggesting that its enhanced phosphorylation in wild-type cells is MEK-dependent. Thus, as T207/188-mutated Erk1/2 and Mpk1 recruit MEKs, the role of T207/188 is to impede MEKs' ability to phosphorylate Erks. T207/188 also impedes autophosphorylation as recombinant Erk2 mutated at T188 is spontaneously autophosphorylated, although exclusively on Y185. The role of T207/188 in regulating activation loop phosphorylation may be common to most Ser/Thr kinases, as 86% of them (in the human kinome) possess T207/188 orthologs, and 160 of them were already reported to be phosphorylated on this residue.
Collapse
Affiliation(s)
- Alexey Baskin
- Department of Biological Chemistry, The Institute of Life Science, The Hebrew University of Jerusalem, Jerusalem, Israel
| | - Nadine Soudah
- Department of Biological Chemistry, The Institute of Life Science, The Hebrew University of Jerusalem, Jerusalem, Israel
| | - Nechama Gilad
- Department of Biological Chemistry, The Institute of Life Science, The Hebrew University of Jerusalem, Jerusalem, Israel; Singapore-HUJ Alliance for Research and Enterprise, Mechanisms of Liver Inflammatory Diseases Program, National University of Singapore, Singapore
| | - Neriya Halevi
- Department of Biological Chemistry, The Institute of Life Science, The Hebrew University of Jerusalem, Jerusalem, Israel
| | - Ilona Darlyuk-Saadon
- Singapore-HUJ Alliance for Research and Enterprise, Mechanisms of Liver Inflammatory Diseases Program, National University of Singapore, Singapore
| | - Hanan Schoffman
- Stein Family Mass Spectrometry Unit, The Research Infrastructure Center, The Institute of Life Science, The Hebrew University of Jerusalem, Jerusalem, Israel
| | - David Engelberg
- Department of Biological Chemistry, The Institute of Life Science, The Hebrew University of Jerusalem, Jerusalem, Israel; Singapore-HUJ Alliance for Research and Enterprise, Mechanisms of Liver Inflammatory Diseases Program, National University of Singapore, Singapore; Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.
| |
Collapse
|
|
3
|
Ullah MA, Garcillán B, Whitlock E, Figgett WA, Infantino S, Eslami M, Yang S, Rahman MA, Sheng YH, Weber N, Schneider P, Tam CS, Mackay F. An unappreciated cell survival-independent role for BAFF initiating chronic lymphocytic leukemia. Front Immunol 2024; 15:1345515. [PMID: 38469292 PMCID: PMC10927009 DOI: 10.3389/fimmu.2024.1345515] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/27/2023] [Accepted: 01/25/2024] [Indexed: 03/13/2024] Open
Abstract
Background Chronic Lymphocytic Leukemia (CLL) is characterized by the expansion of CD19+ CD5+ B cells but its origin remains debated. Mutated CLL may originate from post-germinal center B cells and unmutated CLL from CD5+ mature B cell precursors. Irrespective of precursor types, events initiating CLL remain unknown. The cytokines BAFF and APRIL each play a significant role in CLL cell survival and accumulation, but their involvement in disease initiation remains unclear. Methods We generated novel CLL models lacking BAFF or APRIL. In vivo experiments were conducted to explore the impact of BAFF or APRIL loss on leukemia initiation, progression, and dissemination. Additionally, RNA-seq and quantitative real-time PCR were performed to unveil the transcriptomic signature influenced by BAFF in CLL. The direct role of BAFF in controlling the expression of tumor-promoting genes was further assessed in patient-derived primary CLL cells ex-vivo. Results Our findings demonstrate a crucial role for BAFF, but not APRIL, in the initiation and dissemination of CLL cells. In the absence of BAFF or its receptor BAFF-R, the TCL1 transgene only increases CLL cell numbers in the peritoneal cavity, without dissemination into the periphery. While BAFF binding to BAFF-R is dispensable for peritoneal CLL cell survival, it is necessary to activate a tumor-promoting gene program, potentially linked to CLL initiation and progression. This direct role of BAFF in controlling the expression of tumor-promoting genes was confirmed in patient-derived primary CLL cells ex-vivo. Conclusions Our study, involving both mouse and human CLL cells, suggests that BAFF might initiate CLL through mechanisms independent of cell survival. Combining current CLL therapies with BAFF inhibition could offer a dual benefit by reducing peripheral tumor burden and suppressing transformed CLL cell output.
Collapse
Affiliation(s)
- Md Ashik Ullah
- Queensland Institute of Medical Research (QIMR) Berghofer Medical Research Institute, Cancer Program, Herston, QLD, Australia
| | - Beatriz Garcillán
- The Department of Microbiology and Immunology, School of Biomedical Sciences, Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne, Parkville, VIC, Australia
| | - Eden Whitlock
- Queensland Institute of Medical Research (QIMR) Berghofer Medical Research Institute, Cancer Program, Herston, QLD, Australia
| | - William A. Figgett
- The Department of Microbiology and Immunology, School of Biomedical Sciences, Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne, Parkville, VIC, Australia
- Garvan Institute of Medical Research, Kinghorn Centre for Clinical Genomics, Darlinghurst, NSW, Australia
| | - Simona Infantino
- The Department of Microbiology and Immunology, School of Biomedical Sciences, Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne, Parkville, VIC, Australia
| | - Mahya Eslami
- Department of Immunobiology, University of Lausanne, Epalinges, Switzerland
- Department of Oncology and Children’s Research Centre, University Children’s Hospital Zürich, Zürich, Switzerland
| | - SiLing Yang
- Queensland Institute of Medical Research (QIMR) Berghofer Medical Research Institute, Cancer Program, Herston, QLD, Australia
| | - M. Arifur Rahman
- Queensland Institute of Medical Research (QIMR) Berghofer Medical Research Institute, Cancer Program, Herston, QLD, Australia
| | - Yong H. Sheng
- Queensland Institute of Medical Research (QIMR) Berghofer Medical Research Institute, Cancer Program, Herston, QLD, Australia
| | - Nicholas Weber
- Cancer Care Services, Royal Brisbane and Women’s Hospital, Herston, QLD, Australia
| | - Pascal Schneider
- Department of Immunobiology, University of Lausanne, Epalinges, Switzerland
| | - Constantine S. Tam
- Department of Haematology, Alfred Hospital, Melbourne, VIC, Australia
- Department of Haematology, Monash University, Melbourne, VIC, Australia
| | - Fabienne Mackay
- Queensland Institute of Medical Research (QIMR) Berghofer Medical Research Institute, Cancer Program, Herston, QLD, Australia
- The Department of Microbiology and Immunology, School of Biomedical Sciences, Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne, Parkville, VIC, Australia
- The Department of Immunology and Pathology, Monash University, VIC, Australia
- Faculty of Medicine, University of Queensland, Brisbane, QLD, Australia
| |
Collapse
|
|
4
|
Hong JY, Han JH, Jeong SH, Kwak C, Kim HH, Jeong CW. Polygenic risk score model for renal cell carcinoma in the Korean population and relationship with lifestyle-associated factors. BMC Genomics 2024; 25:46. [PMID: 38200428 PMCID: PMC10777500 DOI: 10.1186/s12864-024-09974-w] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/01/2023] [Accepted: 01/04/2024] [Indexed: 01/12/2024] Open
Abstract
BACKGROUND The polygenic risk score (PRS) is used to predict the risk of developing common complex diseases or cancers using genetic markers. Although PRS is used in clinical practice to predict breast cancer risk, it is more accurate for Europeans than for non-Europeans because of the sample size of training genome-wide association studies (GWAS). To address this disparity, we constructed a PRS model for predicting the risk of renal cell carcinoma (RCC) in the Korean population. RESULTS Using GWAS analysis, we identified 43 Korean-specific variants and calculated the PRS. Subsequent to plotting receiver operating characteristic (ROC) curves, we selected the 31 best-performing variants to construct an optimal PRS model. The resultant PRS model with 31 variants demonstrated a prediction rate of 77.4%. The pathway analysis indicated that the identified non-coding variants are involved in regulating the expression of genes related to cancer initiation and progression. Notably, favorable lifestyle habits, such as avoiding tobacco and alcohol, mitigated the risk of RCC across PRS strata expressing genetic risk. CONCLUSION A Korean-specific PRS model was established to predict the risk of RCC in the underrepresented Korean population. Our findings suggest that lifestyle-associated factors influencing RCC risk are associated with acquired risk factors indirectly through epigenetic modification, even among individuals in the higher PRS category.
Collapse
Affiliation(s)
- Joo Young Hong
- Biomedical Research Institute, Seoul National University Hospital, Seoul, Republic of Korea
| | - Jang Hee Han
- Department of Urology, Seoul National University Hospital, Seoul, Republic of Korea
- Department of Urology, Seoul National University College of Medicine, Seoul, Republic of Korea
| | - Seung Hwan Jeong
- Department of Urology, Seoul National University Hospital, Seoul, Republic of Korea
- Department of Urology, Seoul National University College of Medicine, Seoul, Republic of Korea
| | - Cheol Kwak
- Department of Urology, Seoul National University Hospital, Seoul, Republic of Korea
- Department of Urology, Seoul National University College of Medicine, Seoul, Republic of Korea
| | - Hyeon Hoe Kim
- Department of Urology, Seoul National University Hospital, Seoul, Republic of Korea
- Department of Urology, Seoul National University College of Medicine, Seoul, Republic of Korea
- Department of Urology, Myongji Hospital, Gyeonggi-do, Republic of Korea
| | - Chang Wook Jeong
- Department of Urology, Seoul National University Hospital, Seoul, Republic of Korea.
- Department of Urology, Seoul National University College of Medicine, Seoul, Republic of Korea.
| |
Collapse
|
|
5
|
Wu X, Deng K, Cai H, Zeng Z, Cao J, Zhang L, Lu Z, Cheng W. Nek6 knockdown polarized macrophages into a pro-inflammatory phenotype via inhibiting STAT3 expression. Int J Exp Pathol 2023; 104:237-246. [PMID: 37431082 PMCID: PMC10500168 DOI: 10.1111/iep.12489] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/05/2023] [Revised: 05/08/2023] [Accepted: 06/18/2023] [Indexed: 07/12/2023] Open
Abstract
Recently macrophage polarization has emerged as playing an essential role in the oathogenesis of atherosclerosis, which is the most important underlying process in many types of cardiovascular diseases. Although Nek6 has been reported to be involved in various cellular processes, the effect of Nek6 on macrophage polarization remains unknown. Macrophages exposed to lipopolysaccharide (LPS) or IL-4 were used to establish an in vitro model for the study of regulation of classically (M1) or alternatively (M2) activated macrophage. Bone marrow-derived macrophages (BMDMs) transfected with short hairpin RNA-targeting Nek6 were then in functional studies. We observed that Nek6 expression was decreased in both peritoneal macrophages (PMs) and BMDMs stimulated by LPS. This effect was seen at both mRNA and protein level. The opposite results were obtained after administration of IL-4. Macrophage-specific Nek6 knockdown significantly exacerbated pro-inflammatory M1 polarized macrophage gene expression in response to LPS challenge, but the anti-inflammatory response gene expression that is related to M2 macrophages was attenuated by Nek6 silencing followed by treatment with IL-4. Mechanistic studies exhibited that Nek6 knockdown inhibited the phosphorylated STAT3 expression that mediated the effect on macrophage polarization regulated by AdshNek6. Moreover, decreased Nek6 expression was also observed in atherosclerotic plaques. Collectively, these evidences suggested that Nek6 acts as a crucial site in macrophage polarization, and that this operates in a STAT3-dependent manner.
Collapse
Affiliation(s)
- Xiaoyan Wu
- Department of Cardiology, Zhongnan HospitalWuhan UniversityWuhanChina
- Institute of Myocardial Injury and RepairWuhan UniversityWuhanChina
| | - Ke‐Qiong Deng
- Department of Cardiology, Zhongnan HospitalWuhan UniversityWuhanChina
- Institute of Myocardial Injury and RepairWuhan UniversityWuhanChina
| | - Huan‐Huan Cai
- Department of Cardiology, Zhongnan HospitalWuhan UniversityWuhanChina
- Institute of Myocardial Injury and RepairWuhan UniversityWuhanChina
| | - Ziyue Zeng
- Department of Cardiology, Zhongnan HospitalWuhan UniversityWuhanChina
- Institute of Myocardial Injury and RepairWuhan UniversityWuhanChina
| | - Jian‐Lei Cao
- Department of Cardiology, Zhongnan HospitalWuhan UniversityWuhanChina
- Institute of Myocardial Injury and RepairWuhan UniversityWuhanChina
| | - Lin Zhang
- Department of Cardiology, Zhongnan HospitalWuhan UniversityWuhanChina
- Institute of Myocardial Injury and RepairWuhan UniversityWuhanChina
| | - Zhibing Lu
- Department of Cardiology, Zhongnan HospitalWuhan UniversityWuhanChina
- Institute of Myocardial Injury and RepairWuhan UniversityWuhanChina
| | - Wen‐Lin Cheng
- Department of Cardiology, Zhongnan HospitalWuhan UniversityWuhanChina
- Institute of Myocardial Injury and RepairWuhan UniversityWuhanChina
| |
Collapse
|
|
6
|
Zhang H, Li B. NIMA-related kinase 6 as an effective target inhibits the hepatocarcinogenesis and progression of hepatocellular carcinoma. Heliyon 2023; 9:e15971. [PMID: 37260886 PMCID: PMC10227323 DOI: 10.1016/j.heliyon.2023.e15971] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2022] [Revised: 04/26/2023] [Accepted: 04/27/2023] [Indexed: 06/02/2023] Open
Abstract
Background NIMA-related kinase 6 (NEK 6) is over-expressed in some tumor cell lines and tissues. However, its expression in hepatocellular carcinoma (HCC) and its correlation with clinical features remain unclear. Methods Total RNA from HCC liver tissues, other liver specimens, and hepatic cell lines was extracted and QPCR was adopted to detect NEK6 expression. The correlation between NEK6 expression and the clinical characteristics of HCC was analyzed. Scratch assay, Transwell assay, and tumor-formation assay were used to evaluate the effects of NEK6 on the HCC progression in vitro and in vivo. Results The expression of NEK6 was up-regulated in HCC tissues and HCC cell lines: Li-7 and HepG2. The overexpression of NEK6 was correlated with hepatitis B virus infection and tumor diameter (P = 0.045). When down-regulated the expression of NEK6, both the migration and invasion capabilities of Li-7 and HepG2 cells and the growth of xenograft tumors were suppressed. (P < 0.05). Conclusions NEK6 expression was up-regulated in HCC and correlated with the progression, suggesting it might be a valuable biomarker and a potential therapeutic target for HCC.
Collapse
Affiliation(s)
- Hao Zhang
- Department of Hepatobiliary Surgery, Sichuan Provincial People's Hospital, University of Electronic Science and Technology of China, Chengdu 610072, China
| | - Bo Li
- Department of Hepatobiliary Surgery, West China Hospital, Sichuan University, Chengdu 610041, China
| |
Collapse
|
|
7
|
Panchal NK, Mohanty S, Prince SE. NIMA-related kinase-6 (NEK6) as an executable target in cancer. CLINICAL & TRANSLATIONAL ONCOLOGY : OFFICIAL PUBLICATION OF THE FEDERATION OF SPANISH ONCOLOGY SOCIETIES AND OF THE NATIONAL CANCER INSTITUTE OF MEXICO 2023; 25:66-77. [PMID: 36074296 DOI: 10.1007/s12094-022-02926-4] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/22/2022] [Accepted: 08/09/2022] [Indexed: 01/07/2023]
Abstract
Cancer is a disease that develops when cells begin to divide uncontrollably and spreads to other parts of the body. Proliferation and invasion of cancerous cells are generally known to be influenced by cell cycle-related proteins in human malignancies. Therefore, in this review, we have emphasized on the serine/threonine kinase named NEK6. NEK6 is been deliberated to play a critical role in mitosis progression that includes mitotic spindle formation, metaphase to anaphase transition, and centrosome separation. Moreover, it has a mechanistic role in DNA repair and can cause apoptosis when inhibited. Past studies have connected NEK6 protein expression to cancer cell senescence. Besides, there are reports relating NEK6 to a range of malignancies including breast, lung, ovarian, prostate, kidney, liver, and others. Given its significance, this review attempts to describe the structural and functional aspects of NEK6 in various cellular processes, as well as how it is linked to different forms of cancer. Lastly, we have accentuated, on some of the plausible inhibitors that have been explored against NEK6 overexpression.
Collapse
Affiliation(s)
- Nagesh Kishan Panchal
- Department of Biomedical Sciences, School of Biosciences and Technology, Vellore Institute of Technology, Vellore, 632014, India
| | - Shruti Mohanty
- Department of Biomedical Sciences, School of Biosciences and Technology, Vellore Institute of Technology, Vellore, 632014, India
| | - Sabina Evan Prince
- Department of Biomedical Sciences, School of Biosciences and Technology, Vellore Institute of Technology, Vellore, 632014, India.
| |
Collapse
|
|
8
|
In Mitosis You Are Not: The NIMA Family of Kinases in Aspergillus, Yeast, and Mammals. Int J Mol Sci 2022; 23:ijms23074041. [PMID: 35409400 PMCID: PMC8999480 DOI: 10.3390/ijms23074041] [Citation(s) in RCA: 9] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/08/2022] [Revised: 03/31/2022] [Accepted: 04/01/2022] [Indexed: 11/17/2022] Open
Abstract
The Never in mitosis gene A (NIMA) family of serine/threonine kinases is a diverse group of protein kinases implicated in a wide variety of cellular processes, including cilia regulation, microtubule dynamics, mitotic processes, cell growth, and DNA damage response. The founding member of this family was initially identified in Aspergillus and was found to play important roles in mitosis and cell division. The yeast family has one member each, Fin1p in fission yeast and Kin3p in budding yeast, also with functions in mitotic processes, but, overall, these are poorly studied kinases. The mammalian family, the main focus of this review, consists of 11 members named Nek1 to Nek11. With the exception of a few members, the functions of the mammalian Neks are poorly understood but appear to be quite diverse. Like the prototypical NIMA, many members appear to play important roles in mitosis and meiosis, but their functions in the cell go well beyond these well-established activities. In this review, we explore the roles of fungal and mammalian NIMA kinases and highlight the most recent findings in the field.
Collapse
|
|
9
|
Zhong S, Peng S, Chen Z, Chen Z, Luo JL. Choosing Kinase Inhibitors for Androgen Deprivation Therapy-Resistant Prostate Cancer. Pharmaceutics 2022; 14:498. [PMID: 35335873 PMCID: PMC8950316 DOI: 10.3390/pharmaceutics14030498] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/01/2021] [Revised: 01/26/2022] [Accepted: 02/22/2022] [Indexed: 11/25/2022] Open
Abstract
Androgen deprivation therapy (ADT) is a systemic therapy for advanced prostate cancer (PCa). Although most patients initially respond to ADT, almost all cancers eventually develop castration resistance. Castration-resistant PCa (CRPC) is associated with a very poor prognosis, and the treatment of which is a serious clinical challenge. Accumulating evidence suggests that abnormal expression and activation of various kinases are associated with the emergence and maintenance of CRPC. Many efforts have been made to develop small molecule inhibitors to target the key kinases in CRPC. These inhibitors are designed to suppress the kinase activity or interrupt kinase-mediated signal pathways that are associated with PCa androgen-independent (AI) growth and CRPC development. In this review, we briefly summarize the roles of the kinases that are abnormally expressed and/or activated in CRPC and the recent advances in the development of small molecule inhibitors that target kinases for the treatment of CRPC.
Collapse
Affiliation(s)
- Shangwei Zhong
- Department of General Surgery, Xiangya Hospital, Central South University, Hunan 410008, China; (S.Z.); (S.P.); (Z.C.)
- Department of Molecular Medicine, The Scripps Research Institute, Jupiter, FL 33459, USA
| | - Shoujiao Peng
- Department of General Surgery, Xiangya Hospital, Central South University, Hunan 410008, China; (S.Z.); (S.P.); (Z.C.)
- Department of Molecular Medicine, The Scripps Research Institute, Jupiter, FL 33459, USA
| | - Zihua Chen
- Department of General Surgery, Xiangya Hospital, Central South University, Hunan 410008, China; (S.Z.); (S.P.); (Z.C.)
| | - Zhikang Chen
- Department of General Surgery, Xiangya Hospital, Central South University, Hunan 410008, China; (S.Z.); (S.P.); (Z.C.)
| | - Jun-Li Luo
- Department of Molecular Medicine, The Scripps Research Institute, Jupiter, FL 33459, USA
| |
Collapse
|
|
10
|
MicroRNA-323a-3p Negatively Regulates NEK6 in Colon Adenocarcinoma Cells. JOURNAL OF ONCOLOGY 2022; 2022:7007718. [PMID: 35096064 PMCID: PMC8791743 DOI: 10.1155/2022/7007718] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 08/21/2021] [Revised: 12/31/2021] [Accepted: 01/05/2022] [Indexed: 11/18/2022]
Abstract
Objective. The activity of NEK6 is enhanced in several cancer cells, including colon adenocarcinoma (COAD) cells. However, there are few reports on the microRNA (miRNA/miR) regulation of NEK6. In this study, we aimed to investigate the effects of miRNAs targeting NEK6 in COAD cells. Methods. Public data and online analysis sites were used to analyze the expression levels of NEK6 and miR-323a-3p in COAD tissues as well as the relationship between NEK6 or miR-323a-3p levels and survival in patients with COAD and to predict miRNAs targeting NEK6. Real-time polymerase chain reaction and western blotting were performed to determine the levels of NEK6 and miR-323a-3p in COAD cells. The targeting of NEK6 by miR-323a-3p was verified using a dual-luciferase reporter assay. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, 5-ethynyl-2′-deoxyuridine assay, propidium iodide (PI) staining, annexin V-fluorescein isothiocyanate/PI staining, and transwell assay were employed to test the proliferation, apoptosis, migration ability, and invasiveness of COAD cells. Results. In COAD cells, NEK6 was highly expressed, whereas miR-323a-3p was expressed at low levels and negatively regulated NEK6. Upregulating the level of miR-323a-3p impaired the proliferation, migration, and invasion of COAD cells and promoted apoptosis, whereas supplementing NEK6 alleviated the damage of the proliferation, migration, and invasion of COAD cells caused by miR-323a-3p and inhibited miR-323a-3p-induced apoptosis. These findings indicate that miR-323a-3p regulates the proliferation, migration, invasion, and apoptosis of COAD cells by targeting NEK6. Conclusion. miR-323a-3p downregulates NEK6 in COAD cells; this provides a novel basis for further understanding the occurrence and development of COAD.
Collapse
|
|
11
|
Liu Y, Fu W, Yin F, Xia L, Zhang Y, Wang B, Li T, Zhang T, Cheng L, Wei Y, Gao B. miR-141-3p suppresses development of clear cell renal cell carcinoma by regulating NEK6. Anticancer Drugs 2022; 33:e125-e133. [PMID: 34387594 DOI: 10.1097/cad.0000000000001158] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
Abstract
Currently, there have been few studies on the function and molecular mechanism of miR-141-3p in the development of clear cell renal cell carcinoma (CCRCC). This study aimed to explore the relationship between miR-141-3p and NIMA (never in mitosis, gene A)-related kinase-6 (NEK6) and investigate the role of the interaction in CCRCC cell proliferation, migration, invasion and apoptosis.Starbase database was used to predict the target gene of miR-141-3p in CCRCC and dual-luciferase reporter assay was performed to verify the targeting relationship between miR-141-3p and the target gene. Real-time quantitative PCR was conducted to detect the expression of miR-141-3p and NEK6 mRNA in cells. Western blot was carried out to detect the protein level of NEK6 in cells. Cell Counting Kit-8 assay, transwell assay and wound healing assay were conducted to detect CCRCC cell proliferation, invasion and migration abilities. Flow cytometry was performed to detect CCRCC cell apoptosis. miR-141-3p was markedly lowly expressed, and NEK6 was a target of miR-141-3p and was remarkably highly expressed in CCRCC cells. Over-expressing miR-141-3p could inhibit CCRCC cell proliferation, migration, invasion and promote apoptosis. The inhibitory effect of miR-141-3p over-expression on cell proliferation, migration and invasion was significantly weakened by over-expressing NEK6. miR-141-3p could regulate CCRCC cell proliferation, migration, invasion and apoptosis by targeting NEK6. This study lays the basis for the exploration of the molecular mechanism underlying CCRCC pathogenesis and research on targeted therapies for CCRCC.
Collapse
Affiliation(s)
- Yifei Liu
- Department of Urology, Tangshan Central Hospital, Tangshan
| | - Wenqiang Fu
- Department of Urology, Tangshan Central Hospital, Tangshan
| | - Feng Yin
- Department of Urology, Tangshan Central Hospital, Tangshan
| | - Lianjie Xia
- Department of Urology, Tangshan Central Hospital, Tangshan
| | - Yubo Zhang
- Department of Urology, Tangshan Central Hospital, Tangshan
| | - Baocun Wang
- Department of Urology, Tangshan Central Hospital, Tangshan
| | - Tengfei Li
- Department of Urology, Tangshan Central Hospital, Tangshan
| | - Tingting Zhang
- Department of Urology, Tangshan Central Hospital, Tangshan
| | - Ling Cheng
- Shanghai Engineering Research Center of Pharmaceutical Translation, Shanghai, China
| | - Yanbing Wei
- Shanghai Engineering Research Center of Pharmaceutical Translation, Shanghai, China
| | - Bin Gao
- Department of Urology, Tangshan Central Hospital, Tangshan
| |
Collapse
|
|
12
|
Yu Y, Shen T, Zhong X, Wang LL, Tai W, Zou Y, Qin J, Zhang Z, Zhang CL. NEK6 is an injury-responsive kinase cooperating with STAT3 in regulation of reactive astrogliosis. Glia 2021; 70:273-286. [PMID: 34643969 DOI: 10.1002/glia.24104] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/04/2021] [Revised: 09/29/2021] [Accepted: 09/30/2021] [Indexed: 11/08/2022]
Abstract
In response to brain injury, resident astrocytes become reactive and play dynamic roles in neural repair and regeneration. The signaling pathways underlying such reactive astrogliosis remain largely unclear. We here show that NEK6, a NIMA-related serine/threonine protein kinase, is rapidly induced following pathological stimulations and plays critical roles in reactive astrogliosis. Enhanced NEK6 expression promotes reactive astrogliosis and exacerbates brain lesions; and conversely, NEK6 downregulation dampens injury-induced astrocyte reactivity and reduces lesion size. Mechanistically, NEK6 associates with and phosphorylates STAT3. Kinase activity of NEK6 is required for induction of GFAP and PCNA, markers of reactive astrogliosis. Interestingly, NEK6 is also localized in the nucleus and binds to STAT3-responsive genomic elements in astrocytes. These results indicate that NEK6 constitutes a molecular target for the regulation of reactive astrogliosis.
Collapse
Affiliation(s)
- Ying Yu
- Department of Neurology, Renmin Hospital of Wuhan University, Wuhan, China.,Department of Molecular Biology, Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, Texas, USA
| | - Tianjin Shen
- Department of Molecular Biology, Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, Texas, USA
| | - Xiaoling Zhong
- Department of Molecular Biology, Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, Texas, USA
| | - Lei-Lei Wang
- Department of Molecular Biology, Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, Texas, USA
| | - Wenjiao Tai
- Department of Molecular Biology, Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, Texas, USA
| | - Yuhua Zou
- Department of Molecular Biology, Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, Texas, USA
| | - Jun Qin
- Department of Orthopedic Surgery, Zhongnan Hospital of Wuhan University, Wuhan, China
| | - Zhaohui Zhang
- Department of Neurology, Renmin Hospital of Wuhan University, Wuhan, China
| | - Chun-Li Zhang
- Department of Molecular Biology, Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, Texas, USA
| |
Collapse
|
|
13
|
The Multifaceted Role and Utility of MicroRNAs in Indolent B-Cell Non-Hodgkin Lymphomas. Biomedicines 2021; 9:biomedicines9040333. [PMID: 33806113 PMCID: PMC8064455 DOI: 10.3390/biomedicines9040333] [Citation(s) in RCA: 17] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/08/2021] [Revised: 03/14/2021] [Accepted: 03/18/2021] [Indexed: 02/07/2023] Open
Abstract
Normal B-cell development is a tightly regulated complex procedure, the deregulation of which can lead to lymphomagenesis. One common group of blood cancers is the B-cell non-Hodgkin lymphomas (NHLs), which can be categorized according to the proliferation and spread rate of cancer cells into indolent and aggressive ones. The most frequent indolent B-cell NHLs are follicular lymphoma and marginal zone lymphoma. MicroRNAs (miRNAs) are small non-coding RNAs that can greatly influence protein expression. Based on the multiple interactions among miRNAs and their targets, complex networks of gene expression regulation emerge, which normally are essential for proper B-cell development. Multiple miRNAs have been associated with B-cell lymphomas, as the deregulation of these complex networks can lead to such pathological states. The aim of the present review is to summarize the existing information regarding the multifaceted role of miRNAs in indolent B-cell NHLs, affecting the main B-cell subpopulations. We attempt to provide insight into their biological function, the complex miRNA-mRNA interactions, and their biomarker utility in these malignancies. Lastly, we address the limitations that hinder the investigation of the role of miRNAs in these lymphomas and discuss ways that these problems could be overcome in the future.
Collapse
|
|
14
|
Pavan ICB, Peres de Oliveira A, Dias PRF, Basei FL, Issayama LK, Ferezin CDC, Silva FR, Rodrigues de Oliveira AL, Alves dos Reis Moura L, Martins MB, Simabuco FM, Kobarg J. On Broken Ne(c)ks and Broken DNA: The Role of Human NEKs in the DNA Damage Response. Cells 2021; 10:507. [PMID: 33673578 PMCID: PMC7997185 DOI: 10.3390/cells10030507] [Citation(s) in RCA: 23] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2021] [Revised: 02/04/2021] [Accepted: 02/22/2021] [Indexed: 02/07/2023] Open
Abstract
NIMA-related kinases, or NEKs, are a family of Ser/Thr protein kinases involved in cell cycle and mitosis, centrosome disjunction, primary cilia functions, and DNA damage responses among other biological functional contexts in vertebrate cells. In human cells, there are 11 members, termed NEK1 to 11, and the research has mainly focused on exploring the more predominant roles of NEKs in mitosis regulation and cell cycle. A possible important role of NEKs in DNA damage response (DDR) first emerged for NEK1, but recent studies for most NEKs showed participation in DDR. A detailed analysis of the protein interactions, phosphorylation events, and studies of functional aspects of NEKs from the literature led us to propose a more general role of NEKs in DDR. In this review, we express that NEK1 is an activator of ataxia telangiectasia and Rad3-related (ATR), and its activation results in cell cycle arrest, guaranteeing DNA repair while activating specific repair pathways such as homology repair (HR) and DNA double-strand break (DSB) repair. For NEK2, 6, 8, 9, and 11, we found a role downstream of ATR and ataxia telangiectasia mutated (ATM) that results in cell cycle arrest, but details of possible activated repair pathways are still being investigated. NEK4 shows a connection to the regulation of the nonhomologous end-joining (NHEJ) repair of DNA DSBs, through recruitment of DNA-PK to DNA damage foci. NEK5 interacts with topoisomerase IIβ, and its knockdown results in the accumulation of damaged DNA. NEK7 has a regulatory role in the detection of oxidative damage to telomeric DNA. Finally, NEK10 has recently been shown to phosphorylate p53 at Y327, promoting cell cycle arrest after exposure to DNA damaging agents. In summary, this review highlights important discoveries of the ever-growing involvement of NEK kinases in the DDR pathways. A better understanding of these roles may open new diagnostic possibilities or pharmaceutical interventions regarding the chemo-sensitizing inhibition of NEKs in various forms of cancer and other diseases.
Collapse
Affiliation(s)
- Isadora Carolina Betim Pavan
- Graduate Program in “Ciências Farmacêuticas”, School of Pharmaceutical Sciences, Faculty of Pharmaceutical Sciences, State University of Campinas (UNICAMP), R. Cândido Portinari 200, Prédio 2, Campinas CEP 13083-871, Brazil; (I.C.B.P.); (A.P.d.O.); (P.R.F.D.); (F.L.B.); (L.K.I.); (F.R.S.); (A.L.R.d.O.); (L.A.d.R.M.); (M.B.M.)
| | - Andressa Peres de Oliveira
- Graduate Program in “Ciências Farmacêuticas”, School of Pharmaceutical Sciences, Faculty of Pharmaceutical Sciences, State University of Campinas (UNICAMP), R. Cândido Portinari 200, Prédio 2, Campinas CEP 13083-871, Brazil; (I.C.B.P.); (A.P.d.O.); (P.R.F.D.); (F.L.B.); (L.K.I.); (F.R.S.); (A.L.R.d.O.); (L.A.d.R.M.); (M.B.M.)
| | - Pedro Rafael Firmino Dias
- Graduate Program in “Ciências Farmacêuticas”, School of Pharmaceutical Sciences, Faculty of Pharmaceutical Sciences, State University of Campinas (UNICAMP), R. Cândido Portinari 200, Prédio 2, Campinas CEP 13083-871, Brazil; (I.C.B.P.); (A.P.d.O.); (P.R.F.D.); (F.L.B.); (L.K.I.); (F.R.S.); (A.L.R.d.O.); (L.A.d.R.M.); (M.B.M.)
| | - Fernanda Luisa Basei
- Graduate Program in “Ciências Farmacêuticas”, School of Pharmaceutical Sciences, Faculty of Pharmaceutical Sciences, State University of Campinas (UNICAMP), R. Cândido Portinari 200, Prédio 2, Campinas CEP 13083-871, Brazil; (I.C.B.P.); (A.P.d.O.); (P.R.F.D.); (F.L.B.); (L.K.I.); (F.R.S.); (A.L.R.d.O.); (L.A.d.R.M.); (M.B.M.)
| | - Luidy Kazuo Issayama
- Graduate Program in “Ciências Farmacêuticas”, School of Pharmaceutical Sciences, Faculty of Pharmaceutical Sciences, State University of Campinas (UNICAMP), R. Cândido Portinari 200, Prédio 2, Campinas CEP 13083-871, Brazil; (I.C.B.P.); (A.P.d.O.); (P.R.F.D.); (F.L.B.); (L.K.I.); (F.R.S.); (A.L.R.d.O.); (L.A.d.R.M.); (M.B.M.)
| | - Camila de Castro Ferezin
- Graduate Program in “Biologia Funcional e Molecular”, Department of Biochemistry and Tissue Biology, Institute of Biology, State University of Campinas (UNICAMP), Campinas 13083-857, Brazil;
| | - Fernando Riback Silva
- Graduate Program in “Ciências Farmacêuticas”, School of Pharmaceutical Sciences, Faculty of Pharmaceutical Sciences, State University of Campinas (UNICAMP), R. Cândido Portinari 200, Prédio 2, Campinas CEP 13083-871, Brazil; (I.C.B.P.); (A.P.d.O.); (P.R.F.D.); (F.L.B.); (L.K.I.); (F.R.S.); (A.L.R.d.O.); (L.A.d.R.M.); (M.B.M.)
| | - Ana Luisa Rodrigues de Oliveira
- Graduate Program in “Ciências Farmacêuticas”, School of Pharmaceutical Sciences, Faculty of Pharmaceutical Sciences, State University of Campinas (UNICAMP), R. Cândido Portinari 200, Prédio 2, Campinas CEP 13083-871, Brazil; (I.C.B.P.); (A.P.d.O.); (P.R.F.D.); (F.L.B.); (L.K.I.); (F.R.S.); (A.L.R.d.O.); (L.A.d.R.M.); (M.B.M.)
| | - Lívia Alves dos Reis Moura
- Graduate Program in “Ciências Farmacêuticas”, School of Pharmaceutical Sciences, Faculty of Pharmaceutical Sciences, State University of Campinas (UNICAMP), R. Cândido Portinari 200, Prédio 2, Campinas CEP 13083-871, Brazil; (I.C.B.P.); (A.P.d.O.); (P.R.F.D.); (F.L.B.); (L.K.I.); (F.R.S.); (A.L.R.d.O.); (L.A.d.R.M.); (M.B.M.)
| | - Mariana Bonjiorno Martins
- Graduate Program in “Ciências Farmacêuticas”, School of Pharmaceutical Sciences, Faculty of Pharmaceutical Sciences, State University of Campinas (UNICAMP), R. Cândido Portinari 200, Prédio 2, Campinas CEP 13083-871, Brazil; (I.C.B.P.); (A.P.d.O.); (P.R.F.D.); (F.L.B.); (L.K.I.); (F.R.S.); (A.L.R.d.O.); (L.A.d.R.M.); (M.B.M.)
- Graduate Program in “Biologia Funcional e Molecular”, Department of Biochemistry and Tissue Biology, Institute of Biology, State University of Campinas (UNICAMP), Campinas 13083-857, Brazil;
| | | | - Jörg Kobarg
- Graduate Program in “Ciências Farmacêuticas”, School of Pharmaceutical Sciences, Faculty of Pharmaceutical Sciences, State University of Campinas (UNICAMP), R. Cândido Portinari 200, Prédio 2, Campinas CEP 13083-871, Brazil; (I.C.B.P.); (A.P.d.O.); (P.R.F.D.); (F.L.B.); (L.K.I.); (F.R.S.); (A.L.R.d.O.); (L.A.d.R.M.); (M.B.M.)
- Graduate Program in “Biologia Funcional e Molecular”, Department of Biochemistry and Tissue Biology, Institute of Biology, State University of Campinas (UNICAMP), Campinas 13083-857, Brazil;
| |
Collapse
|
|
15
|
Xu X, Li M, Deng Z, Hu J, Jiang Z, Liu Y, Chang K, Hu C. Grass Carp ( Ctenopharyngodon idellus) NIMA-Related Kinase 6 Blocks dsRNA-Induced IFN I Response by Targeting IRF3. Front Immunol 2021; 11:597775. [PMID: 33488591 PMCID: PMC7820699 DOI: 10.3389/fimmu.2020.597775] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/22/2020] [Accepted: 11/25/2020] [Indexed: 02/05/2023] Open
Abstract
Accumulating evidence indicates that mammalian NIMA (never in mitosis, gene A)-related kinase 6 (NEK6) plays potential roles during the course of tumorigenesis, but little is known about NEK6 in lower vertebrates. Herein, we reported a mammalian ortholog of NEK6 in grass carp (Ctenopharyngodon idellus) (CiNEK6). Multiple alignment of amino acid sequences and phylogenetic analysis showed that CiNEK6 shares a high level of sequence similarity with its counterparts in birds. CiNEK6 was ubiquitously expressed in all tested tissues, and its expression level was increased under treatment with GCRV (dsRNA virus) or poly I:C (dsRNA analog). Q-PCR and dual-luciferase assays suggested that CiNEK6 overexpression suppressed IFN I activity in CIK cells treated with poly I:C. Knockdown of CiNEK6 resulted in a higher level of IFN I expression in CIK cells treated with poly I:C compared to those which received PBS. Interestingly, analysis of subcellular localization demonstrated that CiNEK6 protein scattered throughout the cytoplasm is gradually congregated together at the edges of karyotheca upon stimulation with poly I:C. Co-IP and co-localization assays suggested that CiNEK6 interacts with CiIRF3 after poly I:C challenge. In poly I:C-treated cells, the phosphorylation of CiIRF3 was increased by CiNEK6 knockdown, but was suppressed by CiNEK6 overexpression, suggesting that CiNEK6 decreases IFN I expression through inhibiting CiIRF3 activity. Cell viability assay, crystal violet staining, and detection of Vp5 also showed that CiNEK6 plays an inhibitory role in IRF3-mediated antiviral responses.
Collapse
Affiliation(s)
- Xiaowen Xu
- College of Life Science, Nanchang University, Nanchang, China
| | - Meifeng Li
- College of Life Science, Nanchang University, Nanchang, China
| | - Zeyuan Deng
- State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, China
| | - Jihuan Hu
- College of Life Science, Nanchang University, Nanchang, China
| | - Zeyin Jiang
- College of Life Science, Nanchang University, Nanchang, China
| | - Yapeng Liu
- College of Life Science, Nanchang University, Nanchang, China
| | - Kaile Chang
- College of Life Science, Nanchang University, Nanchang, China
| | - Chengyu Hu
- College of Life Science, Nanchang University, Nanchang, China
| |
Collapse
|
|
16
|
Hong J, Won M, Ro H. The Molecular and Pathophysiological Functions of Members of the LNX/PDZRN E3 Ubiquitin Ligase Family. Molecules 2020; 25:E5938. [PMID: 33333989 PMCID: PMC7765395 DOI: 10.3390/molecules25245938] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/28/2020] [Revised: 12/10/2020] [Accepted: 12/10/2020] [Indexed: 12/27/2022] Open
Abstract
The ligand of Numb protein-X (LNX) family, also known as the PDZRN family, is composed of four discrete RING-type E3 ubiquitin ligases (LNX1, LNX2, LNX3, and LNX4), and LNX5 which may not act as an E3 ubiquitin ligase owing to the lack of the RING domain. As the name implies, LNX1 and LNX2 were initially studied for exerting E3 ubiquitin ligase activity on their substrate Numb protein, whose stability was negatively regulated by LNX1 and LNX2 via the ubiquitin-proteasome pathway. LNX proteins may have versatile molecular, cellular, and developmental functions, considering the fact that besides these proteins, none of the E3 ubiquitin ligases have multiple PDZ (PSD95, DLGA, ZO-1) domains, which are regarded as important protein-interacting modules. Thus far, various proteins have been isolated as LNX-interacting proteins. Evidence from studies performed over the last two decades have suggested that members of the LNX family play various pathophysiological roles primarily by modulating the function of substrate proteins involved in several different intracellular or intercellular signaling cascades. As the binding partners of RING-type E3s, a large number of substrates of LNX proteins undergo degradation through ubiquitin-proteasome system (UPS) dependent or lysosomal pathways, potentially altering key signaling pathways. In this review, we highlight recent and relevant findings on the molecular and cellular functions of the members of the LNX family and discuss the role of the erroneous regulation of these proteins in disease progression.
Collapse
Affiliation(s)
- Jeongkwan Hong
- Department of Biological Sciences, College of Bioscience and Biotechnology, Chungnam National University, Daejeon 305-764, Korea;
| | - Minho Won
- Biotechnology Process Engineering Center, Korea Research Institute of Bioscience & Biotechnology (KRIBB), 30 Yeongudanji-ro, Cheongwon-gu, Cheongju 28116, Korea
| | - Hyunju Ro
- Department of Biological Sciences, College of Bioscience and Biotechnology, Chungnam National University, Daejeon 305-764, Korea;
| |
Collapse
|
|
17
|
Fu B, Xue W, Zhang H, Zhang R, Feldman K, Zhao Q, Zhang S, Shi L, Pavani KC, Nian W, Lin X, Wu H. MicroRNA-325-3p Facilitates Immune Escape of Mycobacterium tuberculosis through Targeting LNX1 via NEK6 Accumulation to Promote Anti-Apoptotic STAT3 Signaling. mBio 2020; 11:e00557-20. [PMID: 32487755 PMCID: PMC7267881 DOI: 10.1128/mbio.00557-20] [Citation(s) in RCA: 30] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/07/2020] [Accepted: 04/30/2020] [Indexed: 12/12/2022] Open
Abstract
Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis that poses threats to the public. M. tuberculosis survives in macrophages by escaping from immune surveillance and clearance, which exacerbates the bacterial proliferation. However, the molecular mechanisms of this immune escape have not yet been fully understood. Using multiple cell and mouse models, we found that microRNA-325-3p (miR-325-3p) is upregulated after M. tuberculosis infection and Mir325-deficient mice show resistance to M. tuberculosis We demonstrated that miR-325-3p directly targets LNX1, an E3 ubiquitin ligase of NEK6, and that this hampers the proteasomal degradation of NEK6 in macrophages. The abnormal accumulation of NEK6 leads to the activation of STAT3 signaling, thus inhibiting the process of apoptosis and promoting the intracellular survival of M. tuberculosis Our findings not only reveal a new immune escape pathway of M. tuberculosis but also may provide new insights into the development of therapeutic approaches for drug-resistant TB.IMPORTANCE Intracellular survival of Mycobacterium tuberculosis results in bacterial proliferation and the spread of infection in lungs, consequently deteriorating the conditions of tuberculosis (TB) patients. This research discovers a new immune escape pathway of M. tuberculosis by modulating host miR-325-3p expression, thus leading to the intracellular survival of M. tuberculosis These findings make a contribution to the understanding of the immune escape of M. tuberculosis, and they provide a theoretical basis for the development of therapeutic approaches for drug-resistant TB.
Collapse
Affiliation(s)
- Beibei Fu
- School of Life Sciences, Chongqing University, Chongqing, China
| | - Weiwei Xue
- School of Pharmaceutical Sciences, Chongqing University, Chongqing, China
| | - Haiwei Zhang
- Chongqing Key Laboratory of Translational Research for Cancer Metastasis and Individualized Treatment, Chongqing University Cancer Hospital, Chongqing, China
| | - Rui Zhang
- Department of Respiratory Medicine, The First Affiliated Hospital of Chongqing Medical University, Chongqing, China
| | - Kelly Feldman
- Department of Molecular and Cell Biology, University of California, Berkeley, California, USA
| | - Qingting Zhao
- School of Life Sciences, Chongqing University, Chongqing, China
| | - Shanfu Zhang
- School of Life Sciences, Chongqing University, Chongqing, China
| | - Lei Shi
- School of Life Sciences, Chongqing University, Chongqing, China
| | | | - Weiqi Nian
- Chongqing Key Laboratory of Translational Research for Cancer Metastasis and Individualized Treatment, Chongqing University Cancer Hospital, Chongqing, China
| | - Xiaoyuan Lin
- School of Life Sciences, Chongqing University, Chongqing, China
| | - Haibo Wu
- School of Life Sciences, Chongqing University, Chongqing, China
| |
Collapse
|
|
18
|
Joseph BB, Wang Y, Edeen P, Lažetić V, Grant BD, Fay DS. Control of clathrin-mediated endocytosis by NIMA family kinases. PLoS Genet 2020; 16:e1008633. [PMID: 32069276 PMCID: PMC7048319 DOI: 10.1371/journal.pgen.1008633] [Citation(s) in RCA: 30] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/06/2019] [Revised: 02/28/2020] [Accepted: 01/28/2020] [Indexed: 12/16/2022] Open
Abstract
Endocytosis, the process by which cells internalize plasma membrane and associated cargo, is regulated extensively by posttranslational modifications. Previous studies suggested the potential involvement of scores of protein kinases in endocytic control, of which only a few have been validated in vivo. Here we show that the conserved NIMA-related kinases NEKL-2/NEK8/9 and NEKL-3/NEK6/7 (the NEKLs) control clathrin-mediated endocytosis in C. elegans. Loss of NEKL-2 or NEKL-3 activities leads to penetrant larval molting defects and to the abnormal localization of trafficking markers in arrested larvae. Using an auxin-based degron system, we also find that depletion of NEKLs in adult-stage C. elegans leads to gross clathrin mislocalization and to a dramatic reduction in clathrin mobility at the apical membrane. Using a non-biased genetic screen to identify suppressors of nekl molting defects, we identified several components and regulators of AP2, the major clathrin adapter complex acting at the plasma membrane. Strikingly, reduced AP2 activity rescues both nekl mutant molting defects as well as associated trafficking phenotypes, whereas increased levels of active AP2 exacerbate nekl defects. Moreover, in a unique example of mutual suppression, NEKL inhibition alleviates defects associated with reduced AP2 activity, attesting to the tight link between NEKL and AP2 functions. We also show that NEKLs are required for the clustering and internalization of membrane cargo required for molting. Notably, we find that human NEKs can rescue molting and trafficking defects in nekl mutant worms, suggesting that the control of intracellular trafficking is an evolutionarily conserved function of NEK family kinases. In order to function properly, cells must continually import materials from the outside. This process, termed endocytosis, is necessary for the uptake of nutrients and for interpreting signals coming from the external environment or from within the body. These signals are critical during animal development but also affect many types of cell behaviors throughout life. In our current work, we show that several highly conserved proteins in the nematode Caenorhabditis elegans, NEKL-2 and NEKL-3, regulate endocytosis. The human counterparts of NEKL-2 and NEKL-3 have been implicated in cardiovascular and renal diseases as well as many types of cancers. However, their specific functions within cells is incompletely understood and very little is known about their role in endocytosis or how this role might impact disease processes. Here we use several complementary approaches to characterize the specific functions of C. elegans NEKL-2 and NEKL-3 in endocytosis and show that their human counterparts likely have very similar functions. This work paves the way to a better understanding of fundamental biological processes and to determining the cellular functions of proteins connected to human diseases.
Collapse
Affiliation(s)
- Braveen B. Joseph
- Department of Molecular Biology, College of Agriculture and Natural Resources, University of Wyoming, Laramie, Wyoming, United States of America
| | - Yu Wang
- Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, New Jersey, United States of America
- Department of Biochemistry and Molecular Biology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China
| | - Phil Edeen
- Department of Molecular Biology, College of Agriculture and Natural Resources, University of Wyoming, Laramie, Wyoming, United States of America
| | - Vladimir Lažetić
- Department of Molecular Biology, College of Agriculture and Natural Resources, University of Wyoming, Laramie, Wyoming, United States of America
| | - Barth D. Grant
- Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, New Jersey, United States of America
| | - David S. Fay
- Department of Molecular Biology, College of Agriculture and Natural Resources, University of Wyoming, Laramie, Wyoming, United States of America
- * E-mail:
| |
Collapse
|
|
19
|
He Z, Ni X, Xia L, Shao Z. Overexpression of NIMA-related kinase 6 (NEK6) contributes to malignant growth and dismal prognosis in Human Breast Cancer. Pathol Res Pract 2018; 214:1648-1654. [PMID: 30153958 DOI: 10.1016/j.prp.2018.07.030] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/07/2018] [Revised: 07/16/2018] [Accepted: 07/25/2018] [Indexed: 12/16/2022]
Abstract
NIMA Related Kinase 6 (Nek6) is a protein kinase involved in various cellular processes, including cell cycle regulation, apopotosis, senescence, telomere maintainance and chemoresistance. In the present study, we investigated the role of Nek6 in breast tumorigenesis and the prognostic merit of Nek6 expression in breast cancer. Immunohistochemistry and Western blot analyses was conducted to determine the expression profile of Nek6 in 133 breast cancer specimens. Nek6 was overexpressed in a majority of breast cancer specimens, compared with the adjacent non-tumorous tissues. Furthermore, we revealed that high expression of Nek6 was associated with histologic grade, tumor size and TNM stage in breast cancer. Liner regression analysis showed a significant association between the levels of Nek6 and Ki-67. Cox regression analysis indicated that Nek6 expression was an independent prognostic predictor for breast cancer. Moreover, intracellular level of Nek6 was remarkably increased following the release from serum starvation in MCF-7 cells. EdU incorporation assay indicated that depletion of Nek6 remarkably impaired the proliferation of MCF-7 cells. Finally, spheroid formation assay revealed that interference of Nek6 led to diminished oncospheroid-forming capacity of breast cancer cells. In conclusion, our results imply that Nek6 plays a facilitating role in breast cancer cell proliferation and may serve as a promising therapeutic target for breast cancer.
Collapse
Affiliation(s)
- Zhixian He
- Department of Breast Surgery, Fudan University Shanghai Cancer Center, Shanghai 200032, PR China; Department of General Surgery, Affiliated Hospital of Nantong University, Nantong, 226001 Jiangsu, PR China
| | - Xiaojian Ni
- Department of General Surgery, Fudan University Zhongshan Hospital, Shanghai 200032, PR China
| | - Liuwan Xia
- Department of General Surgery, Affiliated Hospital of Nantong University, Nantong, 226001 Jiangsu, PR China
| | - Zhimin Shao
- Department of Breast Surgery, Fudan University Shanghai Cancer Center, Shanghai 200032, PR China.
| |
Collapse
|
|
20
|
Lee HJ, Jedrychowski MP, Vinayagam A, Wu N, Shyh-Chang N, Hu Y, Min-Wen C, Moore JK, Asara JM, Lyssiotis CA, Perrimon N, Gygi SP, Cantley LC, Kirschner MW. Proteomic and Metabolomic Characterization of a Mammalian Cellular Transition from Quiescence to Proliferation. Cell Rep 2018; 20:721-736. [PMID: 28723573 DOI: 10.1016/j.celrep.2017.06.074] [Citation(s) in RCA: 30] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/20/2017] [Revised: 05/22/2017] [Accepted: 06/25/2017] [Indexed: 12/28/2022] Open
Abstract
There exist similarities and differences in metabolism and physiology between normal proliferative cells and tumor cells. Once a cell enters the cell cycle, metabolic machinery is engaged to facilitate various processes. The kinetics and regulation of these metabolic changes have not been properly evaluated. To correlate the orchestration of these processes with the cell cycle, we analyzed the transition from quiescence to proliferation of a non-malignant murine pro-B lymphocyte cell line in response to IL-3. Using multiplex mass-spectrometry-based proteomics, we show that the transition to proliferation shares features generally attributed to cancer cells: upregulation of glycolysis, lipid metabolism, amino-acid synthesis, and nucleotide synthesis and downregulation of oxidative phosphorylation and the urea cycle. Furthermore, metabolomic profiling of this transition reveals similarities to cancer-related metabolic pathways. In particular, we find that methionine is consumed at a higher rate than that of other essential amino acids, with a potential link to maintenance of the epigenome.
Collapse
Affiliation(s)
- Ho-Joon Lee
- Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA
| | | | | | - Ning Wu
- Center for Cancer and Cell Biology, Van Andel Research Institute, Grand Rapids, MI 49503, USA
| | - Ng Shyh-Chang
- Stem Cell & Regenerative Biology, Genome Institute of Singapore, S138672 Singapore, Singapore
| | - Yanhui Hu
- Department of Genetics, Harvard Medical School, Boston, MA 02115, USA
| | - Chua Min-Wen
- Stem Cell & Regenerative Biology, Genome Institute of Singapore, S138672 Singapore, Singapore
| | - Jodene K Moore
- Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA
| | - John M Asara
- Division of Signal Transduction, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02115, USA
| | - Costas A Lyssiotis
- Division of Gastroenterology, Department of Molecular and Integrative Physiology and Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI 48109, USA
| | - Norbert Perrimon
- Department of Genetics, Harvard Medical School, Boston, MA 02115, USA
| | - Steven P Gygi
- Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA
| | - Lewis C Cantley
- Meyer Cancer Center, Department of Medicine, Weill Cornell Medical College, New York, NY 10065, USA
| | - Marc W Kirschner
- Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA.
| |
Collapse
|
|
21
|
Inhibition of never in mitosis A (NIMA)-related kinase-4 reduces survivin expression and sensitizes cancer cells to TRAIL-induced cell death. Oncotarget 2018; 7:65957-65967. [PMID: 27602754 PMCID: PMC5323206 DOI: 10.18632/oncotarget.11781] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/19/2016] [Accepted: 08/09/2016] [Indexed: 01/17/2023] Open
Abstract
The tumor necrosis factor-related apoptosis inducing ligand (TRAIL) preferentially induces apoptosis in cancer cells. However, many tumors are resistant to TRAIL-induced apoptosis, and resistance mechanisms are not fully understood. To identify novel regulatory molecules of TRAIL resistance, we screened a siRNA library targeting the human kinome, and NEK4 (NIMA-related kinase-4) was identified. Knockdown of NEK4 sensitized TRAIL-resistant cancer cells and in vivo xenografts to cell death. In contrast, over expression of NEK4 suppressed TRAIL-induced cell death in TRAIL-sensitive cancer cells. In addition, loss of NEK4 resulted in decrease of the anti-apoptotic protein survivin, but an increase in apoptotic cell death. Interestingly, NEK4 was highly upregulated in tumor tissues derived from patients with lung cancer and colon cancer. These results suggest that inhibition of NEK4 sensitizes cancer cells to TRAIL-induced apoptosis by regulation of survivin expression.
Collapse
|
|
22
|
Wells CI, Kapadia NR, Couñago RM, Drewry DH. In depth analysis of kinase cross screening data to identify chemical starting points for inhibition of the Nek family of kinases. MEDCHEMCOMM 2018; 9:44-66. [PMID: 30108900 PMCID: PMC6071746 DOI: 10.1039/c7md00510e] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/09/2017] [Accepted: 11/01/2017] [Indexed: 01/11/2023]
Abstract
Potent, selective, and cell active small molecule kinase inhibitors are useful tools to help unravel the complexities of kinase signaling. As the biological functions of individual kinases become better understood, they can become targets of drug discovery efforts. The small molecules used to shed light on function can also then serve as chemical starting points in these drug discovery efforts. The Nek family of kinases has received very little attention, as judged by number of citations in PubMed, yet they appear to play many key roles and have been implicated in disease. Here we present our work to identify high quality chemical starting points that have emerged due to the increased incidence of broad kinome screening. We anticipate that this analysis will allow the community to progress towards the generation of chemical probes and eventually drugs that target members of the Nek family.
Collapse
Affiliation(s)
- C I Wells
- Structural Genomics Consortium , Eshelman School of Pharmacy , University of North Carolina at Chapel Hill , Chapel Hill , NC , 27599 USA .
| | - N R Kapadia
- Structural Genomics Consortium , Eshelman School of Pharmacy , University of North Carolina at Chapel Hill , Chapel Hill , NC , 27599 USA .
| | - R M Couñago
- Structural Genomics Consortium , Universidade Estadual de Campinas - UNICAMP , Campinas , SP , 13083 Brazil
| | - D H Drewry
- Structural Genomics Consortium , Eshelman School of Pharmacy , University of North Carolina at Chapel Hill , Chapel Hill , NC , 27599 USA .
| |
Collapse
|
|
23
|
Choudhury AD, Schinzel AC, Cotter MB, Lis RT, Labella K, Lock YJ, Izzo F, Guney I, Bowden M, Li YY, Patel J, Hartman E, Carr SA, Schenone M, Jaffe JD, Kantoff PW, Hammerman PS, Hahn WC. Castration Resistance in Prostate Cancer Is Mediated by the Kinase NEK6. Cancer Res 2017; 77:753-765. [PMID: 27899381 PMCID: PMC5290202 DOI: 10.1158/0008-5472.can-16-0455] [Citation(s) in RCA: 25] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/11/2016] [Revised: 10/09/2016] [Accepted: 11/04/2016] [Indexed: 01/16/2023]
Abstract
In prostate cancer, the development of castration resistance is pivotal in progression to aggressive disease. However, understanding of the pathways involved remains incomplete. In this study, we performed a high-throughput genetic screen to identify kinases that enable tumor formation by androgen-dependent prostate epithelial (LHSR-AR) cells under androgen-deprived conditions. In addition to the identification of known mediators of castration resistance, which served to validate the screen, we identified a mitotic-related serine/threonine kinase, NEK6, as a mediator of androgen-independent tumor growth. NEK6 was overexpressed in a subset of human prostate cancers. Silencing NEK6 in castration-resistant cancer cells was sufficient to restore sensitivity to castration in a mouse xenograft model system. Tumors in which castration resistance was conferred by NEK6 were predominantly squamous in histology with no evidence of AR signaling. Gene expression profiling suggested that NEK6 overexpression stimulated cytoskeletal, differentiation, and immune signaling pathways and maintained gene expression patterns normally decreased by castration. Phosphoproteome profiling revealed the transcription factor FOXJ2 as a novel NEK6 substrate, with FOXJ2 phosphorylation associated with increased expression of newly identified NEK6 transcriptional targets. Overall, our studies establish NEK6 signaling as a central mechanism mediating castration-resistant prostate cancer. Cancer Res; 77(3); 753-65. ©2016 AACR.
Collapse
Affiliation(s)
- Atish D Choudhury
- Dana-Farber Cancer Institute, Boston, Massachusetts
- Harvard Medical School, Boston, Massachusetts
- Broad Institute of Harvard and MIT, Cambridge, Massachusetts
| | - Anna C Schinzel
- Dana-Farber Cancer Institute, Boston, Massachusetts
- Broad Institute of Harvard and MIT, Cambridge, Massachusetts
| | | | - Rosina T Lis
- Dana-Farber Cancer Institute, Boston, Massachusetts
- Brigham and Women's Hospital, Boston, Massachusetts
| | | | | | - Francesca Izzo
- Dana-Farber Cancer Institute, Boston, Massachusetts
- Broad Institute of Harvard and MIT, Cambridge, Massachusetts
| | - Isil Guney
- Dana-Farber Cancer Institute, Boston, Massachusetts
- Harvard Medical School, Boston, Massachusetts
| | | | - Yvonne Y Li
- Dana-Farber Cancer Institute, Boston, Massachusetts
| | - Jinal Patel
- Broad Institute of Harvard and MIT, Cambridge, Massachusetts
| | - Emily Hartman
- Broad Institute of Harvard and MIT, Cambridge, Massachusetts
| | - Steven A Carr
- Broad Institute of Harvard and MIT, Cambridge, Massachusetts
| | - Monica Schenone
- Broad Institute of Harvard and MIT, Cambridge, Massachusetts
| | - Jacob D Jaffe
- Broad Institute of Harvard and MIT, Cambridge, Massachusetts
| | - Philip W Kantoff
- Dana-Farber Cancer Institute, Boston, Massachusetts
- Harvard Medical School, Boston, Massachusetts
| | - Peter S Hammerman
- Dana-Farber Cancer Institute, Boston, Massachusetts
- Harvard Medical School, Boston, Massachusetts
- Broad Institute of Harvard and MIT, Cambridge, Massachusetts
| | - William C Hahn
- Dana-Farber Cancer Institute, Boston, Massachusetts.
- Harvard Medical School, Boston, Massachusetts
- Broad Institute of Harvard and MIT, Cambridge, Massachusetts
| |
Collapse
|
|
24
|
McSkimming DI, Dastgheib S, Baffi TR, Byrne DP, Ferries S, Scott ST, Newton AC, Eyers CE, Kochut KJ, Eyers PA, Kannan N. KinView: a visual comparative sequence analysis tool for integrated kinome research. MOLECULAR BIOSYSTEMS 2016; 12:3651-3665. [PMID: 27731453 PMCID: PMC5508867 DOI: 10.1039/c6mb00466k] [Citation(s) in RCA: 35] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/29/2022]
Abstract
Multiple sequence alignments (MSAs) are a fundamental analysis tool used throughout biology to investigate relationships between protein sequence, structure, function, evolutionary history, and patterns of disease-associated variants. However, their widespread application in systems biology research is currently hindered by the lack of user-friendly tools to simultaneously visualize, manipulate and query the information conceptualized in large sequence alignments, and the challenges in integrating MSAs with multiple orthogonal data such as cancer variants and post-translational modifications, which are often stored in heterogeneous data sources and formats. Here, we present the Multiple Sequence Alignment Ontology (MSAOnt), which represents a profile or consensus alignment in an ontological format. Subsets of the alignment are easily selected through the SPARQL Protocol and RDF Query Language for downstream statistical analysis or visualization. We have also created the Kinome Viewer (KinView), an interactive integrative visualization that places eukaryotic protein kinase cancer variants in the context of natural sequence variation and experimentally determined post-translational modifications, which play central roles in the regulation of cellular signaling pathways. Using KinView, we identified differential phosphorylation patterns between tyrosine and serine/threonine kinases in the activation segment, a major kinase regulatory region that is often mutated in proliferative diseases. We discuss cancer variants that disrupt phosphorylation sites in the activation segment, and show how KinView can be used as a comparative tool to identify differences and similarities in natural variation, cancer variants and post-translational modifications between kinase groups, families and subfamilies. Based on KinView comparisons, we identify and experimentally characterize a regulatory tyrosine (Y177PLK4) in the PLK4 C-terminal activation segment region termed the P+1 loop. To further demonstrate the application of KinView in hypothesis generation and testing, we formulate and validate a hypothesis explaining a novel predicted loss-of-function variant (D523NPKCβ) in the regulatory spine of PKCβ, a recently identified tumor suppressor kinase. KinView provides a novel, extensible interface for performing comparative analyses between subsets of kinases and for integrating multiple types of residue specific annotations in user friendly formats.
Collapse
Affiliation(s)
| | - Shima Dastgheib
- Department of Computer Science, University of Georgia, Athens, GA 30602, USA
| | - Timothy R Baffi
- Department of Pharmacology, University of California at San Diego, La Jolla, CA 92093, USA
| | - Dominic P Byrne
- Department of Biochemistry, Institute of Integrative Biology, University of Liverpool, Liverpool, UK
| | - Samantha Ferries
- Department of Biochemistry, Institute of Integrative Biology, University of Liverpool, Liverpool, UK
| | - Steven Thomas Scott
- Department of Biochemistry & Molecular Biology, University of Georgia, Athens, GA 30602, USA
| | - Alexandra C Newton
- Department of Pharmacology, University of California at San Diego, La Jolla, CA 92093, USA
| | - Claire E Eyers
- Department of Biochemistry, Institute of Integrative Biology, University of Liverpool, Liverpool, UK
| | - Krzysztof J Kochut
- Department of Computer Science, University of Georgia, Athens, GA 30602, USA
| | - Patrick A Eyers
- Department of Biochemistry, Institute of Integrative Biology, University of Liverpool, Liverpool, UK
| | - Natarajan Kannan
- Institute of Bioinformatics, University of Georgia, Athens, GA 30602, USA. and Department of Biochemistry & Molecular Biology, University of Georgia, Athens, GA 30602, USA
| |
Collapse
|
|
25
|
Zuo J, Ma H, Cai H, Wu Y, Jiang W, Yu L. An inhibitory role of NEK6 in TGFβ/Smad signaling pathway. BMB Rep 2015; 48:473-478. [PMID: 25523445 PMCID: PMC4576956 DOI: 10.5483/bmbrep.2015.48.8.225] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/26/2014] [Revised: 11/17/2014] [Accepted: 12/09/2014] [Indexed: 11/20/2022] Open
Abstract
The NEK6 (NIMA-related kinases 6) is reported to play potential roles in tumorigenesis. Although it is suggested to function in several cellular pathways, the underlying mechanism in tumorigenesis is still largely unknown. In the present study, we discovered interaction of NEK6 with Smad4, a key member of transforming growth factor beta (TGFβ) pathway. Over-expression of NEK6 in hepatocellular carcinoma (HCC) cell lines suppresses TGFβ-mediated transcription activity in a kinase activity-dependent manner. In addition, NEK6 suppresses the cell growth arrest induced by TGFβ. Mechanically, NEK6 blocks nuclear translocation of Smad4, which is essential for TGFβ function. Moreover, we identified that NEK6 could be regulated by TGFβ and hypoxia. Our study sheds new light on the roles of NEK6 in canonical TGFβ/Smad pathway and tumorigenesis.
Collapse
Affiliation(s)
- Jie Zuo
- State Key Laboratory of Genetic Engineering, Institute of Genetics, Fudan University, Shanghai 200433, PR China
| | - Haijie Ma
- Cell and Molecular Biology Laboratory, Zhoushan Hospital, Zhejiang, China
| | - Hao Cai
- State Key Laboratory of Genetic Engineering, Institute of Genetics, Fudan University, Shanghai 200433, PR China
| | - Yanhua Wu
- State Key Laboratory of Genetic Engineering, Institute of Genetics, Fudan University, Shanghai 200433, PR China
| | - Wei Jiang
- State Key Laboratory of Genetic Engineering, Institute of Genetics, Fudan University, Shanghai 200433, PR China
| | - Long Yu
- State Key Laboratory of Genetic Engineering, Institute of Genetics, Fudan University, Shanghai 200433, PR China
| |
Collapse
|
|
26
|
Wang N, Zhu M, Wang X, Tan HY, Tsao SW, Feng Y. Berberine-induced tumor suppressor p53 up-regulation gets involved in the regulatory network of MIR-23a in hepatocellular carcinoma. BIOCHIMICA ET BIOPHYSICA ACTA 2014; 1839:849-857. [PMID: 24942805 DOI: 10.1016/j.bbagrm.2014.05.027] [Citation(s) in RCA: 76] [Impact Index Per Article: 6.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/21/2014] [Revised: 05/02/2014] [Accepted: 05/27/2014] [Indexed: 11/24/2022]
Abstract
AIM OF THE STUDY To investigate the involvement of p53 in the regulatory network of microRNA-23a (miR-23a) in berberine-treated hepatocellular carcinoma (HCC) cells. METHODS The biogenesis of miR-23a upon berberine treatment was monitored by detecting the transcript expression of primary precursor, precursor and mature forms of miR-23a. Protein expression was detected with immunoblotting. The binding capacity between p53 and chromatin DNA was determined by chromatin immunoprecipitation. The role of miR-23a in mediating suppression of HCC by berberine was determined both in vitro and in vivo. RESULTS miR-23a was up-regulated upon berberine treatment in human HCC cells, and berberine could increase the expression of primary precursor, precursor and mature forms of miR-23a. The up-regulation of miR-23a by berberine is p53-dependent. Inhibition of p53 expression and activity could block the up-regulation of miR-23a induced by berberine. Furthermore, berberine-induced miR-23a expression may mediate the transcription activation of p53-related tumor suppressive genes p21 and GADD45α. Inhibition of miR-23a abolishes the binding of p53 onto chromatin and attenuates transcription activation of p21 and GADD45α. Target prediction and experimental validation demonstrate that berberine-induced miR-23a may target to Nek6 to suppress its expression. Berberine-induced G2/M cell cycle arrest in HCC was attenuated when miR-23a was inhibited. Berberine-induced cell death and in vivo tumor growth inhibition are attenuated upon inhibition of miR-23a. CONCLUSION Our study reveals that miR-23a may be involved in regulating the anti-HCC effect of berberine by mediating the regulation of p53.
Collapse
Affiliation(s)
- Ning Wang
- School of Chinese Medicine, The University of Hong Kong, 10 Sassoon Road, Pokfulam, Hong Kong, PR China
| | - Meifen Zhu
- School of Chinese Medicine, The University of Hong Kong, 10 Sassoon Road, Pokfulam, Hong Kong, PR China
| | - Xuanbin Wang
- School of Chinese Medicine, The University of Hong Kong, 10 Sassoon Road, Pokfulam, Hong Kong, PR China; Laboratory of Chinese Herbal Pharmacology, Renmin Hospital, School of Pharmacy, Hubei University of Medicine, 442000, PR China
| | - Hor-Yue Tan
- School of Chinese Medicine, The University of Hong Kong, 10 Sassoon Road, Pokfulam, Hong Kong, PR China
| | - Sai-Wah Tsao
- Department of Anatomy, The University of Hong Kong, 21 Sassoon Road, Pokfulam, Hong Kong, PR China
| | - Yibin Feng
- School of Chinese Medicine, The University of Hong Kong, 10 Sassoon Road, Pokfulam, Hong Kong, PR China.
| |
Collapse
|
|
27
|
ZHANG BIAO, ZHANG HAI, WANG DONG, HAN SHENG, WANG KE, YAO AIHUA, LI XIANGCHENG. Never in mitosis gene A-related kinase 6 promotes cell proliferation of hepatocellular carcinoma via cyclin B modulation. Oncol Lett 2014; 8:1163-1168. [PMID: 25120679 PMCID: PMC4114597 DOI: 10.3892/ol.2014.2300] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/06/2013] [Accepted: 06/12/2014] [Indexed: 01/12/2023] Open
Abstract
Never in mitosis gene A-related kinase (Nek) 6 is a recently identified Nek that is required for mitotic cell cycle progression; however, the role and mechanism of Nek6 activity during hepatocarcinogenesis is not well known. The aim of this study was to investigate the potential roles and internal mechanism of Nek6 in hepatocellular carcinoma (HCC) development. In the present study, Nek6 was found to be overexpressed in HCC samples and cell lines by florescent real-time quantitative polymerase chain reaction, immunohistochemistry and western blot analysis. Furthermore, it was evidenced to contribute to oncogenesis and progression. The ectopic overexpression of Nek6 promoted cell proliferation and colony formation, whereas gene silencing of Nek6 inhibited these phenotypes, as documented in Huh7, PLC/PRF/5, Hep3B and HepG2 HCC cell lines. Mechanistic analyses indicated that Nek6 regulates the transcription of cyclin B through cdc2 activation, and promotes the accumulation of G0/G1-phase cells. In conclusion, the findings of the current study suggested that Nek6 contributes to the oncogenic potential of HCC, and may present as a potential therapeutic target in this disease.
Collapse
Affiliation(s)
- BIAO ZHANG
- Liver Transplantation Center, First Affiliated Hospital of Nanjing Medical University, Key Laboratory of Living Donor Liver Transplantation, Ministry of Public Health, Nanjing, Jiangsu 210029, P.R. China
| | - HAI ZHANG
- Department of Hepatobiliary Surgery, Affiliated Hospital of Jiangsu University, Zhenjiang, Jiangsu 212000, P.R. China
| | - DONG WANG
- Liver Transplantation Center, First Affiliated Hospital of Nanjing Medical University, Key Laboratory of Living Donor Liver Transplantation, Ministry of Public Health, Nanjing, Jiangsu 210029, P.R. China
| | - SHENG HAN
- Liver Transplantation Center, First Affiliated Hospital of Nanjing Medical University, Key Laboratory of Living Donor Liver Transplantation, Ministry of Public Health, Nanjing, Jiangsu 210029, P.R. China
| | - KE WANG
- Liver Transplantation Center, First Affiliated Hospital of Nanjing Medical University, Key Laboratory of Living Donor Liver Transplantation, Ministry of Public Health, Nanjing, Jiangsu 210029, P.R. China
| | - AIHUA YAO
- Liver Transplantation Center, First Affiliated Hospital of Nanjing Medical University, Key Laboratory of Living Donor Liver Transplantation, Ministry of Public Health, Nanjing, Jiangsu 210029, P.R. China
| | - XIANGCHENG LI
- Liver Transplantation Center, First Affiliated Hospital of Nanjing Medical University, Key Laboratory of Living Donor Liver Transplantation, Ministry of Public Health, Nanjing, Jiangsu 210029, P.R. China
| |
Collapse
|
|
28
|
Li X, Lian L, Zhang D, Qu L, Yang N. gga-miR-26a targets NEK6 and suppresses Marek's disease lymphoma cell proliferation. Poult Sci 2014; 93:1097-105. [PMID: 24795301 DOI: 10.3382/ps.2013-03656] [Citation(s) in RCA: 34] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
MicroRNA (miRNA) are a class of highly conserved, small noncoding RNA that emerge as key posttranscriptional regulators in various neoplastic transformations. Our previous study profiling the miRNA transcriptome in Marek's disease virus (MDV)-induced lymphoma revealed many novel and differentially expressed miRNA, including gga-miR-26a, which was downregulated in MDV-infected spleens of chickens. In this study, differential expression of gga-miR-26a between MDV-infected and noninfected spleens at 4, 7, 14, 21, and 28 d postinfection was analyzed by real-time PCR. The results showed gga-miR-26a were downregulated in MDV-infected spleens at cytolytic infection, latency, and tumor transformation phases. Subsequent cell proliferation assay revealed cell viability was lower in gga-miR-26a mimic transfection group than that in negative controls. Target genes of gga-miR-26a were identified by luciferase reporter gene assay. The results showed significant interaction between gga-miR-26a and Never In Mitosis Gene A (NIMA)-related kinase 6 (NEK6) gene. Subsequent gain of function experiment and Western blot assay showed that mRNA and protein levels of NEK6 were downregulated after gga-miR-26 mimic was transfected into MDV-transformed lymphoid cell line (MSB-1), indicating that NEK6 was modulated by gga-miR-26a. The expression of NEK6 showed a higher trend in MDV-infected samples including tumorous spleen and MD lymphoma from liver than that in noninfected controls. The results suggested that gga-miR-26a inhibited MSB-1 cell proliferation. Gga-miR-26a and its direct target, NEK6, might play important roles in MDV infection.
Collapse
Affiliation(s)
- Xin Li
- Department of Animal Genetics and Breeding, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing 100193, China
| | | | | | | | | |
Collapse
|
|
29
|
Meirelles GV, Perez AM, de Souza EE, Basei FL, Papa PF, Melo Hanchuk TD, Cardoso VB, Kobarg J. “Stop Ne(c)king around”: How interactomics contributes to functionally characterize Nek family kinases. World J Biol Chem 2014; 5:141-160. [PMID: 24921005 PMCID: PMC4050109 DOI: 10.4331/wjbc.v5.i2.141] [Citation(s) in RCA: 41] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/23/2013] [Revised: 01/07/2014] [Accepted: 02/18/2014] [Indexed: 02/05/2023] Open
Abstract
Aside from Polo and Aurora, a third but less studied kinase family involved in mitosis regulation is the never in mitosis-gene A (NIMA)-related kinases (Neks). The founding member of this family is the sole member NIMA of Aspergillus nidulans, which is crucial for the initiation of mitosis in that organism. All 11 human Neks have been functionally assigned to one of the three core functions established for this family in mammals: (1) centrioles/mitosis; (2) primary ciliary function/ciliopathies; and (3) DNA damage response (DDR). Recent findings, especially on Nek 1 and 8, showed however, that several Neks participate in parallel in at least two of these contexts: primary ciliary function and DDR. In the core section of this in-depth review, we report the current detailed functional knowledge on each of the 11 Neks. In the discussion, we return to the cross-connections among Neks and point out how our and other groups’ functional and interactomics studies revealed that most Neks interact with protein partners associated with two if not all three of the functional contexts. We then raise the hypothesis that Neks may be the connecting regulatory elements that allow the cell to fine tune and synchronize the cellular events associated with these three core functions. The new and exciting findings on the Nek family open new perspectives and should allow the Neks to finally claim the attention they deserve in the field of kinases and cell cycle biology.
Collapse
|
|
30
|
Bian Z, Liao H, Zhang Y, Wu Q, Zhou H, Yang Z, Fu J, Wang T, Yan L, Shen D, Li H, Tang Q. Never in mitosis gene A related kinase-6 attenuates pressure overload-induced activation of the protein kinase B pathway and cardiac hypertrophy. PLoS One 2014; 9:e96095. [PMID: 24763737 PMCID: PMC3999101 DOI: 10.1371/journal.pone.0096095] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/25/2013] [Accepted: 04/02/2014] [Indexed: 12/31/2022] Open
Abstract
Cardiac hypertrophy appears to be a specialized form of cellular growth that involves the proliferation control and cell cycle regulation. NIMA (never in mitosis, gene A)-related kinase-6 (Nek6) is a cell cycle regulatory gene that could induce centriole duplication, and control cell proliferation and survival. However, the exact effect of Nek6 on cardiac hypertrophy has not yet been reported. In the present study, the loss- and gain-of-function experiments were performed in Nek6 gene-deficient (Nek6−/−) mice and Nek6 overexpressing H9c2 cells to clarify whether Nek6 which promotes the cell cycle also mediates cardiac hypertrophy. Cardiac hypertrophy was induced by transthoracic aorta constriction (TAC) and then evaluated by echocardiography, pathological and molecular analyses in vivo. We got novel findings that the absence of Nek6 promoted cardiac hypertrophy, fibrosis and cardiac dysfunction, which were accompanied by a significant activation of the protein kinase B (Akt) signaling in an experimental model of TAC. Consistent with this, the overexpression of Nek6 prevented hypertrophy in H9c2 cells induced by angiotonin II and inhibited Akt signaling in vitro. In conclusion, our results demonstrate that the cell cycle regulatory gene Nek6 is also a critical signaling molecule that helps prevent cardiac hypertrophy and inhibits the Akt signaling pathway.
Collapse
Affiliation(s)
- Zhouyan Bian
- Department of Cardiology, Renmin Hospital of Wuhan University, Wuhan, Hubei Province, P. R. China
- Cardiovascular Research Institute of Wuhan University, Wuhan, Hubei Province, P. R. China
| | - Haihan Liao
- Department of Cardiology, Renmin Hospital of Wuhan University, Wuhan, Hubei Province, P. R. China
- Cardiovascular Research Institute of Wuhan University, Wuhan, Hubei Province, P. R. China
| | - Yan Zhang
- Department of Cardiology, Renmin Hospital of Wuhan University, Wuhan, Hubei Province, P. R. China
- Cardiovascular Research Institute of Wuhan University, Wuhan, Hubei Province, P. R. China
| | - Qingqing Wu
- Department of Cardiology, Renmin Hospital of Wuhan University, Wuhan, Hubei Province, P. R. China
- Cardiovascular Research Institute of Wuhan University, Wuhan, Hubei Province, P. R. China
| | - Heng Zhou
- Department of Cardiology, Renmin Hospital of Wuhan University, Wuhan, Hubei Province, P. R. China
| | - Zheng Yang
- Department of Cardiology, Renmin Hospital of Wuhan University, Wuhan, Hubei Province, P. R. China
- Cardiovascular Research Institute of Wuhan University, Wuhan, Hubei Province, P. R. China
| | - Jinrong Fu
- Department of Cardiology, Renmin Hospital of Wuhan University, Wuhan, Hubei Province, P. R. China
| | - Teng Wang
- Department of Cardiology, Renmin Hospital of Wuhan University, Wuhan, Hubei Province, P. R. China
- Cardiovascular Research Institute of Wuhan University, Wuhan, Hubei Province, P. R. China
| | - Ling Yan
- Department of Cardiology, Renmin Hospital of Wuhan University, Wuhan, Hubei Province, P. R. China
| | - Difei Shen
- Department of Cardiology, Renmin Hospital of Wuhan University, Wuhan, Hubei Province, P. R. China
| | - Hongliang Li
- Department of Cardiology, Renmin Hospital of Wuhan University, Wuhan, Hubei Province, P. R. China
- Cardiovascular Research Institute of Wuhan University, Wuhan, Hubei Province, P. R. China
| | - Qizhu Tang
- Department of Cardiology, Renmin Hospital of Wuhan University, Wuhan, Hubei Province, P. R. China
- Cardiovascular Research Institute of Wuhan University, Wuhan, Hubei Province, P. R. China
- * E-mail:
| |
Collapse
|
|
31
|
Glant TT, Besenyei T, Kádár A, Kurkó J, Tryniszewska B, Gál J, Soós G, Szekanecz Z, Hoffmann G, Block JA, Katz RS, Mikecz K, Rauch TA. Differentially expressed epigenome modifiers, including aurora kinases A and B, in immune cells in rheumatoid arthritis in humans and mouse models. ACTA ACUST UNITED AC 2013; 65:1725-35. [PMID: 23653330 DOI: 10.1002/art.37986] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/25/2012] [Accepted: 04/16/2013] [Indexed: 12/24/2022]
Abstract
OBJECTIVE To identify epigenetic factors that are implicated in the pathogenesis of rheumatoid arthritis (RA), and to explore the therapeutic potential of the targeted inhibition of these factors. METHODS Polymerase chain reaction (PCR) arrays were used to investigate the expression profile of genes that encode key epigenetic regulator enzymes. Mononuclear cells from RA patients and mice were monitored for gene expression changes, in association with arthritis development in murine models of RA. Selected genes were further characterized by quantitative reverse transcription-PCR, Western blot, and flow cytometry methods. The targeted inhibition of the up-regulated enzymes was studied in arthritic mice. RESULTS A set of genes with arthritis-specific expression was identified by the PCR arrays. Aurora kinases A and B, both of which were highly expressed in arthritic mice and treatment-naive RA patients, were selected for detailed analysis. Elevated aurora kinase expression was accompanied by increased phosphorylation of histone H3, which promotes proliferation of T lymphocytes. Treatment with VX-680, a pan-aurora kinase inhibitor, promoted B cell apoptosis, provided significant protection against disease onset, and attenuated inflammatory reactions in arthritic mice. CONCLUSION Arthritis development is accompanied by changes in expression of a number of epigenome-modifying enzymes. Drug-induced down-regulation of the aurora kinases, among other targets, seems to be sufficient to treat experimental arthritis. Development of new therapeutics that target aurora kinases can potentially improve RA management.
Collapse
|
|
32
|
Dodson CA, Yeoh S, Haq T, Bayliss R. A kinetic test characterizes kinase intramolecular and intermolecular autophosphorylation mechanisms. Sci Signal 2013; 6:ra54. [PMID: 23821772 DOI: 10.1126/scisignal.2003910] [Citation(s) in RCA: 47] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023]
Abstract
Many protein kinases catalyze their own activation by autophosphorylation. The mechanism of this is generally considered to be intermolecular and similar to that used in substrate phosphorylation. We derived the kinetic signatures of the four simplest autophosphorylation reactions and developed a test to determine the autoactivation mechanism of individual kinases. Whereas autophosphorylation of Nek7 and Plk4 occurred through an intermolecular mechanism, the kinases Aurora-A and Chk2 followed an intramolecular mechanism. Autophosphorylation of Aurora-A was accelerated in the presence of its protein activator TPX2. Nek9, the binding partner for Nek7, had a concentration-dependent effect such that low amounts enhanced autoactivation of Nek7 and high amounts were inhibitory. A structural model of Aurora-A undergoing autophosphorylation confirmed that an intramolecular mechanism is physically possible, and provided an explanation for how TPX2 could stimulate both autophosphorylation and substrate phosphorylation. The distinct mechanisms of autoactivation have consequences for cellular regulation because each molecule of a kinase that undergoes intramolecular autophosphorylation is activated individually, whereas the activity of kinases that undergo intermolecular autophosphorylation can be rapidly self-amplified in the cell. Local control of individual molecules, such as Aurora-A, may be particularly advantageous for a kinase with multiple, distinct cellular roles.
Collapse
Affiliation(s)
- Charlotte A Dodson
- Division of Structural Biology, The Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, UK.
| | | | | | | |
Collapse
|
|
33
|
The inhibition of Nek6 function sensitizes human cancer cells to premature senescence upon serum reduction or anticancer drug treatment. Cancer Lett 2013; 335:175-82. [PMID: 23416273 DOI: 10.1016/j.canlet.2013.02.012] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/01/2012] [Revised: 02/01/2013] [Accepted: 02/05/2013] [Indexed: 12/30/2022]
Abstract
The induction of premature senescence in cancer cells was proposed as an effective cancer treatment strategy. In this paper, we show that the inhibition of Nek6 expression by Nek6 siRNA-mediated knockdown or the overexpression of a dominant negative form of Nek6 (Nek6KM) induced premature senescence as well as cell death under reduced serum conditions in multiple cancer cell lines, including both p53 wild-type and p53 mutant/null backgrounds. Moreover, cancer cells expressing Nek6KM exhibited significantly increased premature senescence upon treatment with the anticancer drugs doxorubicin (DOX) and camptothecin (CPT). Significantly, the overexpression of Nek6KM also inhibited tumor growth and promoted premature senescence in vivo in a xenograft mouse model. Taken together, our results further confirm that Nek6 plays an important role in the premature senescence of cancer cells, suggesting that Nek6 may be a potential therapeutic target for human cancers.
Collapse
|
|
34
|
Fry AM, O'Regan L, Sabir SR, Bayliss R. Cell cycle regulation by the NEK family of protein kinases. J Cell Sci 2012; 125:4423-33. [PMID: 23132929 DOI: 10.1242/jcs.111195] [Citation(s) in RCA: 260] [Impact Index Per Article: 20.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023] Open
Abstract
Genetic screens for cell division cycle mutants in the filamentous fungus Aspergillus nidulans led to the discovery of never-in-mitosis A (NIMA), a serine/threonine kinase that is required for mitotic entry. Since that discovery, NIMA-related kinases, or NEKs, have been identified in most eukaryotes, including humans where eleven genetically distinct proteins named NEK1 to NEK11 are expressed. Although there is no evidence that human NEKs are essential for mitotic entry, it is clear that several NEK family members have important roles in cell cycle control. In particular, NEK2, NEK6, NEK7 and NEK9 contribute to the establishment of the microtubule-based mitotic spindle, whereas NEK1, NEK10 and NEK11 have been implicated in the DNA damage response. Roles for NEKs in other aspects of mitotic progression, such as chromatin condensation, nuclear envelope breakdown, spindle assembly checkpoint signalling and cytokinesis have also been proposed. Interestingly, NEK1 and NEK8 also function within cilia, the microtubule-based structures that are nucleated from basal bodies. This has led to the current hypothesis that NEKs have evolved to coordinate microtubule-dependent processes in both dividing and non-dividing cells. Here, we review the functions of the human NEKs, with particular emphasis on those family members that are involved in cell cycle control, and consider their potential as therapeutic targets in cancer.
Collapse
Affiliation(s)
- Andrew M Fry
- Department of Biochemistry, University of Leicester, Lancaster Road, Leicester LE1 9HN, UK.
| | | | | | | |
Collapse
|
|
35
|
Abstract
The basic biology of the cell division cycle and its control by protein kinases was originally studied through genetic and biochemical studies in yeast and other model organisms. The major regulatory mechanisms identified in this pioneer work are conserved in mammals. However, recent studies in different cell types or genetic models are now providing a new perspective on the function of these major cell cycle regulators in different tissues. Here, we review the physiological relevance of mammalian cell cycle kinases such as cyclin-dependent kinases (Cdks), Aurora and Polo-like kinases, and mitotic checkpoint regulators (Bub1, BubR1, and Mps1) as well as other less-studied enzymes such as Cdc7, Nek proteins, or Mastl and their implications in development, tissue homeostasis, and human disease. Among these functions, the control of self-renewal or asymmetric cell division in stem/progenitor cells and the ability to regenerate injured tissues is a central issue in current research. In addition, many of these proteins play previously unexpected roles in metabolism, cardiovascular function, or neuron biology. The modulation of their enzymatic activity may therefore have multiple therapeutic benefits in human disease.
Collapse
Affiliation(s)
- Marcos Malumbres
- Cell Division and Cancer Group, Spanish National Cancer Research Centre, Madrid, Spain.
| |
Collapse
|
|
36
|
Škalamera D, Ranall MV, Wilson BM, Leo P, Purdon AS, Hyde C, Nourbakhsh E, Grimmond SM, Barry SC, Gabrielli B, Gonda TJ. A high-throughput platform for lentiviral overexpression screening of the human ORFeome. PLoS One 2011; 6:e20057. [PMID: 21629697 PMCID: PMC3101218 DOI: 10.1371/journal.pone.0020057] [Citation(s) in RCA: 41] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/23/2010] [Accepted: 04/24/2011] [Indexed: 11/22/2022] Open
Abstract
In response to the growing need for functional analysis of the human genome, we have developed a platform for high-throughput functional screening of genes overexpressed from lentiviral vectors. Protein-coding human open reading frames (ORFs) from the Mammalian Gene Collection were transferred into lentiviral expression vector using the highly efficient Gateway recombination cloning. Target ORFs were inserted into the vector downstream of a constitutive promoter and upstream of an IRES controlled GFP reporter, so that their transfection, transduction and expression could be monitored by fluorescence. The expression plasmids and viral packaging plasmids were combined and transfected into 293T cells to produce virus, which was then used to transduce the screening cell line. We have optimised the transfection and transduction procedures so that they can be performed using robotic liquid handling systems in arrayed 96-well microplate, one-gene-per-well format, without the need to concentrate the viral supernatant. Since lentiviruses can infect both dividing and non-dividing cells, this system can be used to overexpress human ORFs in a broad spectrum of experimental contexts. We tested the platform in a 1990 gene pilot screen for genes that can increase proliferation of the non-tumorigenic mammary epithelial cell line MCF-10A after removal of growth factors. Transduced cells were labelled with the nucleoside analogue 5-ethynyl-2′-deoxyuridine (EdU) to detect cells progressing through S phase. Hits were identified using high-content imaging and statistical analysis and confirmed with vectors using two different promoters (CMV and EF1α). The screen demonstrates the reliability, versatility and utility of our screening platform, and identifies novel cell cycle/proliferative activities for a number of genes.
Collapse
Affiliation(s)
- Dubravka Škalamera
- University of Queensland Diamantina Institute, Princess Alexandra Hospital, Brisbane, Queensland, Australia.
| | | | | | | | | | | | | | | | | | | | | |
Collapse
|
|
37
|
Jee HJ, Kim HJ, Kim AJ, Song N, Kim M, Yun J. Nek6 suppresses the premature senescence of human cancer cells induced by camptothecin and doxorubicin treatment. Biochem Biophys Res Commun 2011; 408:669-73. [PMID: 21539811 DOI: 10.1016/j.bbrc.2011.04.083] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/16/2011] [Accepted: 04/18/2011] [Indexed: 01/24/2023]
Abstract
Cellular senescence plays an important role in tumor suppression. The mitotic kinase Nek6 has recently been shown to be overexpressed in various cancers and has been implicated in tumorigenesis. Previously, we reported that the down-regulation of Nek6 expression was required for p53-induced senescence. In this study, we examined the effect of Nek6 overexpression on the premature senescence of cancer cells induced by the anticancer drugs camptothecin (CPT) and doxorubicin (DOX). We found that CPT- and DOX-induced morphology changes and increases in senescence-associated β-galactosidase staining were significantly inhibited in EJ human bladder cancer cells and H1299 human lung cancer cells overexpressing HA-Nek6. DOX-induced G2/M cell cycle arrest and the reduction in cyclin B and cdc2 levels after DOX treatment were significantly reduced by Nek6 overexpression. In addition, an increase in the intracellular levels of ROS in response to DOX was also inhibited in cells overexpressing Nek6. These results suggest that the increased expression of Nek6 renders cancer cells resistant to premature senescence, and targeting Nek6 could be an efficient strategy for cancer treatment.
Collapse
Affiliation(s)
- Hye Jin Jee
- Department of Biochemistry, College of Medicine, Dong-A University, Busan, South Korea
| | | | | | | | | | | |
Collapse
|
|
38
|
Regué L, Sdelci S, Bertran MT, Caelles C, Reverter D, Roig J. DYNLL/LC8 protein controls signal transduction through the Nek9/Nek6 signaling module by regulating Nek6 binding to Nek9. J Biol Chem 2011; 286:18118-29. [PMID: 21454704 DOI: 10.1074/jbc.m110.209080] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/15/2023] Open
Abstract
The NIMA family protein kinases Nek9/Nercc1 and the highly similar Nek6 and Nek7 form a signaling module activated in mitosis, when they are involved in the control of spindle organization and function. Here we report that Nek9, the module upstream kinase, binds to DYNLL/LC8, a highly conserved protein originally described as a component of the dynein complex. LC8 is a dimer that interacts with different proteins and has been suggested to act as a dimerization hub promoting the organization and oligomerization of partially disorganized partners. We find that the interaction of LC8 with Nek9 depends on a (K/R)XTQT motif adjacent to the Nek9 C-terminal coiled coil motif, results in Nek9 multimerization, and increases the rate of Nek9 autoactivation. LC8 binding to Nek9 is regulated by Nek9 activity through the autophosphorylation of Ser(944), a residue immediately N-terminal to the (K/R)XTQT motif. Remarkably, LC8 binding interferes with the interaction of Nek9 with its downstream partner Nek6 as well as with Nek6 activation, thus controlling both processes. Our work sheds light into the control of signal transduction through the module formed by Nek9 and Nek6/7 and uncovers a novel manner in which LC8 can regulate partner physiology by interfering with protein complex formation. We suggest that this and other LC8 functions can be specifically regulated by partner phosphorylation.
Collapse
Affiliation(s)
- Laura Regué
- Cell Signaling Group, Molecular Medicine Program, Institute for Research in Biomedicine (IRB Barcelona), 08028 Barcelona, Spain
| | | | | | | | | | | |
Collapse
|
|
39
|
Radisky DC. Function following form: functional differentiation of mammary epithelial cells requires laminin-induced polarization of PI3-kinase. Cell Cycle 2011; 10:15. [PMID: 21200145 PMCID: PMC3233478 DOI: 10.4161/cc.10.1.14218] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/15/2023] Open
|