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1
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Vujovic F, Farahani RM. Thyroid Hormones and Brain Development: A Focus on the Role of Mitochondria as Regulators of Developmental Time. Cells 2025; 14:150. [PMID: 39936942 PMCID: PMC11816491 DOI: 10.3390/cells14030150] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/27/2024] [Revised: 01/16/2025] [Accepted: 01/19/2025] [Indexed: 02/13/2025] Open
Abstract
Thyroid hormones (THs) regulate metabolism in a homeostatic state in an adult organism. During the prenatal period, prior to the establishment of homeostatic mechanisms, THs assume additional functions as key regulators of brain development. Here, we focus on reviewing the role of THs in orchestrating cellular dynamics in a developing brain. The evidence from the reviewed scientific literature suggests that the developmental roles of the hormones are predominantly mediated by non-genomic mitochondrial effects of THs due to attenuation of genomic effects of THs that antagonise non-genomic impacts. We argue that the key function of TH signalling during brain development is to orchestrate the tempo of self-organisation of neural progenitor cells. Further, evidence is provided that major neurodevelopmental consequences of hypothyroidism stem from an altered tempo of cellular self-organisation.
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Affiliation(s)
- Filip Vujovic
- IDR/WSLHD Research and Education Network, Sydney, NSW 2145, Australia
- School of Medical Sciences, Faculty of Medicine and Health, University of Sydney, Sydney, NSW 2006, Australia
| | - Ramin M Farahani
- IDR/WSLHD Research and Education Network, Sydney, NSW 2145, Australia
- School of Medical Sciences, Faculty of Medicine and Health, University of Sydney, Sydney, NSW 2006, Australia
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2
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Khan S, Patra PH, Somerfield H, Benya-Aphikul H, Upadhya M, Zhang X. IQGAP1 promotes chronic pain by regulating the trafficking and sensitization of TRPA1 channels. Brain 2022:6881565. [PMID: 36477832 DOI: 10.1093/brain/awac462] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/26/2022] [Revised: 11/14/2022] [Accepted: 11/24/2022] [Indexed: 12/12/2022] Open
Abstract
TRPA1 channels have been implicated in mechanical and cold hypersensitivity in chronic pain. But how TRPA1 mediates this process is unclear. Here we show that IQ-motif containing GTPase activating protein 1 (IQGAP1) is responsible using a combination of biochemical, molecular, Ca2+ imaging and behavioural approaches. TRPA1 and IQGAP1 bind to each other and are highly colocalised in sensory DRG neurons in mice. The expression of IQGAP1 but not TRPA1 is increased in chronic inflammatory and neuropathic pain. However, TRPA1 undergoes increased trafficking to the membrane of DRG neurons catalysed by the small GTPase Cdc42 associated with IQGAP1, leading to functional sensitization of the channel. Activation of PKA is also sufficient to evoke TRPA1 trafficking and sensitization. All these responses are, however, completely prevented in the absence of IQGAP1. Concordantly, deletion of IQGAP1 markedly reduces mechanical and cold hypersensitivity in chronic inflammatory and neuropathic pain in mice. IQGAP1 thus promotes chronic pain by coupling the trafficking and signalling machineries to TRPA1 channels.
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Affiliation(s)
- Shakil Khan
- School of Health & Life Sciences, Aston University, Birmingham B4 7ET, UK
| | - Pabitra H Patra
- School of Health & Life Sciences, Aston University, Birmingham B4 7ET, UK
| | - Hannah Somerfield
- School of Health & Life Sciences, Aston University, Birmingham B4 7ET, UK
| | | | - Manoj Upadhya
- School of Health & Life Sciences, Aston University, Birmingham B4 7ET, UK
| | - Xuming Zhang
- School of Health & Life Sciences, Aston University, Birmingham B4 7ET, UK
- School of Life Sciences, University of Warwick, Coventry CV4 7AL, UK
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3
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Kryszczuk M, Kowalczuk O. Significance of NRF2 in physiological and pathological conditions an comprehensive review. Arch Biochem Biophys 2022; 730:109417. [DOI: 10.1016/j.abb.2022.109417] [Citation(s) in RCA: 21] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/13/2022] [Revised: 09/22/2022] [Accepted: 09/24/2022] [Indexed: 11/30/2022]
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4
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Muehlbauer LK, Wei T, Shishkova E, Coon JJ, Lambert PF. IQGAP1 and RNA Splicing in the Context of Head and Neck via Phosphoproteomics. J Proteome Res 2022; 21:2211-2223. [PMID: 35980772 PMCID: PMC9833422 DOI: 10.1021/acs.jproteome.2c00309] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
IQGAP1 (IQ motif-containing GTPase-activating protein 1) scaffolds several signaling pathways in mammalian cells that are implicated in carcinogenesis, including the RAS and PI3K pathways that involve multiple protein kinases. IQGAP1 has been shown to promote head and neck squamous cell carcinoma (HNSCC); however, the underlying mechanism(s) remains unclear. Here, we report a mass spectrometry-based analysis identifying differences in phosphorylation of cellular proteins in vivo and in vitro in the presence or absence of IQGAP1. By comparing the esophageal phosphoproteome profiles between Iqgap1+/+ and Iqgap1-/- mice, we identified RNA splicing as one of the most altered cellular processes. Serine/arginine-rich splicing factor 6 (SRSF6) was the protein with the most downregulated levels of phosphorylation in Iqgap1-/- tissue. We confirmed that the absence of IQGAP1 reduced SRSF6 phosphorylation both in vivo and in vitro. We then expanded our analysis to human normal oral keratinocytes. Again, we found factors involved in RNA splicing to be highly altered in the phosphoproteome profile upon genetic disruption of IQGAP1. Both the Clinical Proteomic Tumor Analysis Consortium (CPTAC) and the Cancer Genome Atlas (TCGA) data sets indicate that phosphorylation of splicing-related proteins is important in HNSCC prognosis. The Biological General Repository for Interaction Datasets (BioGRID) repository also suggested multiple interactions between IQGAP1 and splicing-related proteins. Based on these collective observations, we propose that IQGAP1 regulates the phosphorylation of splicing proteins, which potentially affects their splicing activities and, therefore, contributes to HNSCC. Raw data are available from the MassIVE database with identifier MSV000087770.
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Affiliation(s)
- Laura K. Muehlbauer
- Department of Chemistry, University of Wisconsin-Madison, Madison, WI, 53706, USA
| | - Tao Wei
- McArdle Laboratory for Cancer Research, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI, 53705, USA
| | - Evgenia Shishkova
- Department of Biomolecular Chemistry, University of Wisconsin-Madison, Madison, WI, 53706, USA
- National Center for Quantitative Biology of Complex Systems, University of Wisconsin-Madison, Madison, WI 53706, USA
| | - Joshua J. Coon
- Department of Chemistry, University of Wisconsin-Madison, Madison, WI, 53706, USA
- Department of Biomolecular Chemistry, University of Wisconsin-Madison, Madison, WI, 53706, USA
- National Center for Quantitative Biology of Complex Systems, University of Wisconsin-Madison, Madison, WI 53706, USA
- Morgridge Institute for Research, Madison, WI 53706, USA
| | - Paul F. Lambert
- McArdle Laboratory for Cancer Research, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI, 53705, USA
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5
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Chhichholiya Y, Suryan AK, Suman P, Munshi A, Singh S. SNPs in miRNAs and Target Sequences: Role in Cancer and Diabetes. Front Genet 2021; 12:793523. [PMID: 34925466 PMCID: PMC8673831 DOI: 10.3389/fgene.2021.793523] [Citation(s) in RCA: 25] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/12/2021] [Accepted: 10/28/2021] [Indexed: 12/27/2022] Open
Abstract
miRNAs are fascinating molecular players for gene regulation as individual miRNA can control multiple targets and a single target can be regulated by multiple miRNAs. Loss of miRNA regulated gene expression is often reported to be implicated in various human diseases like diabetes and cancer. Recently, geneticists across the world started reporting single nucleotide polymorphism (SNPs) in seed sequences of miRNAs. Similarly, SNPs are also reported in various target sequences of these miRNAs. Both the scenarios lead to dysregulated gene expression which may result in the progression of diseases. In the present paper, we explore SNPs in various miRNAs and their target sequences reported in various human cancers as well as diabetes. Similarly, we also present evidence of these mutations in various other human diseases.
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Affiliation(s)
- Yogita Chhichholiya
- Department of Human Genetics and Molecular Medicine, Central University of Punjab, Bathinda, India
| | - Aman Kumar Suryan
- Department of Human Genetics and Molecular Medicine, Central University of Punjab, Bathinda, India
| | - Prabhat Suman
- Department of Human Genetics and Molecular Medicine, Central University of Punjab, Bathinda, India
| | - Anjana Munshi
- Department of Human Genetics and Molecular Medicine, Central University of Punjab, Bathinda, India
| | - Sandeep Singh
- Department of Human Genetics and Molecular Medicine, Central University of Punjab, Bathinda, India
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6
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Wei T, Lambert PF. Role of IQGAP1 in Carcinogenesis. Cancers (Basel) 2021; 13:3940. [PMID: 34439095 PMCID: PMC8391515 DOI: 10.3390/cancers13163940] [Citation(s) in RCA: 31] [Impact Index Per Article: 7.8] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/20/2021] [Revised: 07/28/2021] [Accepted: 08/02/2021] [Indexed: 12/31/2022] Open
Abstract
Scaffolding proteins can play important roles in cell signaling transduction. IQ motif-containing GTPase-activating protein 1 (IQGAP1) influences many cellular activities by scaffolding multiple key signaling pathways, including ones involved in carcinogenesis. Two decades of studies provide evidence that IQGAP1 plays an essential role in promoting cancer development. IQGAP1 is overexpressed in many types of cancer, and its overexpression in cancer is associated with lower survival of the cancer patient. Here, we provide a comprehensive review of the literature regarding the oncogenic roles of IQGAP1. We start by describing the major cancer-related signaling pathways scaffolded by IQGAP1 and their associated cellular activities. We then describe clinical and molecular evidence for the contribution of IQGAP1 in different types of cancers. In the end, we review recent evidence implicating IQGAP1 in tumor-related immune responses. Given the critical role of IQGAP1 in carcinoma development, anti-tumor therapies targeting IQGAP1 or its associated signaling pathways could be beneficial for patients with many types of cancer.
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Affiliation(s)
| | - Paul F. Lambert
- McArdle Laboratory for Cancer Research, Department of Oncology, University of Wisconsin School of Medicine and Public Health, Madison, WI 53705, USA;
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7
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Interaction Network Provides Clues on the Role of BCAR1 in Cellular Response to Changes in Gravity. COMPUTATION 2021. [DOI: 10.3390/computation9080081] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/22/2023]
Abstract
When culturing cells in space or under altered gravity conditions on Earth to investigate the impact of gravity, their adhesion and organoid formation capabilities change. In search of a target where the alteration of gravity force could have this impact, we investigated p130cas/BCAR1 and its interactions more thoroughly, particularly as its activity is sensitive to applied forces. This protein is well characterized regarding its role in growth stimulation and adhesion processes. To better understand BCAR1′s force-dependent scaffolding of other proteins, we studied its interactions with proteins we had detected by proteome analyses of MCF-7 breast cancer and FTC-133 thyroid cancer cells, which are both sensitive to exposure to microgravity and express BCAR1. Using linked open data resources and our experiments, we collected comprehensive information to establish a semantic knowledgebase and analyzed identified proteins belonging to signaling pathways and their networks. The results show that the force-dependent phosphorylation and scaffolding of BCAR1 influence the structure, function, and degradation of intracellular proteins as well as the growth, adhesion and apoptosis of cells similarly to exposure of whole cells to altered gravity. As BCAR1 evidently plays a significant role in cell responses to gravity changes, this study reveals a clear path to future research performing phosphorylation experiments on BCAR1.
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8
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Sackmann E, Tanaka M. Critical role of lipid membranes in polarization and migration of cells: a biophysical view. Biophys Rev 2021; 13:123-138. [PMID: 33747247 PMCID: PMC7930189 DOI: 10.1007/s12551-021-00781-1] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/19/2020] [Accepted: 01/03/2021] [Indexed: 12/15/2022] Open
Abstract
Cell migration plays vital roles in many biologically relevant processes such as tissue morphogenesis and cancer metastasis, and it has fascinated biophysicists over the past several decades. However, despite an increasing number of studies highlighting the orchestration of proteins involved in different signaling pathways, the functional roles of lipid membranes have been essentially overlooked. Lipid membranes are generally considered to be a functionless two-dimensional matrix of proteins, although many proteins regulating cell migration gain functions only after they are recruited to the membrane surface and self-organize their functional domains. In this review, we summarize how the logistical recruitment and release of proteins to and from lipid membranes coordinates complex spatiotemporal molecular processes. As predicted from the classical framework of the Smoluchowski equation of diffusion, lipid/protein membranes serve as a 2D reaction hub that contributes to the effective and robust regulation of polarization and migration of cells involving several competing pathways.
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Affiliation(s)
- Erich Sackmann
- Physics Department E22/E27, Technical University of Munich, James-Franck-Strasse, 85747 Garching, Germany
| | - Motomu Tanaka
- Physical Chemistry of Biosystems, Institute of Physical Chemistry, Heidelberg University, 69120 Heidelberg, Germany.,Center for Integrative Medicine and Physics, Institute for Advanced Study, Kyoto University, Kyoto, 606-8501 Japan
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9
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Hedman AC, Li Z, Gorisse L, Parvathaneni S, Morgan CJ, Sacks DB. IQGAP1 binds AMPK and is required for maximum AMPK activation. J Biol Chem 2020; 296:100075. [PMID: 33191271 PMCID: PMC7948462 DOI: 10.1074/jbc.ra120.016193] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/25/2020] [Accepted: 11/15/2020] [Indexed: 12/25/2022] Open
Abstract
AMP-activated protein kinase (AMPK) is a fundamental component of a protein kinase cascade that is an energy sensor. AMPK maintains energy homeostasis in the cell by promoting catabolic and inhibiting anabolic pathways. Activation of AMPK requires phosphorylation by the liver kinase B1 or by the Ca2+/calmodulin-dependent protein kinase 2 (CaMKK2). The scaffold protein IQGAP1 regulates intracellular signaling pathways, such as the mitogen-activated protein kinase and AKT signaling cascades. Recent work implicates the participation of IQGAP1 in metabolic function, but the molecular mechanisms underlying these effects are poorly understood. Here, using several approaches including binding analysis with fusion proteins, siRNA-mediated gene silencing, RT-PCR, and knockout mice, we investigated whether IQGAP1 modulates AMPK signaling. In vitro analysis reveals that IQGAP1 binds directly to the α1 subunit of AMPK. In addition, we observed a direct interaction between IQGAP1 and CaMKK2, which is mediated by the IQ domain of IQGAP1. Both CaMKK2 and AMPK associate with IQGAP1 in cells. The ability of metformin and increased intracellular free Ca2+ concentrations to activate AMPK is reduced in cells lacking IQGAP1. Importantly, Ca2+-stimulated AMPK phosphorylation was rescued by re-expression of IQGAP1 in IQGAP1-null cell lines. Comparison of the fasting response in wild-type and IQGAP1-null mice revealed that transcriptional regulation of the gluconeogenesis genes PCK1 and G6PC and the fatty acid synthesis genes FASN and ACC1 is impaired in IQGAP1-null mice. Our data disclose a previously unidentified functional interaction between IQGAP1 and AMPK and suggest that IQGAP1 modulates AMPK signaling.
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Affiliation(s)
- Andrew C Hedman
- Department of Laboratory Medicine, National Institutes of Health, Bethesda, Maryland, USA
| | - Zhigang Li
- Department of Laboratory Medicine, National Institutes of Health, Bethesda, Maryland, USA
| | - Laëtitia Gorisse
- Department of Laboratory Medicine, National Institutes of Health, Bethesda, Maryland, USA
| | - Swetha Parvathaneni
- Department of Laboratory Medicine, National Institutes of Health, Bethesda, Maryland, USA
| | - Chase J Morgan
- Department of Laboratory Medicine, National Institutes of Health, Bethesda, Maryland, USA
| | - David B Sacks
- Department of Laboratory Medicine, National Institutes of Health, Bethesda, Maryland, USA.
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10
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Nakajima K, Arora PD, Plaha A, McCulloch CA. Role of the small GTPase activating protein IQGAP1 in collagen phagocytosis. J Cell Physiol 2020; 236:1270-1280. [PMID: 32643295 DOI: 10.1002/jcp.29933] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/27/2020] [Accepted: 07/01/2020] [Indexed: 01/23/2023]
Abstract
Many adult connective tissues undergo continuous remodeling to maintain matrix homeostasis. Physiological remodeling involves the degradation of collagen fibers by the intracellular cathepsin-dependent phagocytic pathway. We considered that a multidomain, small GTPase activating protein, IQGAP1, which is involved in the generation of cell extensions, is required for collagen phagocytosis, possibly arising from its interactions with cdc42 and the actin-binding protein Flightless I (FliI). We examined the role of IQGAP1 in collagen phagocytosis by human gingival fibroblasts (HGFs) and by IQGAP1+/+ and IQGAP1-/- mouse embryonic fibroblasts. IQGAP1 was strongly expressed by HGFs, localized to vinculin-stained cell adhesions and sites where cell extensions are initiated, and colocalized with FliI. Immunoprecipitation showed that IQGAP1 associated with FliI. HGFs showed 10-fold increases of collagen binding, 6-fold higher internalization, and 3-fold higher β1 integrin activation between 30 and 180 min after incubation with collagen. Compared with IQGAP1+/+ fibroblasts, deletion of IQGAP1 reduced collagen binding (1.4-fold), collagen internalization (3-fold), β1 integrin activation (2-fold), and collagen degradation (1.8-fold). We conclude that IQGAP1 affects collagen remodeling through its regulation of phagocytic degradation pathways, which may involve the interaction of IQGAP1 with FliI.
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Affiliation(s)
- Kei Nakajima
- Department of Clinical Pathophysiology, Tokyo Dental College, Chiyoda-ku, Tokyo, Japan.,Faculty of Dentistry, University of Toronto, Toronto, Ontario, Canada
| | - Pamela D Arora
- Faculty of Dentistry, University of Toronto, Toronto, Ontario, Canada
| | - Ajay Plaha
- Faculty of Dentistry, University of Toronto, Toronto, Ontario, Canada
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11
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Pleiotropic Roles of Calmodulin in the Regulation of KRas and Rac1 GTPases: Functional Diversity in Health and Disease. Int J Mol Sci 2020; 21:ijms21103680. [PMID: 32456244 PMCID: PMC7279331 DOI: 10.3390/ijms21103680] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/17/2020] [Revised: 05/18/2020] [Accepted: 05/21/2020] [Indexed: 12/21/2022] Open
Abstract
Calmodulin is a ubiquitous signalling protein that controls many biological processes due to its capacity to interact and/or regulate a large number of cellular proteins and pathways, mostly in a Ca2+-dependent manner. This complex interactome of calmodulin can have pleiotropic molecular consequences, which over the years has made it often difficult to clearly define the contribution of calmodulin in the signal output of specific pathways and overall biological response. Most relevant for this review, the ability of calmodulin to influence the spatiotemporal signalling of several small GTPases, in particular KRas and Rac1, can modulate fundamental biological outcomes such as proliferation and migration. First, direct interaction of calmodulin with these GTPases can alter their subcellular localization and activation state, induce post-translational modifications as well as their ability to interact with effectors. Second, through interaction with a set of calmodulin binding proteins (CaMBPs), calmodulin can control the capacity of several guanine nucleotide exchange factors (GEFs) to promote the switch of inactive KRas and Rac1 to an active conformation. Moreover, Rac1 is also an effector of KRas and both proteins are interconnected as highlighted by the requirement for Rac1 activation in KRas-driven tumourigenesis. In this review, we attempt to summarize the multiple layers how calmodulin can regulate KRas and Rac1 GTPases in a variety of cellular events, with biological consequences and potential for therapeutic opportunities in disease settings, such as cancer.
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12
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Grb7-derived calmodulin-binding peptides inhibit proliferation, migration and invasiveness of tumor cells while they enhance attachment to the substrate. Heliyon 2020; 6:e03922. [PMID: 32420488 PMCID: PMC7215194 DOI: 10.1016/j.heliyon.2020.e03922] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2019] [Revised: 02/05/2020] [Accepted: 04/30/2020] [Indexed: 12/28/2022] Open
Abstract
The growth factor receptor bound protein 7 (Grb7) is a Ca2+-dependent calmodulin (CaM)-binding adaptor protein implicated, among other functions, in cell proliferation, migration and tumor-associated angiogenesis. The goal of this study was to determine whether a peptide based on the CaM binding site of Grb7 disrupts cellular processes, relevant for the malignancy of tumor cells, in which this adaptor protein is implicated. We designed synthetic myristoylated and non-myristoylated peptides corresponding to the CaM-binding domain of human Grb7 with the sequence 243RKLWKRFFCFLRRS256 and a variant peptide with the mutated sequence RKLERFFCFLRRE (W246E-ΔK247-S256E). The two non-myristoylated peptides bind dansyl-CaM with higher efficiency in the presence than in the absence of Ca2+ and they enter into the cell, as tested with 5(6)-carboxytetramethylrhodamine (TAMRA)-labeled peptides. The myristoylated and non-myristoylated peptides inhibit the proliferation, migration and invasiveness of A431 tumor cells while they enhance their adhesion to the substrate. The myristoylated peptides have stronger inhibitory effect than the non-myristoylated counterparts, in agreement with their expected higher cell-permeant capacity. The myristoylated and non-myristoylated W246E-ΔK247-S256E mutant peptide has a lesser inhibitory effect on cell proliferation as compared to the wild-type peptide. We also demonstrated that the myristoylated peptides were more efficient than the CaM antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) inhibiting cell migration and equally efficient inhibiting cell proliferation.
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13
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Xu L, Shao Y, Ren L, Liu X, Li Y, Xu J, Ye Y. IQGAP2 Inhibits Migration and Invasion of Gastric Cancer Cells via Elevating SHIP2 Phosphatase Activity. Int J Mol Sci 2020; 21:ijms21061968. [PMID: 32183047 PMCID: PMC7139352 DOI: 10.3390/ijms21061968] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/23/2020] [Revised: 03/09/2020] [Accepted: 03/12/2020] [Indexed: 12/30/2022] Open
Abstract
Previous studies have shown reduced expression of Src homology 2-containing inositol 5-phosphatase 2 (SHIP2) and its tumor-suppressive role in gastric cancer (GC). However, the precise role of SHIP2 in the migration and invasion of GC cells remains unclear. Here, an IQ motif containing the GTPase-activating protein 2 (IQGAP2) as a SHIP2 binding partner, was screened and identified by co-immunoprecipitation and mass spectrometry studies. While IQGAP2 ubiquitously expressed in GC cells, IQGAP2 and SHIP2 co-localized in the cytoplasm of GC cells, and this physical association was confirmed by the binding of IQGAP2 to PRD and SAM domains of SHIP2. The knockdown of either SHIP2 or IQGAP2 promoted cell migration and invasion by inhibiting SHIP2 phosphatase activity, activating Akt and subsequently increasing epithelial–mesenchymal transition (EMT). Furthermore, knockdown of IQGAP2 in SHIP2-overexpressing GC cells reversed the inhibition of cell migration and invasion by SHIP2 induction, which was associated with the suppression of elevated SHIP2 phosphatase activity. Moreover, the deletion of PRD and SAM domains of SHIP2 abrogated the interaction and restored cell migration and invasion. Collectively, these results indicate that IQGAP2 interacts with SHIP2, leading to the increment of SHIP2 phosphatase activity, and thereby inhibiting the migration and invasion of GC cells via the inactivation of Akt and reduction in EMT.
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Affiliation(s)
| | | | | | | | | | | | - Yan Ye
- Correspondence: ; Tel.: +86-551-65161139
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14
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Villalobo A, Berchtold MW. The Role of Calmodulin in Tumor Cell Migration, Invasiveness, and Metastasis. Int J Mol Sci 2020; 21:ijms21030765. [PMID: 31991573 PMCID: PMC7037201 DOI: 10.3390/ijms21030765] [Citation(s) in RCA: 55] [Impact Index Per Article: 11.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/31/2019] [Revised: 01/18/2020] [Accepted: 01/21/2020] [Indexed: 12/12/2022] Open
Abstract
Calmodulin (CaM) is the principal Ca2+ sensor protein in all eukaryotic cells, that upon binding to target proteins transduces signals encoded by global or subcellular-specific changes of Ca2+ concentration within the cell. The Ca2+/CaM complex as well as Ca2+-free CaM modulate the activity of a vast number of enzymes, channels, signaling, adaptor and structural proteins, and hence the functionality of implicated signaling pathways, which control multiple cellular functions. A basic and important cellular function controlled by CaM in various ways is cell motility. Here we discuss the role of CaM-dependent systems involved in cell migration, tumor cell invasiveness, and metastasis development. Emphasis is given to phosphorylation/dephosphorylation events catalyzed by myosin light-chain kinase, CaM-dependent kinase-II, as well as other CaM-dependent kinases, and the CaM-dependent phosphatase calcineurin. In addition, the role of the CaM-regulated small GTPases Rac1 and Cdc42 (cell division cycle protein 42) as well as CaM-binding adaptor/scaffold proteins such as Grb7 (growth factor receptor bound protein 7), IQGAP (IQ motif containing GTPase activating protein) and AKAP12 (A kinase anchoring protein 12) will be reviewed. CaM-regulated mechanisms in cancer cells responsible for their greater migratory capacity compared to non-malignant cells, invasion of adjacent normal tissues and their systemic dissemination will be discussed, including closely linked processes such as the epithelial–mesenchymal transition and the activation of metalloproteases. This review covers as well the role of CaM in establishing metastatic foci in distant organs. Finally, the use of CaM antagonists and other blocking techniques to downregulate CaM-dependent systems aimed at preventing cancer cell invasiveness and metastasis development will be outlined.
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Affiliation(s)
- Antonio Villalobo
- Cancer and Human Molecular Genetics Area—Oto-Neurosurgery Research Group, University Hospital La Paz Research Institute (IdiPAZ), Paseo de la Castellana 261, E-28046 Madrid, Spain
- Correspondence: (A.V.); (M.W.B.)
| | - Martin W. Berchtold
- Department of Biology, University of Copenhagen, 13 Universitetsparken, DK-2100 Copenhagen, Denmark
- Correspondence: (A.V.); (M.W.B.)
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15
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Zhang M, Li Z, Jang H, Hedman AC, Sacks DB, Nussinov R. Ca 2+-Dependent Switch of Calmodulin Interaction Mode with Tandem IQ Motifs in the Scaffolding Protein IQGAP1. Biochemistry 2019; 58:4903-4911. [PMID: 31724397 PMCID: PMC8195445 DOI: 10.1021/acs.biochem.9b00854] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022]
Abstract
IQ domain GTPase-activating scaffolding protein 1 (IQGAP1) mediates cytoskeleton, cell migration, proliferation, and apoptosis events. Calmodulin (CaM) modulates IQGAP1 functions by binding to its four tandem IQ motifs. Exactly how CaM binds the IQ motifs and which functions of IQGAP1 CaM regulates and how are fundamental mechanistic questions. We combine experimental pull-down assays, mutational data, and molecular dynamics simulations to understand the IQ-CaM complexes with and without Ca2+ at the atomic level. Apo-CaM favors the IQ3 and IQ4 motifs but not the IQ1 and IQ2 motifs that lack two hydrophobic residues for interactions with apo-CaM's hydrophobic pocket. Ca2+-CaM binds all four IQ motifs, with both N- and C-lobes tightly wrapped around each motif. Ca2+ promotes IQ-CaM interactions and increases the amount of IQGAP1-loaded CaM for IQGAP1-mediated signaling. Collectively, we describe IQ-CaM binding in atomistic detail and feature the emergence of Ca2+ as a key modulator of the CaM-IQGAP1 interactions.
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Affiliation(s)
- Mingzhen Zhang
- Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, Maryland 20892, United States
| | - Zhigang Li
- Department of Laboratory Medicine, National Institutes of Health, Bethesda, Maryland 20892, United States
| | - Hyunbum Jang
- Department of Laboratory Medicine, National Institutes of Health, Bethesda, Maryland 20892, United States
| | - Andrew C. Hedman
- Department of Laboratory Medicine, National Institutes of Health, Bethesda, Maryland 20892, United States
| | - David B. Sacks
- Department of Laboratory Medicine, National Institutes of Health, Bethesda, Maryland 20892, United States
| | - Ruth Nussinov
- Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, Maryland 20892, United States
- Sackler Institute of Molecular Medicine, Department of Human Genetics and Molecular Medicine, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel
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16
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Okuyama Y, Nagashima H, Ushio-Fukai M, Croft M, Ishii N, So T. IQGAP1 restrains T-cell cosignaling mediated by OX40. FASEB J 2019; 34:540-554. [PMID: 31914585 DOI: 10.1096/fj.201900879rr] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/05/2019] [Revised: 10/25/2019] [Accepted: 11/01/2019] [Indexed: 12/17/2022]
Abstract
A costimulatory signal from the tumor necrosis factor receptor (TNFR) family molecule OX40 (CD134), which is induced on activated T cells, is important for T-cell immunity. Aberrant OX40 cosignaling has been implicated in autoimmune and inflammatory disorders. However, the molecular mechanism by which the OX40 cosignaling regulates the T-cell response remains obscure. We found that OX40 associated with a scaffold protein, IQ motif-containing GTPase-activating protein 1 (IQGAP1) after ligation by its ligand OX40L. Naïve CD4+ T cells from Iqgap1-/- mice displayed enhanced proliferation and cytokine secretion upon receiving OX40 cosignaling. A C-terminal IQGAP1 region was responsible for its association with OX40, and TNFR-associated factor 2 (TRAF2) bridged these two proteins. The enhanced cytokine response in Iqgap1-/- T cells was restored by the expression of the C-terminal IQGAP1. Thus, the IQGAP1 binding limits the OX40 cosignaling. Disease severity of experimental autoimmune encephalomyelitis (EAE) was significantly exacerbated in Iqgap1-/- mice as compared to wild-type mice. Additionally, recipient mice with Iqgap1-/- donor CD4+ T cells exhibited significantly higher EAE scores than those with their wild-type counterparts, and OX40 blockade led to a significant reduction in the EAE severity. Thus, our study defines an important component of the OX40 cosignaling that restricts inflammation driven by antigen-activated T cells.
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Affiliation(s)
- Yuko Okuyama
- Department of Microbiology and Immunology, Tohoku University Graduate School of Medicine, Sendai, Japan
| | - Hiroyuki Nagashima
- Department of Microbiology and Immunology, Tohoku University Graduate School of Medicine, Sendai, Japan
| | - Masuko Ushio-Fukai
- Vascular Biology Center, Medical College of Georgia at Augusta University, Augusta, GA, USA
| | - Michael Croft
- Division of Immune Regulation, La Jolla Institute for Allergy and Immunology, La Jolla, CA, USA
| | - Naoto Ishii
- Department of Microbiology and Immunology, Tohoku University Graduate School of Medicine, Sendai, Japan
| | - Takanori So
- Department of Microbiology and Immunology, Tohoku University Graduate School of Medicine, Sendai, Japan.,Laboratory of Molecular Cell Biology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan
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17
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Hu G, Liu H, Wang M, Peng W. IQ Motif Containing GTPase-Activating Protein 3 (IQGAP3) Inhibits Kaempferol-Induced Apoptosis in Breast Cancer Cells by Extracellular Signal-Regulated Kinases 1/2 (ERK1/2) Signaling Activation. Med Sci Monit 2019; 25:7666-7674. [PMID: 31605603 PMCID: PMC6807529 DOI: 10.12659/msm.915642] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/12/2019] [Accepted: 04/23/2019] [Indexed: 01/11/2023] Open
Abstract
BACKGROUND Breast cancer (BC), a prevalent and heterogeneous disease of glandular breast tissue, is the most common cancer in women. The interaction between Kaempferol and IQ motif containing GTPase-activating protein 3 (IQGAP3) in BC and its underlying mechanism are poorly defined. MATERIAL AND METHODS After natural phytochemicals treatment, the expression of IQGAP3 in BC cells (ZR-75-30 and BT474) was detected by real-time PCR. Then, the proliferation and apoptosis in BC cells with different gradient concentrations (10, 25, 50, and 100 µmol/l) of Kaempferol treatment were detected. After treatment with Kaempferol or epidermal growth factor (EGF), we assessed apoptosis and expression of related genes. RESULTS We found that natural phytochemicals, especially Kaempferol, decreased IQGAP3 expression in BC cells. Kaempferol significantly induced proliferation inhibition and apoptosis in BC cells, concurrent with decreased IQGAP3 expression. Upregulation of IQGAP3 inhibited apoptosis in BC cells, along with increased expression of phosphorylated extracellular signal-regulated kinases 1/2 (p-ERK1/2) and B cell lymphoma 2 (Bcl2) and decreased Bcl-2-associated X protein (Bax) expression, which was counteracted by Kaempferol treatment. EGF markedly inhibited Kaempferol-induced apoptosis in BC cells, and ERK1/2 inhibitor PD98059 had an effect similar to that of Kaempferol. CONCLUSIONS IQGAP3 may be a potential target gene for Kaempferol in the treatment of BC, and upregulation of IQGAP3 inhibits Kaempferol-induced apoptosis in BC cells by ERK1/2 signaling activation. Targeting IQGAP3 may contribute to the study of natural phytochemicals as anti-tumor drugs in BC.
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Affiliation(s)
- Gaowu Hu
- Department of General Surgery, Shanghai Traditional Chinese Medicine-Integrated Hospital, Shanghai, P.R. China
| | - Huajiang Liu
- Department of Chinese Medicine, Jinshan Hospital of Fudan University, Shanghai, P.R. China
| | - Ming Wang
- Department of General Surgery, Shanghai Traditional Chinese Medicine-Integrated Hospital, Shanghai, P.R. China
| | - Wei Peng
- Department of General Surgery, Shanghai Traditional Chinese Medicine-Integrated Hospital, Shanghai, P.R. China
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18
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Sullivan DP, Dalal PJ, Jaulin F, Sacks DB, Kreitzer G, Muller WA. Endothelial IQGAP1 regulates leukocyte transmigration by directing the LBRC to the site of diapedesis. J Exp Med 2019; 216:2582-2601. [PMID: 31395618 PMCID: PMC6829592 DOI: 10.1084/jem.20190008] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/02/2019] [Revised: 06/10/2019] [Accepted: 07/03/2019] [Indexed: 01/09/2023] Open
Abstract
The function of endothelial cell IQGAP1 during diapedesis requires its actin-binding domain and IQ motifs to recruit the lateral border recycling compartment. Genetic ablation of endothelial cell IQGAP1 expression in vivo causes significant disruption of diapedesis in two models of inflammation. Transendothelial migration (TEM) of leukocytes across the endothelium is critical for inflammation. In the endothelium, TEM requires the coordination of membrane movements and cytoskeletal interactions, including, prominently, recruitment of the lateral border recycling compartment (LBRC). The scaffold protein IQGAP1 was recently identified in a screen for LBRC-interacting proteins. Knockdown of endothelial IQGAP1 disrupted the directed movement of the LBRC and substantially reduced leukocyte TEM. Expression of truncated IQGAP1 constructs demonstrated that the calponin homology domain is required for IQGAP1 localization to endothelial borders and that the IQ domain, on the same IQGAP1 polypeptide, is required for its function in TEM. This is the first reported function of IQGAP1 requiring two domains to be present on the same polypeptide. Additionally, we show for the first time that IQGAP1 in the endothelium is required for efficient TEM in vivo. These findings reveal a novel function for IQGAP1 and demonstrate that IQGAP1 in endothelial cells facilitates TEM by directing the LBRC to the site of TEM.
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Affiliation(s)
- David P Sullivan
- Department of Pathology, Feinberg School of Medicine, Northwestern University, Chicago, IL
| | - Prarthana J Dalal
- Department of Pathology, Feinberg School of Medicine, Northwestern University, Chicago, IL
| | | | - David B Sacks
- Department of Laboratory Medicine, National Institutes of Health, Bethesda, MD
| | - Geri Kreitzer
- Department of Molecular, Cellular and Biomedical Sciences, City University of New York School of Medicine, The City College of New York, New York, NY
| | - William A Muller
- Department of Pathology, Feinberg School of Medicine, Northwestern University, Chicago, IL
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19
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Erickson HL, Anakk S. Identification of IQ motif-containing GTPase-activating protein 1 as a regulator of long-term ketosis. JCI Insight 2018; 3:99866. [PMID: 30385723 DOI: 10.1172/jci.insight.99866] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/02/2018] [Accepted: 09/26/2018] [Indexed: 12/18/2022] Open
Abstract
IQ motif-containing GTPase-activating protein 1 (IQGAP1) is a ubiquitously expressed scaffolding protein that integrates multiple cellular processes, including motility, adhesion, and proliferation, but its role in metabolism is unknown. Here, we show that IQGAP1 is induced upon fasting and regulates β-oxidation of fatty acids and synthesis of ketone bodies in the liver. IQGAP1-null (Iqgap1-/-) mice exhibit reduced hepatic PPARα transcriptional activity, as evidenced during fasting, after ketogenic diet, and upon pharmacological activation. Conversely, we found that the activity of fed-state sensor mTORC1 is enhanced in Iqgap1-/- livers, but acute inhibition of mTOR in Iqgap1-/- mice was unable to rescue the defect in ketone body synthesis. However, reexpressing IQGAP1 in the livers of Iqgap1-/- mice was sufficient to promote ketone body synthesis, increase PPARα signaling, and suppress mTORC1 activity. Taken together, we uncover what we believe to be a previously unidentified role for IQGAP1 in regulating PPARα activity and ketogenesis.
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20
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IQGAP1 binds the Axl receptor kinase and inhibits its signaling. Biochem J 2018; 475:3073-3086. [PMID: 30185434 DOI: 10.1042/bcj20180594] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/03/2018] [Revised: 08/31/2018] [Accepted: 09/05/2018] [Indexed: 02/06/2023]
Abstract
Axl is a tyrosine kinase receptor that is important for hematopoiesis, the innate immune response, platelet aggregation, engulfment of apoptotic cells and cell survival. Binding of growth arrest-specific protein 6 (Gas6) activates Axl signaling, but the mechanism of inactivation of the Axl receptor is poorly understood. In the present study, we show that IQGAP1 modulates Axl signaling. IQGAP1 is a scaffold protein that integrates cell signaling pathways by binding several growth factor receptors and intracellular signaling molecules. Our in vitro analysis revealed a direct interaction between the IQ domain of IQGAP1 and Axl. Analysis by both immunoprecipitation and proximity ligation assays demonstrated an association between Axl and IQGAP1 in cells and this interaction was decreased by Gas6. Unexpectedly, reducing IQGAP1 levels in cells significantly enhanced the ability of Gas6 to stimulate both Axl phosphorylation and activation of Akt. Moreover, IQGAP1 regulates the interaction of Axl with the epidermal growth factor receptor. Our data identify IQGAP1 as a previously undescribed suppressor of Axl and provide insight into regulation of Axl function.
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21
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Beyrakhova K, Li L, Xu C, Gagarinova A, Cygler M. Legionella pneumophila effector Lem4 is a membrane-associated protein tyrosine phosphatase. J Biol Chem 2018; 293:13044-13058. [PMID: 29976756 DOI: 10.1074/jbc.ra118.003845] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/07/2018] [Revised: 07/02/2018] [Indexed: 01/16/2023] Open
Abstract
Legionella pneumophila is a Gram-negative pathogenic bacterium that causes severe pneumonia in humans. It establishes a replicative niche called Legionella-containing vacuole (LCV) that allows bacteria to survive and replicate inside pulmonary macrophages. To hijack host cell defense systems, L. pneumophila injects over 300 effector proteins into the host cell cytosol. The Lem4 effector (lpg1101) consists of two domains: an N-terminal haloacid dehalogenase (HAD) domain with unknown function and a C-terminal phosphatidylinositol 4-phosphate-binding domain that anchors Lem4 to the membrane of early LCVs. Herein, we demonstrate that the HAD domain (Lem4-N) is structurally similar to mouse MDP-1 phosphatase and displays phosphotyrosine phosphatase activity. Substrate specificity of Lem4 was probed using a tyrosine phosphatase substrate set, which contained a selection of 360 phosphopeptides derived from human phosphorylation sites. This assay allowed us to identify a consensus pTyr-containing motif. Based on the localization of Lem4 to lysosomes and to some extent to plasma membrane when expressed in human cells, we hypothesize that this protein is involved in protein-protein interactions with an LCV or plasma membrane-associated tyrosine-phosphorylated host target.
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Affiliation(s)
- Ksenia Beyrakhova
- From the Department of Biochemistry, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E5 and
| | - Lei Li
- From the Department of Biochemistry, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E5 and
| | - Caishuang Xu
- From the Department of Biochemistry, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E5 and
| | - Alla Gagarinova
- From the Department of Biochemistry, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E5 and
| | - Miroslaw Cygler
- From the Department of Biochemistry, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E5 and .,the Department of Biochemistry, McGill University, Montreal, Quebec H3G 1Y6, Canada
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22
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Kim WT, Kim YH, Jeong P, Seo SP, Kang HW, Kim YJ, Yun SJ, Lee SC, Moon SK, Choi YH, Lee GT, Kim IY, Kim WJ. Urinary cell-free nucleic acid IQGAP3: a new non-invasive diagnostic marker for bladder cancer. Oncotarget 2018; 9:14354-14365. [PMID: 29581849 PMCID: PMC5865675 DOI: 10.18632/oncotarget.24436] [Citation(s) in RCA: 32] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/26/2017] [Accepted: 07/12/2017] [Indexed: 12/23/2022] Open
Abstract
Background There is growing interest in developing new non-invasive diagnostic tools for
bladder cancer (BC) that have better sensitivity and specificity than cystoscopy
and cytology. This study examined the value of urinary cell-free nucleic acid (NA)
as a diagnostic marker for BC. Material and methods A total of 81 patients (74 BC and 7 normal controls) were used for a tissue set,
and 212 patients (92 BC and 120 normal controls) were used as a urine set.
Expression of tissue mRNA and urinary cell-free NAs was then examined. Results Four candidate genes were top-ranked in the tissue microarray. Expression levels
of two of these (IQGAP3 and TOP2A) in BC tissue and urine samples from BC patients
were significantly higher than those in samples from the control groups. Binary
logistic regression analysis of cell-free NA levels in urine samples revealed that
IQGAP3 was significantly associated with BC: PicoGreen-adjusted odds ratio (OR),
3.434; confidence interval (CI), 2.999–4.180;
P<0.001; RiboGreen-adjusted OR, 2.242; CI,
1.793–2.840; P<0.001. Further analysis of IQGAP3
urinary cell-free NAs with respect to tumor invasiveness and grade also yielded a
high AUC, suggesting that IQGAP3 can discriminate between BC patients and
non-cancer patients with hematuria. Conclusions Levels of IQGAP3 urinary cell-free NA in BC patients were significantly higher
than those in normal controls or patients with hematuria. High levels of IQGAP3
urinary cell-free NA also reflected high expression in BC tissues. Therefore,
IQGAP3 urinary cell-free NA may be a complementary diagnostic biomarker for
BC.
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Affiliation(s)
- Won Tae Kim
- Department of Urology, Chungbuk National University College of Medicine, Cheongju, Chungbuk, South Korea.,Department of Urology, Chungbuk National University Hospital, Cheongju, Chungbuk, South Korea.,Section of Urological Oncology, The Cancer Institute of New Jersey, Robert Wood Johnson Medical School, New Brunswick, NJ, USA
| | - Ye Hwan Kim
- Department of Urology, Chungbuk National University College of Medicine, Cheongju, Chungbuk, South Korea
| | - Pildu Jeong
- Department of Urology, Chungbuk National University College of Medicine, Cheongju, Chungbuk, South Korea
| | - Sung-Pil Seo
- Department of Urology, Chungbuk National University College of Medicine, Cheongju, Chungbuk, South Korea.,Department of Urology, Chungbuk National University Hospital, Cheongju, Chungbuk, South Korea
| | - Ho-Won Kang
- Department of Urology, Chungbuk National University College of Medicine, Cheongju, Chungbuk, South Korea.,Department of Urology, Chungbuk National University Hospital, Cheongju, Chungbuk, South Korea
| | - Yong-June Kim
- Department of Urology, Chungbuk National University College of Medicine, Cheongju, Chungbuk, South Korea.,Department of Urology, Chungbuk National University Hospital, Cheongju, Chungbuk, South Korea
| | - Seok Joong Yun
- Department of Urology, Chungbuk National University College of Medicine, Cheongju, Chungbuk, South Korea.,Department of Urology, Chungbuk National University Hospital, Cheongju, Chungbuk, South Korea
| | - Sang-Cheol Lee
- Department of Urology, Chungbuk National University College of Medicine, Cheongju, Chungbuk, South Korea.,Department of Urology, Chungbuk National University Hospital, Cheongju, Chungbuk, South Korea
| | - Sung-Kwon Moon
- School of Food Science and Technology, Chung-Ang University, Anseong, South Korea
| | - Yung-Hyun Choi
- Department of Biochemistry, Dongeui University College of Oriental Medicine, Busan, South Korea
| | - Geun Taek Lee
- Section of Urological Oncology, The Cancer Institute of New Jersey, Robert Wood Johnson Medical School, New Brunswick, NJ, USA
| | - Isaac Yi Kim
- Section of Urological Oncology, The Cancer Institute of New Jersey, Robert Wood Johnson Medical School, New Brunswick, NJ, USA
| | - Wun-Jae Kim
- Department of Urology, Chungbuk National University College of Medicine, Cheongju, Chungbuk, South Korea.,Department of Urology, Chungbuk National University Hospital, Cheongju, Chungbuk, South Korea
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23
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Shinsuke K, Junya K, Tomonobu U, Yoshiko K, Izumo N, Takahiko S. Chronic irradiation with low-dose-rate 137Cs-γ rays inhibits NGF-induced neurite extension of PC12 cells via Ca2+/calmodulin-dependent kinase II activation. JOURNAL OF RADIATION RESEARCH 2017; 58:809-815. [PMID: 29106600 PMCID: PMC5710646 DOI: 10.1093/jrr/rrx032] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 11/25/2016] [Revised: 02/27/2017] [Indexed: 06/07/2023]
Abstract
Chronic irradiation with low-dose-rate 137Cs-γ rays inhibits the differentiation of human neural progenitor cells and influences the expression of proteins associated with several cellular functions. We aimed to determine whether such chronic irradiation influences the expression of proteins associated with PC12 cells. Chronic irradiation at 0.027 mGy/min resulted in inhibition of NGF-induced neurite extension. Furthermore, irradiation enhanced the nerve growth factor (NGF)-induced increase in the phosphorylation of extracellular signal-regulated kinase (ERK), but did not affect the phosphorylation of NGF receptors, suggesting that irradiation influences pathways unassociated with the activation of ERK. We then examined whether irradiation influenced the Akt-Rac1 pathway, which is unaffected by ERK activation. Chronic irradiation also enhanced the NGF-induced increase in Akt phosphorylation, but markedly inhibited the NGF-induced increase in Rac1 activity that is associated with neurite extension. These results suggest that the inhibitory effect of irradiation on neurite extension influences pathways unassociated with Akt activation. As Ca2+/calmodulin-dependent kinase II (CaMKII) is known to inhibit the NGF-induced neurite extension in PC12 cells, independent of ERK and Akt activation, we next examined the effects of irradiation on CaMKII activation. Chronic irradiation induced CaMKII activation, while application of KN-62 (a specific inhibitor of CaMKII), attenuated increases in CaMKII activation and recovered neurite extension and NGF-induced increases in Rac1 activity that was inhibited by irradiation. Our results suggest that chronic irradiation with low-dose-rate γ-rays inhibits Rac1 activity via CaMKII activation, thereby inhibiting NGF-induced neurite extension.
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Affiliation(s)
- Katoh Shinsuke
- Research Center for Radiation Science, Yokohama University of Pharmacy, 601 Matano-cho, Totsuka-ku, Yokohama 245-0066, Japan
| | - Kobayashi Junya
- Radiation Biology Center, Kyoto University, Yoshidakonoe-cho, Sakyo-ku, Kyoto 606-8501, Japan
| | - Umeda Tomonobu
- Research Center for Radiation Science, Yokohama University of Pharmacy, 601 Matano-cho, Totsuka-ku, Yokohama 245-0066, Japan
| | - Kobayashi Yoshiko
- Research Center for Radiation Science, Yokohama University of Pharmacy, 601 Matano-cho, Totsuka-ku, Yokohama 245-0066, Japan
| | - Nobuo Izumo
- General Health Medical Center, Yokohama University of Pharmacy, 601 Matano-cho, Totsuka-ku, Yokohama 245-0066, Japan
| | - Suzuki Takahiko
- Clinical Radiology, Faculty of Medical Technology, Teikyo University, 2-11-1 Kaga, Itabashi 173-8605, Japan
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24
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Liang Z, Yang Y, He Y, Yang P, Wang X, He G, Zhang P, Zhu H, Xu N, Zhao X, Liang S. SUMOylation of IQGAP1 promotes the development of colorectal cancer. Cancer Lett 2017; 411:90-99. [PMID: 28987385 DOI: 10.1016/j.canlet.2017.09.046] [Citation(s) in RCA: 38] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/10/2017] [Revised: 09/23/2017] [Accepted: 09/25/2017] [Indexed: 02/05/2023]
Abstract
IQGAP1 is a conserved multifunctional protein implicated in tumorigenesis. An aberrant expression of IQGAP1 widely exists in many cancers, but the SUMOylation modification of IQGAP1 in carcinogenesis is unknown by now. Here we first time explore biological functions of IQGAP1 SUMOylation in promoting colorectal cancer progression in vitro and in vivo. The expression of IQGAP1 and its SUMOylation level are both increased in human colorectal carcinoma (CRC) cells and tissues. IQGAP1 is mainly SUMOylated by SUMO1 at the K1445 residue, which could stabilize IQGAP1 by reducing protein ubiquitination. IQGAP1 SUMOylation improves CRC cell growth, cell migration and tumorigenesis in vivo through activating the phosphorylation of ERK, MEK and AKT. While the SUMOylation site mutation at K1445 of IQGAP1 greatly reduces CRC cell proliferation, migration ability and tumor growth of CRC-xenograft mice by suppressing phosphorylation of ERK, MEK and AKT. Our findings discover the IQGAP1 SUMOylation is a novel regulatory mechanism to enhance tumorigenesis and development of CRC in vitro and in vivo.
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Affiliation(s)
- Ziwei Liang
- State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, and Collaborative Innovation Center for Biotherapy, No.17, 3rd Section of People's South Road, Chengdu, 610041, PR China
| | - Yanfang Yang
- State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, and Collaborative Innovation Center for Biotherapy, No.17, 3rd Section of People's South Road, Chengdu, 610041, PR China
| | - Yu He
- State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, and Collaborative Innovation Center for Biotherapy, No.17, 3rd Section of People's South Road, Chengdu, 610041, PR China
| | - Pengbo Yang
- State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, and Collaborative Innovation Center for Biotherapy, No.17, 3rd Section of People's South Road, Chengdu, 610041, PR China
| | - Xixi Wang
- State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, and Collaborative Innovation Center for Biotherapy, No.17, 3rd Section of People's South Road, Chengdu, 610041, PR China
| | - Gu He
- State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, and Collaborative Innovation Center for Biotherapy, No.17, 3rd Section of People's South Road, Chengdu, 610041, PR China
| | - Peng Zhang
- Department of Urinary Surgery, West China Hospital, Sichuan University, Chengdu, 610041, Sichuan, PR China
| | - Hongxia Zhu
- Laboratory of Cell and Molecular Biology & State Key Laboratory of Molecular Oncology, Cancer Institute & Cancer Hospital, Chinese Academy of Medical Sciences, Beijing, 100034, PR China
| | - Ningzhi Xu
- State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, and Collaborative Innovation Center for Biotherapy, No.17, 3rd Section of People's South Road, Chengdu, 610041, PR China; Laboratory of Cell and Molecular Biology & State Key Laboratory of Molecular Oncology, Cancer Institute & Cancer Hospital, Chinese Academy of Medical Sciences, Beijing, 100034, PR China
| | - Xia Zhao
- State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, and Collaborative Innovation Center for Biotherapy, No.17, 3rd Section of People's South Road, Chengdu, 610041, PR China; Department of Gynecology and Obstetrics, West China Second Hospital, Sichuan University, Chengdu, 610041, PR China
| | - Shufang Liang
- State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, and Collaborative Innovation Center for Biotherapy, No.17, 3rd Section of People's South Road, Chengdu, 610041, PR China.
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25
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Saito N, Mine N, Kufe DW, Von Hoff DD, Kawabe T. CBP501 inhibits EGF-dependent cell migration, invasion and epithelial-to-mesenchymal transition of non-small cell lung cancer cells by blocking KRas to calmodulin binding. Oncotarget 2017; 8:74006-74018. [PMID: 29088764 PMCID: PMC5650319 DOI: 10.18632/oncotarget.18598] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/21/2017] [Accepted: 06/05/2017] [Indexed: 12/14/2022] Open
Abstract
The anti-cancer agent CBP501 binds to calmodulin (CaM). Recent studies showed that migration and metastasis are inhibited by several CaM antagonists. However, there is no available evidence that CBP501 has similar effects. Here we found that CBP501 inhibits migration of non-small cell lung cancer (NSCLC) cells in vitro, even in the presence of migration inducing factors such as WNT, IL-6, and several growth factors. CBP501 also inhibited epidermal growth factor (EGF) enhanced invasion and the epithelial-to-mesenchymal transition (EMT), and this inhibition was accompanied by (i) suppression of Akt and ERK1/2 phosphorylation, and (ii) suppression of expression of transcription factor Zeb1 and the mesenchymal marker Vimentin. A pull down analysis performed using sepharose-immobilized CaM showed that CBP501 blocks the interaction between CaM and KRas. Furthermore, EGF induced Akt activation and cell migration was effectively suppressed by KRas down-regulation in NSCLC cells. Stable knockdown of KRas also made cells insensitive to CBP501's inhibition of growth factor-induced migration. Taken together, these results indicate that CBP501 inhibits binding of CaM with KRas and thereby suppresses the PI3K/AKT pathway, migration, invasion and EMT. These findings have identified a previously unrecognized effect of CBP501 on downstream KRas signaling mechanisms involving EMT and invasion, and provide support for the further clinical development of this agent.
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Affiliation(s)
| | | | - Donald W Kufe
- Dana-Farber Cancer Institute, Harvard School, Boston, MA, USA
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26
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Abstract
The specific and rapid formation of protein complexes, involving IQGAP family proteins, is essential for diverse cellular processes, such as adhesion, polarization, and directional migration. Although CDC42 and RAC1, prominent members of the RHO GTPase family, have been implicated in binding to and activating IQGAP1, the exact nature of this protein-protein recognition process has remained obscure. Here, we propose a mechanistic framework model that is based on a multiple-step binding process, which is a prerequisite for the dynamic functions of IQGAP1 as a scaffolding protein and a critical mechanism in temporal regulation and integration of cellular pathways.
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Affiliation(s)
- Kazem Nouri
- Institute of Biochemistry and Molecular Biology II, Medical faculty of the Heinrich-Heine University, Düsseldorf, Germany
| | - David J Timson
- School of Pharmacy and Biomolecular Sciences, University of Brighton, Huxley Building, Lewes Road, Brighton, BN2 4GJ, United Kingdom
| | - Mohammad R Ahmadian
- Institute of Biochemistry and Molecular Biology II, Medical faculty of the Heinrich-Heine University, Düsseldorf, Germany
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27
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Ramos CJ, Antonetti DA. The role of small GTPases and EPAC-Rap signaling in the regulation of the blood-brain and blood-retinal barriers. Tissue Barriers 2017. [PMID: 28632993 DOI: 10.1080/21688370.2017.1339768] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/19/2022] Open
Abstract
Maintenance and regulation of the vascular endothelial cell junctional complex is critical for proper barrier function of the blood-brain barrier (BBB) and the highly related blood-retinal barrier (BRB) that help maintain proper neuronal environment. Recent research has demonstrated that the junctional complex is actively maintained and can be dynamically regulated. Studies focusing on the mechanisms of barrier formation, maintenance, and barrier disruption have been of interest to understanding development of the BBB and BRB and identifying a means for therapeutic intervention for diseases ranging from brain tumors and dementia to blinding eye diseases. Research has increasingly revealed that small GTPases play a critical role in both barrier formation and disruption mechanisms. This review will summarize the current data on small GTPases in barrier regulation with an emphasis on the EPAC-Rap1 signaling pathway to Rho in endothelial barriers, as well as explore its potential involvement in paracellular flux and transcytosis regulation.
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Affiliation(s)
- Carla J Ramos
- a Department of Ophthalmology and Visual Sciences , University of Michigan , Ann Arbor , MI USA
| | - David A Antonetti
- a Department of Ophthalmology and Visual Sciences , University of Michigan , Ann Arbor , MI USA
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28
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Hu G, Xu Y, Chen W, Wang J, Zhao C, Wang M. RNA Interference of IQ Motif Containing GTPase-Activating Protein 3 (IQGAP3) Inhibits Cell Proliferation and Invasion in Breast Carcinoma Cells. Oncol Res 2017; 24:455-461. [PMID: 28281966 PMCID: PMC7838651 DOI: 10.3727/096504016x14685034103635] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022] Open
Abstract
Breast cancer is a highly prevalent disease affecting women. The association of IQ motif containing GTPase-activating protein 3 (IQGAP3) and breast cancer is poorly defined. Here we reported that IQGAP3 is a key regulator of cell proliferation and metastasis during breast cancer progression. The expression of IQGAP3 was significantly increased in breast tissues compared to nontumor tissues at both protein and mRNA levels. Furthermore, IQGAP3 had a high expression level in ZR-75-30 and BT474 compared to other breast cancer cell lines. Depletion of IQGAP3 through RNA interference in ZR-75-30 and BT474 significantly inhibited cell proliferation. More importantly, IQGAP3 silencing in breast cancer cells notably repressed cell migration and invasion. Further analysis suggested that inhibition of cell proliferation and metastasis was associated with some proteins, including p53, MMP9, Snail, CDC42, p-ERK1/2, KIF2C, KIF4A, PCNA, and Twist. Since expression of IQGAP3 seems to be associated with the pathogenesis of breast cancer and suppression of it can inhibit cancer cell growth and metastasis, IQGAP3 may be a potential therapeutic target in human breast cancer.
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Affiliation(s)
- Gaowu Hu
- Department of General Surgery, Shanghai Traditional Chinese Medicine-Integrated Hospital, Shanghai, P.R. China
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29
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Socoro-Yuste N, Čokić VP, Mondet J, Plo I, Mossuz P. Quantitative Proteome Heterogeneity in Myeloproliferative Neoplasm Subtypes and Association with JAK2 Mutation Status. Mol Cancer Res 2017; 15:852-861. [PMID: 28314843 DOI: 10.1158/1541-7786.mcr-16-0495] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/28/2016] [Revised: 02/23/2017] [Accepted: 03/10/2017] [Indexed: 11/16/2022]
Abstract
Apart from well-known genetic abnormalities, several studies have reported variations in protein expression in Philadelphia-negative myeloproliferative neoplasm (MPN) patients that could contribute toward their clinical phenotype. In this context, a quantitative mass spectrometry proteomics protocol was used to identify differences in the granulocyte proteome with the goal to characterize the pathogenic role of aberrant protein expression in MPNs. LC/MS-MS (LTQ Orbitrap) coupled to iTRAQ labeling showed significant and quantitative differences in protein content among various MPN subtypes [polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF)], and according to the genetic status of JAK2 (JAK2V617F presence and JAK2V617F allele burden). A number of differentially expressed proteins were identified, with the most frequent being members of the RAS GTPase family and oxidative stress regulatory proteins. Subsequent analysis found that calreticulin (CALR), known to be involved in calcium homeostasis and apoptotic signaling, was overexpressed in JAK2V617F granulocytes compared with JAK2 wild type and independently of the JAK2V617F allele burden. Finally, it was demonstrated, in a Ba/F3 cell model, that increased calreticulin expression was directly linked to JAK2V617F and could be regulated by JAK2 kinase inhibitors.Implications: In conclusion, these results reveal proteome alterations in MPN granulocytes depending on the phenotype and genotype of patients, highlighting new oncogenic mechanisms associated with JAK2 mutations and overexpression of calreticulin. Mol Cancer Res; 15(7); 852-61. ©2017 AACR.
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Affiliation(s)
- Nuria Socoro-Yuste
- TheREx Team (Thérapeutique Recombinante Expérimentale), TIMC-IMAG Laboratory, (Techniques de l'Ingénierie Médicale et de la Complexité - Informatique, Mathématiques et Applications de Grenoble), Grenoble Alpes University, Grenoble, France.
| | - Vladan P Čokić
- Institute for Medical Research, University of Belgrade, Belgrade, Serbia
| | - Julie Mondet
- TheREx Team (Thérapeutique Recombinante Expérimentale), TIMC-IMAG Laboratory, (Techniques de l'Ingénierie Médicale et de la Complexité - Informatique, Mathématiques et Applications de Grenoble), Grenoble Alpes University, Grenoble, France
| | - Isabelle Plo
- INSERM, UMR1170. Gustave Roussy. Université Paris-Sud., Villejuif, France
| | - Pascal Mossuz
- TheREx Team (Thérapeutique Recombinante Expérimentale), TIMC-IMAG Laboratory, (Techniques de l'Ingénierie Médicale et de la Complexité - Informatique, Mathématiques et Applications de Grenoble), Grenoble Alpes University, Grenoble, France.,Laboratoire d'Hématologie Cellulaire, Institut de Biologie et Pathologie, Grenoble Alpes University Hospital, Grenoble, France
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30
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Chawla B, Hedman AC, Sayedyahossein S, Erdemir HH, Li Z, Sacks DB. Absence of IQGAP1 Protein Leads to Insulin Resistance. J Biol Chem 2017; 292:3273-3289. [PMID: 28082684 DOI: 10.1074/jbc.m116.752642] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/08/2016] [Revised: 01/10/2017] [Indexed: 11/06/2022] Open
Abstract
Insulin binds to the insulin receptor (IR) and induces tyrosine phosphorylation of the receptor and insulin receptor substrate-1 (IRS-1), leading to activation of the PKB/Akt and MAPK/ERK pathways. IQGAP1 is a scaffold protein that interacts with multiple binding partners and integrates diverse signaling cascades. Here we show that IQGAP1 associates with both IR and IRS-1 and influences insulin action. In vitro analysis with pure proteins revealed that the IQ region of IQGAP1 binds directly to the intracellular domain of IR. Similarly, the phosphotyrosine-binding domain of IRS-1 mediates a direct interaction with the C-terminal tail of IQGAP1. Consistent with these observations, both IR and IRS-1 co-immunoprecipitated with IQGAP1 from cells. Investigation of the functional effects of the interactions revealed that in the absence of IQGAP1, insulin-stimulated phosphorylation of Akt and ERK, as well as the association of phosphatidylinositol 3-kinase with IRS-1, were significantly decreased. Importantly, loss of IQGAP1 results in impaired insulin signaling and glucose homeostasis in vivo Collectively, these data reveal that IQGAP1 is a scaffold for IR and IRS-1 and implicate IQGAP1 as a participant in insulin signaling.
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Affiliation(s)
- Bhavna Chawla
- Department of Laboratory Medicine, National Institutes of Health, Bethesda, Maryland 20892
| | - Andrew C Hedman
- Department of Laboratory Medicine, National Institutes of Health, Bethesda, Maryland 20892
| | - Samar Sayedyahossein
- Department of Laboratory Medicine, National Institutes of Health, Bethesda, Maryland 20892
| | - Huseyin H Erdemir
- Department of Laboratory Medicine, National Institutes of Health, Bethesda, Maryland 20892
| | - Zhigang Li
- Department of Laboratory Medicine, National Institutes of Health, Bethesda, Maryland 20892
| | - David B Sacks
- Department of Laboratory Medicine, National Institutes of Health, Bethesda, Maryland 20892.
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31
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Liu J, Kurella VB, LeCour L, Vanagunas T, Worthylake DK. The IQGAP1 N-Terminus Forms Dimers, and the Dimer Interface Is Required for Binding F-Actin and Calcium-Bound Calmodulin. Biochemistry 2016; 55:6433-6444. [PMID: 27798963 DOI: 10.1021/acs.biochem.6b00745] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Abstract
IQGAP1 is a multidomain scaffold protein involved in many cellular processes. We have determined the crystal structure of an N-terminal fragment spanning residues 1-191 (CHDF hereafter) that contains the entire calponin homology domain. The structure of the CHDF is very similar to those of other type 3 calponin homology domains like those from calponin, Vav, and the yeast IQGAP1 ortholog Rng2. However, in the crystal, two CHDF molecules form a "head-to-head" or parallel dimer through mostly hydrophobic interactions. Binding experiments indicate that the CHDF binds to both F-actin and Ca2+/calmodulin, but binding is mutually exclusive. On the basis of the structure, two dimer interface substitutions were introduced. While CHDFL157D disrupts the dimer in gel filtration experiments, oxidized CHDFK161C stabilizes the dimer. These results imply that the CHDF forms the same dimer in solution that is seen in the crystal structure. The disulfide-stabilized dimer displays a reduced level of F-actin binding in sedimentation assays and shows no binding to Ca2+/calmodulin in isothermal titration calorimetry (ITC) experiments, indicating that interface residues are utilized for both binding events. The Calmodulin Target Database predicts that residues 93KK94 are important for CaM binding, and indeed, the 93EE94 double mutation displays a reduced level of binding to Ca2+/calmodulin in ITC experiments. Our results indicate that the CHDF dimer interface is used for both F-actin and Ca2+/calmodulin binding, and the 93KK94 pair, near the interface, is also used for Ca2+/calmodulin binding. These results are also consistent with full-length IQGAP1 forming a parallel homodimer.
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Affiliation(s)
- Jing Liu
- Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center-New Orleans , 1901 Perdido Street, New Orleans, Louisiana 70112, United States
| | - Vinodh B Kurella
- Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center-New Orleans , 1901 Perdido Street, New Orleans, Louisiana 70112, United States
| | - Louis LeCour
- Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center-New Orleans , 1901 Perdido Street, New Orleans, Louisiana 70112, United States
| | - Tomas Vanagunas
- Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center-New Orleans , 1901 Perdido Street, New Orleans, Louisiana 70112, United States
| | - David K Worthylake
- Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center-New Orleans , 1901 Perdido Street, New Orleans, Louisiana 70112, United States
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32
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Zang T, Broszczak DA, Broadbent JA, Cuttle L, Lu H, Parker TJ. The biochemistry of blister fluid from pediatric burn injuries: proteomics and metabolomics aspects. Expert Rev Proteomics 2015; 13:35-53. [PMID: 26581649 DOI: 10.1586/14789450.2016.1122528] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
Burn injury is a prevalent and traumatic event for pediatric patients. At present, the diagnosis of burn injury severity is subjective and lacks a clinically relevant quantitative measure. This is due in part to a lack of knowledge surrounding the biochemistry of burn injuries and that of blister fluid. A more complete understanding of the blister fluid biochemistry may open new avenues for diagnostic and prognostic development. Burn insult induces a highly complex network of signaling processes and numerous changes within various biochemical systems, which can ultimately be examined using proteome and metabolome measurements. This review reports on the current understanding of burn wound biochemistry and outlines a technical approach for 'omics' profiling of blister fluid from burn wounds of differing severity.
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Affiliation(s)
- Tuo Zang
- a Tissue Repair and Regeneration Program , Institute of Health and Biomedical Innovation , Kelvin Grove , Australia.,b School of Biomedical Sciences , Queensland University of Technology , Brisbane , Australia.,c Wound Management Innovation Co-operative Research Centre , West End , Australia
| | - Daniel A Broszczak
- a Tissue Repair and Regeneration Program , Institute of Health and Biomedical Innovation , Kelvin Grove , Australia.,b School of Biomedical Sciences , Queensland University of Technology , Brisbane , Australia.,c Wound Management Innovation Co-operative Research Centre , West End , Australia
| | - James A Broadbent
- a Tissue Repair and Regeneration Program , Institute of Health and Biomedical Innovation , Kelvin Grove , Australia.,b School of Biomedical Sciences , Queensland University of Technology , Brisbane , Australia.,c Wound Management Innovation Co-operative Research Centre , West End , Australia
| | - Leila Cuttle
- a Tissue Repair and Regeneration Program , Institute of Health and Biomedical Innovation , Kelvin Grove , Australia.,b School of Biomedical Sciences , Queensland University of Technology , Brisbane , Australia.,d Centre for Children's Burns and Trauma Research , Queensland University of Technology, Institute of Health and Biomedical Innovation at the Centre for Children's Health Research , South Brisbane , Australia
| | - Haitao Lu
- a Tissue Repair and Regeneration Program , Institute of Health and Biomedical Innovation , Kelvin Grove , Australia.,b School of Biomedical Sciences , Queensland University of Technology , Brisbane , Australia
| | - Tony J Parker
- a Tissue Repair and Regeneration Program , Institute of Health and Biomedical Innovation , Kelvin Grove , Australia.,b School of Biomedical Sciences , Queensland University of Technology , Brisbane , Australia
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33
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Arnsten AFT. Stress weakens prefrontal networks: molecular insults to higher cognition. Nat Neurosci 2015; 18:1376-85. [PMID: 26404712 DOI: 10.1038/nn.4087] [Citation(s) in RCA: 468] [Impact Index Per Article: 46.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/17/2015] [Accepted: 07/15/2015] [Indexed: 12/13/2022]
Abstract
A variety of cognitive disorders are worsened by stress exposure and involve dysfunction of the newly evolved prefrontal cortex (PFC). Exposure to acute, uncontrollable stress increases catecholamine release in PFC, reducing neuronal firing and impairing cognitive abilities. High levels of noradrenergic α1-adrenoceptor and dopaminergic D1 receptor stimulation activate feedforward calcium-protein kinase C and cyclic AMP-protein kinase A signaling, which open potassium channels to weaken synaptic efficacy in spines. In contrast, high levels of catecholamines strengthen the primary sensory cortices, amygdala and striatum, rapidly flipping the brain from reflective to reflexive control of behavior. These mechanisms are exaggerated by chronic stress exposure, where architectural changes lead to persistent loss of PFC function. Understanding these mechanisms has led to the successful translation of prazosin and guanfacine for treating stress-related disorders. Dysregulation of stress signaling pathways by genetic insults likely contributes to PFC deficits in schizophrenia, while age-related insults initiate interacting vicious cycles that increase vulnerability to Alzheimer's degeneration.
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Affiliation(s)
- Amy F T Arnsten
- Department of Neurobiology, Yale University School of Medicine, New Haven, Connecticut, USA
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34
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Nussinov R, Muratcioglu S, Tsai CJ, Jang H, Gursoy A, Keskin O. The Key Role of Calmodulin in KRAS-Driven Adenocarcinomas. Mol Cancer Res 2015; 13:1265-73. [PMID: 26085527 PMCID: PMC4572916 DOI: 10.1158/1541-7786.mcr-15-0165] [Citation(s) in RCA: 68] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/02/2015] [Accepted: 06/09/2015] [Indexed: 12/14/2022]
Abstract
KRAS4B is a highly oncogenic splice variant of the KRAS isoform. It is the only isoform associated with initiation of adenocarcinomas. Insight into why and how KRAS4B can mediate ductal adenocarcinomas, particularly of the pancreas, is vastly important for its therapeutics. Here we point out the overlooked critical role of calmodulin (CaM). Calmodulin selectively binds to GTP-bound K-Ras4B; but not to other Ras isoforms. Cell proliferation and growth require the MAPK (Raf/MEK/ERK) and PI3K/Akt pathways. We propose that Ca(2+)/calmodulin promote PI3Kα/Akt signaling, and suggest how. The elevated calcium levels clinically observed in adenocarcinomas may explain calmodulin's involvement in recruiting and stimulating PI3Kα through interaction with its n/cSH2 domains as well as K-Ras4B; importantly, it also explains why K-Ras4B specifically is a key player in ductal carcinomas, such as pancreatic (PDAC), colorectal (CRC), and lung cancers. We hypothesize that calmodulin recruits and helps activate PI3Kα at the membrane, and that this is the likely reason for Ca(2+)/calmodulin dependence in adenocarcinomas. Calmodulin can contribute to initiation/progression of ductal cancers via both PI3Kα/Akt and Raf/MEK/ERK pathways. Blocking the K-Ras4B/MAPK pathway and calmodulin/PI3Kα binding in a K-Ras4B/calmodulin/PI3Kα trimer could be a promising adenocarcinoma-specific therapeutic strategy.
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Affiliation(s)
- Ruth Nussinov
- Cancer and Inflammation Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, NCI at Frederick, Frederick, Maryland. Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel.
| | - Serena Muratcioglu
- Department of Chemical and Biological Engineering, Koc University, Istanbul, Turkey
| | - Chung-Jung Tsai
- Cancer and Inflammation Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, NCI at Frederick, Frederick, Maryland
| | - Hyunbum Jang
- Cancer and Inflammation Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, NCI at Frederick, Frederick, Maryland
| | - Attila Gursoy
- Department of Computer Engineering, Koc University, Istanbul, Turkey
| | - Ozlem Keskin
- Department of Chemical and Biological Engineering, Koc University, Istanbul, Turkey
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35
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Huang Y, Li W, Su ZY, Kong ANT. The complexity of the Nrf2 pathway: beyond the antioxidant response. J Nutr Biochem 2015; 26:1401-13. [PMID: 26419687 DOI: 10.1016/j.jnutbio.2015.08.001] [Citation(s) in RCA: 335] [Impact Index Per Article: 33.5] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/08/2015] [Accepted: 08/03/2015] [Indexed: 12/11/2022]
Abstract
The NF-E2-related factor 2 (Nrf2)-mediated signalling pathway provides living organisms an efficient and pivotal line of defensive to counteract environmental insults and endogenous stressors. Nrf2 coordinates the basal and inducible expression of antioxidant and Phase II detoxification enzymes to adapt to different stress conditions. The stability and cellular distribution of Nrf2 is tightly controlled by its inhibitory binding protein Kelch-like ECH-associated protein 1. Nrf2 signalling is also regulated by posttranslational, transcriptional, translational and epigenetic mechanisms, as well as by other protein partners, including p62, p21 and IQ motif-containing GTPase activating protein 1. Many studies have demonstrated that Nrf2 is a promising target for preventing carcinogenesis and other chronic diseases, including cardiovascular diseases, neurodegenerative diseases and pulmonary injury. However, constitutive activation of Nrf2 in advanced cancer cells may confer drug resistance. Here, we review the molecular mechanisms of Nrf2 signalling, the diverse classes of Nrf2 activators, including bioactive nutrients and other chemicals, and the cellular functions and disease relevance of Nrf2 and discuss the dual role of Nrf2 in different contexts.
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Affiliation(s)
- Ying Huang
- Department of Pharmaceutics, Earnest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA
| | - Wenji Li
- Department of Pharmaceutics, Earnest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA
| | - Zheng-yuan Su
- Department of Pharmaceutics, Earnest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA
| | - Ah-Ng Tony Kong
- Department of Pharmaceutics, Earnest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA.
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36
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The European GWAS-identified risk SNP rs457717 within IQGAP2 is not associated with age-related hearing impairment in Han male Chinese population. Eur Arch Otorhinolaryngol 2015; 273:1677-87. [DOI: 10.1007/s00405-015-3711-9] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/02/2015] [Accepted: 07/03/2015] [Indexed: 11/26/2022]
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37
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Hedman AC, Smith JM, Sacks DB. The biology of IQGAP proteins: beyond the cytoskeleton. EMBO Rep 2015; 16:427-46. [PMID: 25722290 DOI: 10.15252/embr.201439834] [Citation(s) in RCA: 172] [Impact Index Per Article: 17.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/06/2014] [Accepted: 01/07/2015] [Indexed: 01/02/2023] Open
Abstract
IQGAP scaffold proteins are evolutionarily conserved in eukaryotes and facilitate the formation of complexes that regulate cytoskeletal dynamics, intracellular signaling, and intercellular interactions. Fungal and mammalian IQGAPs are implicated in cytokinesis. IQGAP1, IQGAP2, and IQGAP3 have diverse roles in vertebrate physiology, operating in the kidney, nervous system, cardio-vascular system, pancreas, and lung. The functions of IQGAPs can be corrupted during oncogenesis and are usurped by microbial pathogens. Therefore, IQGAPs represent intriguing candidates for novel therapeutic agents. While modulation of the cytoskeletal architecture was initially thought to be the primary function of IQGAPs, it is now clear that they have roles beyond the cytoskeleton. This review describes contributions of IQGAPs to physiology at the organism level.
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Affiliation(s)
- Andrew C Hedman
- Department of Laboratory Medicine, National Institutes of Health, Bethesda, MD, USA
| | - Jessica M Smith
- Department of Laboratory Medicine, National Institutes of Health, Bethesda, MD, USA
| | - David B Sacks
- Department of Laboratory Medicine, National Institutes of Health, Bethesda, MD, USA
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38
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IQGAPs choreograph cellular signaling from the membrane to the nucleus. Trends Cell Biol 2015; 25:171-84. [PMID: 25618329 DOI: 10.1016/j.tcb.2014.12.005] [Citation(s) in RCA: 115] [Impact Index Per Article: 11.5] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/11/2014] [Revised: 12/02/2014] [Accepted: 12/03/2014] [Indexed: 12/18/2022]
Abstract
Since its discovery in 1994, recognized cellular functions for the scaffold protein IQGAP1 have expanded immensely. Over 100 unique IQGAP1-interacting proteins have been identified, implicating IQGAP1 as a critical integrator of cellular signaling pathways. Initial research established functions for IQGAP1 in cell-cell adhesion, cell migration, and cell signaling. Recent studies have revealed additional IQGAP1 binding partners, expanding the biological roles of IQGAP1. These include crosstalk between signaling cascades, regulation of nuclear function, and Wnt pathway potentiation. Investigation of the IQGAP2 and IQGAP3 homologs demonstrates unique functions, some of which differ from those of IQGAP1. Summarized here are recent observations that enhance our understanding of IQGAP proteins in the integration of diverse signaling pathways.
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39
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Yang Y, Zhao W, Xu QW, Wang XS, Zhang Y, Zhang J. IQGAP3 promotes EGFR-ERK signaling and the growth and metastasis of lung cancer cells. PLoS One 2014; 9:e97578. [PMID: 24849319 PMCID: PMC4029748 DOI: 10.1371/journal.pone.0097578] [Citation(s) in RCA: 60] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/20/2014] [Accepted: 04/21/2014] [Indexed: 12/23/2022] Open
Abstract
Proteins of the IQGAP family display complicated and often contradictory activities in tumorigenesis. IQGAP1 has well documented oncogenic potential and IQGAP2 has putative tumor-suppressive function. IQGAP3 is the latest addition to this family and its role in cancer development remains to be defined. Here we demonstrate IQGAP3 expression is markedly increased in lung cancer tissues at both mRNA and protein levels. Overexpression of IQGAP3 promoted tumor cell growth, and migration and invasion, whereas knockdown of IQGAP3 exhibited opposite effects. Moreover, suppression of IQGAP3 in a lung cancer cell line caused a reduction in the tumorigenicity of these cells in lung tissue after intravenous injection. Furthermore, we showed that IQGAP3 is able to interact with ERK1 and enhance its phosphorylation following treatment with EGF. These data suggest that IQGAP3 may contribute to the pathogenesis of lung cancer by modulating EGFR-ERK signaling.
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Affiliation(s)
- Ying Yang
- Department of Immunology, School of Basic Medical Sciences, Key Laboratory of Medical Immunology of Ministry of Public Health, Peking University Health Science Center, Beijing, P. R. China
| | - Wei Zhao
- Department of Immunology, School of Basic Medical Sciences, Key Laboratory of Medical Immunology of Ministry of Public Health, Peking University Health Science Center, Beijing, P. R. China
| | - Qing-Wen Xu
- Department of Immunology, School of Basic Medical Sciences, Key Laboratory of Medical Immunology of Ministry of Public Health, Peking University Health Science Center, Beijing, P. R. China
| | - Xiao-Song Wang
- Department of Immunology, School of Basic Medical Sciences, Key Laboratory of Medical Immunology of Ministry of Public Health, Peking University Health Science Center, Beijing, P. R. China
| | - Yu Zhang
- Department of Immunology, School of Basic Medical Sciences, Key Laboratory of Medical Immunology of Ministry of Public Health, Peking University Health Science Center, Beijing, P. R. China
- * E-mail: (YZ); (JZ)
| | - Jun Zhang
- Department of Immunology, School of Basic Medical Sciences, Key Laboratory of Medical Immunology of Ministry of Public Health, Peking University Health Science Center, Beijing, P. R. China
- * E-mail: (YZ); (JZ)
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40
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Wang Y, Jarrard RE, Pratt EPS, Guerra ML, Salyer AE, Lange AM, Soderling IM, Hockerman GH. Uncoupling of Cav1.2 from Ca(2+)-induced Ca(2+) release and SK channel regulation in pancreatic β-cells. Mol Endocrinol 2014; 28:458-76. [PMID: 24506535 DOI: 10.1210/me.2013-1094] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
We investigated the role of Cav1.2 in pancreatic β-cell function by expressing a Cav1.2 II-III loop/green fluorescent protein fusion in INS-1 cells (Cav1.2/II-III cells) to disrupt channel-protein interactions. Neither block of KATP channels nor stimulation of membrane depolarization by tolbutamide was different in INS-1 cells compared with Cav1.2/II-III cells, but whole-cell Cav current density was significantly increased in Cav1.2/II-III cells. Tolbutamide (200 μM) stimulated insulin secretion and Ca(2+) transients in INS-1 cells, and Cav1.2/II-III cells were completely blocked by nicardipine (2 μM), but thapsigargin (1 μM) blocked tolbutamide-stimulated secretion and Ca(2+) transients only in INS-1 cells. Tolbutamide-stimulated endoplasmic reticulum [Ca(2+)] decrease was reduced in Cav1.2/II-III cells compared with INS-1 cells. However, Ca(2+) transients in both INS-1 cells and Cav1.2/II-III cells were significantly potentiated by 8-pCPT-2'-O-Me-cAMP (5 μM), FPL-64176 (0.5 μM), or replacement of extracellular Ca(2+) with Sr(2+). Glucose (10 mM) + glucagon-like peptide-1 (10 nM) stimulated discrete spikes in [Ca(2+)]i in the presence of verapamil at a higher frequency in INS-1 cells than in Cav1.2/II-II cells. Glucose (18 mM) stimulated more frequent action potentials in Cav1.2/II-III cells and primary rat β-cells expressing the Cav1.2/II-II loop than in control cells. Further, apamin (1 μM) increased glucose-stimulated action potential frequency in INS-1 cells, but not Cav1.2/II-III cells, suggesting that SK channels were not activated under these conditions in Cav1.2/II-III loop-expressing cells. We propose the II-III loop of Cav1.2 as a key molecular determinant that couples the channel to Ca(2+)-induced Ca(2+) release and activation of SK channels in pancreatic β-cells.
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Affiliation(s)
- Yuchen Wang
- Purdue University Life Sciences Graduate Program (R.E.J., E.P.S.P., A.M.L.) and Department of Medicinal Chemistry and Molecular Pharmacology (Y.W., M.L.G., A.E.S., I.M.S., G.H.H.), Purdue University, West Lafayette, Indiana 47907-2091
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Holm A, Vikström E. Quorum sensing communication between bacteria and human cells: signals, targets, and functions. FRONTIERS IN PLANT SCIENCE 2014; 5:309. [PMID: 25018766 PMCID: PMC4071818 DOI: 10.3389/fpls.2014.00309] [Citation(s) in RCA: 76] [Impact Index Per Article: 6.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/30/2014] [Accepted: 06/10/2014] [Indexed: 05/10/2023]
Abstract
Both direct and long-range interactions between pathogenic Pseudomonas aeruginosa bacteria and their eukaryotic hosts are important in the outcome of infections. For cell-to-cell communication, these bacteria employ the quorum sensing (QS) system to pass on information of the density of the bacterial population and collectively switch on virulence factor production, biofilm formation, and resistance development. Thus, QS allows bacteria to behave as a community to perform tasks which would be impossible for individual cells, e.g., to overcome defense and immune systems and establish infections in higher organisms. This review highlights these aspects of QS and our own recent research on how P. aeruginosa communicates with human cells using the small QS signal molecules N-acyl homoserine lactones (AHL). We focus on how this conversation changes the behavior and function of neutrophils, macrophages, and epithelial cells and on how the signaling machinery in human cells responsible for the recognition of AHL. Understanding the bacteria-host relationships at both cellular and molecular levels is essential for the identification of new targets and for the development of novel strategies to fight bacterial infections in the future.
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Affiliation(s)
| | - Elena Vikström
- *Correspondence: Elena Vikström, Division of Medical Microbiology, Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linköping University, Linköping SE-58185, Sweden e-mail:
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Liu J, Guidry JJ, Worthylake DK. Conserved sequence repeats of IQGAP1 mediate binding to Ezrin. J Proteome Res 2013; 13:1156-66. [PMID: 24294828 DOI: 10.1021/pr400787p] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
Mammalian IQGAP proteins all feature multiple ∼50 amino acid sequence repeats near their N-termini, and little is known about the function of these "Repeats". We have expressed and purified the Repeats from human IQGAP1 to identify binding partners. We used mass spectrometry to identify 42 mouse kidney proteins that associate with the IQGAP1 Repeats including the ERM proteins ezrin, radixin, and moesin. ERM proteins have an N-terminal FERM domain (4.1, ezrin, radixin, moesin) through which they bind to protein targets and phosphatidylinositol 4,5-bisphosphate (PIP2) and a C-terminal actin-binding domain and function to link the actin cytoskeleton to distinct locations on the cell cortex. Isothermal titration calorimetry (ITC) reveals that the IQGAP1 Repeats directly bind to the ezrin FERM domain, while no binding is seen for full-length "autoinhibited" ezrin or a version of full-length ezrin intended to mimic the activated protein. ITC also indicates that the ezrin FERM domain binds to the Repeats from IQGAP2 but not the Repeats from IQGAP3. We conclude that IQGAP1 and IQGAP2 are positioned at the cell cortex by ERM proteins. We propose that the IQGAP3 Repeats may likewise bind to FERM domains for signaling purposes.
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Affiliation(s)
- Jing Liu
- Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center , 1901 Perdido Street, New Orleans, Louisiana 70112, United States
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Kim JH, Xu EY, Sacks DB, Lee J, Shu L, Xia B, Kong ANT. Identification and functional studies of a new Nrf2 partner IQGAP1: a critical role in the stability and transactivation of Nrf2. Antioxid Redox Signal 2013; 19:89-101. [PMID: 22793650 PMCID: PMC3689176 DOI: 10.1089/ars.2012.4586] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
AIMS Nuclear factor-erythroid-related factor 2 (Nrf2) is a critical transcriptional factor that is used in regulating cellular defense against oxidative stress. This study is aimed at investigating new interacting protein partners of Nrf2 using One-strep tag pull-down coupled with LTQ Orbitrap LC/MS/MS, and at examining the impact on Nr2 signaling by the newly identified IQ motif containing GTPase activating protein 1 (IQGAP1). RESULTS Using the One-strep tag pull-down and LTQ Orbitrap LC/MS/MS, we identified IQGAP1 as a new Nrf2 interacting partner. Direct interactions between IQGAP1 and Nrf2 proteins were verified using in vitro glutathione S-transferase (GST) pull-down, transcription/translation assays, and in vivo utilizing Nrf2 overexpressing cells. Coexpression of Dsredmono-IQGAP1 and eGFP-Nrf2 increased the stability of eGFP-Nrf2 and enhanced the expression of Nrf2-target gene heme oxygenase-1 (HO-1). To confirm the functional role of IQGAP1 on Nrf2, knock-downed IQGAP1 using siIQGAP1 attenuated the expression of endogenous Nrf2, HO-1 proteins, and Nrf2-target genes GSTpi, GCLC, and NAD(P)H quinone oxidoreductase 1 (NQO-1). Furthermore, the stability of Nrf2 was dramatically decreased in IQGAP1-deficient mouse embryonic fibroblast (MEF) cells. Since IQGAP1 signaling could be mediated by calcium, treating the cells with calcium showed the translocation of IQGAP1/Nrf2 complex into the nucleus, suggesting that IQGAP1 may play a critical role in Nrf2 stability. Interestingly, consistent with calcium signaling for IQGAP1, treating the cells with calcium functionally enhanced Nrf2-mediated antioxidant responsive element-transcription activity and enhanced the expression of the endogenous Nrf2-target gene HO-1. INNOVATION In the aggregate, our current study identifies and functionally characterizes a new Nrf2 partner protein IQGAP1, which may contribute to Nrf2's regulation of antioxidant enzymes such as HO-1. CONCLUSION IQGAP1 may play a critical role in the stability and transactivation of Nrf2.
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Affiliation(s)
- Jung-Hwan Kim
- Graduate Program in Pharmaceutical Science, Center for Cancer Prevention Research, Ernest-Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA
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Cheung KL, Lee JH, Shu L, Kim JH, Sacks DB, Kong ANT. The Ras GTPase-activating-like protein IQGAP1 mediates Nrf2 protein activation via the mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK)-ERK pathway. J Biol Chem 2013; 288:22378-86. [PMID: 23788642 DOI: 10.1074/jbc.m112.444182] [Citation(s) in RCA: 38] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022] Open
Abstract
Nrf2 plays a critical role in the regulation of cellular oxidative stress. MEK-ERK activation has been shown to be one of the major pathways resulting in the activation of Nrf2 and induction of Nrf2 downstream targets, including phase II detoxifying/antioxidant genes in response to oxidative stress and xenobiotics. In this study, IQGAP1 (IQ motif-containing GTPase-activating protein 1), a new Nrf2 interaction partner that we have published previously, was found to modulate MEK-ERK-mediated Nrf2 activation and induction of phase II detoxifying/antioxidant genes. Nrf2 binds directly to the IQ domain (amino acids 699-905) of IQGAP1. Knockdown of IQGAP1 significantly attenuated phenethyl isothiocyanate- or MEK-mediated activation of the MEK-ERK-Nrf2 pathway. Knockdown of IQGAP1 also attenuated MEK-mediated increased stability of Nrf2, which in turn was associated with a decrease in the nuclear translocation of Nrf2 and a decrease in the expression of phase II detoxifying/antioxidant genes. In the aggregate, these results suggest that IQGAP1 may play an important role in the MEK-ERK-Nrf2 signaling pathway.
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Affiliation(s)
- Ka Lung Cheung
- Graduate Program in Pharmaceutical Science, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, New Jersey 08854, USA
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Wu Y, Tao Y, Chen Y, Xu W. RhoC regulates the proliferation of gastric cancer cells through interaction with IQGAP1. PLoS One 2012; 7:e48917. [PMID: 23145020 PMCID: PMC3492142 DOI: 10.1371/journal.pone.0048917] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/26/2012] [Accepted: 10/02/2012] [Indexed: 01/29/2023] Open
Abstract
Our previous research results showed that both Ras homolog family member C (RhoC) and IQ-domain GTPase-activating protein 1 (IQGAP1) were over-expressed in gastric cancer tissues and cells, but their role in tumorigenensis has not been addressed clearly. Herein we reported the proliferation stimulating effect of RhoC and IQGAP1 on gastric cancer cells and the interaction between two proteins in regulating the proliferation of gastric cancer cells. Plasmids and viral constructs encoding target siRNA and DNA were used to alter the expression of RhoC and IQGAP1. MTT method and BrdU incorporation assay were used for analyzing the effect of RhoC and different structures of IQGAP1 on proliferation. Protein levels of IQGAP1 and RhoC in cell lines were detected by Western blotting. Immunofluorescence and Co-Immunoprecipitation assays were applied to investigate the localization and binding between RhoC and IQGAP1. The results showed that RhoC, IQGAP1 and the C-terminal fragment of IQGAP1 significantly stimulated the proliferation of gastric cancer cells, and enhanced the expression of cyclin E and cyclin D1. By contrast, reduction of endogenous IQGAP1 or RhoC by siRNA attenuated cell proliferation. The depletion of IQGAP1 expression by siRNA significantly blocked the proliferative activity of constitutively active RhoC, while RhoC silencing by siRNA had no effect on IQGAP1-induced proliferation in gastric cancer cells. Co-immunoprecipitation and Immunofluorescence assays showed that RhoC and IQGAP1 bound each other. In conclusion, our results suggest that RhoC stimulates the proliferation of gastric cancer cells through recruiting IQGAP1 as an effector.
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Affiliation(s)
- Yan Wu
- School of Medical Science and Laboratory Medicine, Jiangsu University, Zhenjiang, Jiangsu, China.
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Abstract
Our previous research results showed that both Ras homolog family member C (RhoC) and IQ-domain GTPase-activating protein 1 (IQGAP1) were over-expressed in gastric cancer tissues and cells, but their role in tumorigenensis has not been addressed clearly. Herein we reported the proliferation stimulating effect of RhoC and IQGAP1 on gastric cancer cells and the interaction between two proteins in regulating the proliferation of gastric cancer cells. Plasmids and viral constructs encoding target siRNA and DNA were used to alter the expression of RhoC and IQGAP1. MTT method and BrdU incorporation assay were used for analyzing the effect of RhoC and different structures of IQGAP1 on proliferation. Protein levels of IQGAP1 and RhoC in cell lines were detected by Western blotting. Immunofluorescence and Co-Immunoprecipitation assays were applied to investigate the localization and binding between RhoC and IQGAP1. The results showed that RhoC, IQGAP1 and the C-terminal fragment of IQGAP1 significantly stimulated the proliferation of gastric cancer cells, and enhanced the expression of cyclin E and cyclin D1. By contrast, reduction of endogenous IQGAP1 or RhoC by siRNA attenuated cell proliferation. The depletion of IQGAP1 expression by siRNA significantly blocked the proliferative activity of constitutively active RhoC, while RhoC silencing by siRNA had no effect on IQGAP1-induced proliferation in gastric cancer cells. Co-immunoprecipitation and Immunofluorescence assays showed that RhoC and IQGAP1 bound each other. In conclusion, our results suggest that RhoC stimulates the proliferation of gastric cancer cells through recruiting IQGAP1 as an effector.
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Affiliation(s)
- Yan Wu
- School of Medical Science and Laboratory Medicine, Jiangsu University, Zhenjiang, Jiangsu, China.
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Jang DJ. Characterization for calmodulin binding activity of IQ motifs on the IQGAP3. ANALYTICAL SCIENCE AND TECHNOLOGY 2012. [DOI: 10.5806/ast.2012.25.5.333] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
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Karlsson T, Turkina MV, Yakymenko O, Magnusson KE, Vikström E. The Pseudomonas aeruginosa N-acylhomoserine lactone quorum sensing molecules target IQGAP1 and modulate epithelial cell migration. PLoS Pathog 2012; 8:e1002953. [PMID: 23071436 PMCID: PMC3469656 DOI: 10.1371/journal.ppat.1002953] [Citation(s) in RCA: 71] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/16/2011] [Accepted: 08/23/2012] [Indexed: 01/01/2023] Open
Abstract
Quorum sensing (QS) signaling allows bacteria to control gene expression once a critical population density is achieved. The Gram-negative human pathogen Pseudomonas aeruginosa uses N-acylhomoserine lactones (AHL) as QS signals, which coordinate the production of virulence factors and biofilms. These bacterial signals can also modulate human cell behavior. Little is known about the mechanisms of the action of AHL on their eukaryotic targets. Here, we found that N-3-oxo-dodecanoyl-L-homoserine lactone 3O-C12-HSL modulates human intestinal epithelial Caco-2 cell migration in a dose- and time-dependent manner. Using new 3O-C12-HSL biotin and fluorescently-tagged probes for LC-MS/MS and confocal imaging, respectively, we demonstrated for the first time that 3O-C12-HSL interacts and co-localizes with the IQ-motif-containing GTPase-activating protein IQGAP1 in Caco-2 cells. The interaction between IQGAP1 and 3O-C12-HSL was further confirmed by pull-down assay using a GST-tagged protein with subsequent Western blot of IQGAP1 and by identifying 3O-C12-HSL with a sensor bioassay. Moreover, 3O-C12-HSL induced changes in the phosphorylation status of Rac1 and Cdc42 and the localization of IQGAP1 as evidenced by confocal and STED microscopy and Western blots. Our findings suggest that the IQGAP1 is a novel partner for P.aeruginosa 3O-C12-HSL and likely the integrator of Rac1 and Cdc42- dependent altered cell migration. We propose that the targeting of IQGAP1 by 3O-C12-HSL can trigger essential changes in the cytoskeleton network and be an essential component in bacterial – human cell communication. The human pathogen Pseudomonas aeruginosa and other bacteria communicate with each other using quorum sensing (QS). This is important for their growth, virulence, motility and the formation of biofilms. Furthermore, eukaryotic cells “listen and respond” to QS signaling, but the exact mechanisms and receptors on mammalian cells have not been identified. We have previously shown that N-acylhomoserine lactones (AHL) alter epithelial barrier functions and increase chemotaxis in human neutrophils. We show here that 3O-C12-HSL modulates the migration of epithelial cells in a dose- and time-dependent manner. Using newly designed and validated biotin- and fluorescein-based 3O-C12-HSL probes we identified the IQ-motif-containing GTPase-activating protein IQGAP1 as a human target of 3O-C12-HSL. We propose that the interaction between IQGAP1 and 3O-C12-HSL provides a novel mechanism for its mode of action on eukaryotic cells, and by affecting the distribution of IQGAP1 and phosphorylation of Rac1 and Cdc42, upstream effectors of filamentous actin remodeling, also cell migration. We suggest that recognition of IQGAP1 by 3O-C12-HSL is a very early event in the communication between bacteria and human epithelial cells.
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Affiliation(s)
- Thommie Karlsson
- Division of Medical Microbiology, Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linköping University, Linköping, Sweden
| | - Maria V. Turkina
- Division of Cell Biology, Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linköping University, Linköping, Sweden
| | - Olena Yakymenko
- Division of Medical Microbiology, Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linköping University, Linköping, Sweden
| | - Karl-Eric Magnusson
- Division of Medical Microbiology, Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linköping University, Linköping, Sweden
| | - Elena Vikström
- Division of Medical Microbiology, Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linköping University, Linköping, Sweden
- * E-mail:
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Malarkannan S, Awasthi A, Rajasekaran K, Kumar P, Schuldt KM, Bartoszek A, Manoharan N, Goldner NK, Umhoefer CM, Thakar MS. IQGAP1: a regulator of intracellular spacetime relativity. THE JOURNAL OF IMMUNOLOGY 2012; 188:2057-63. [PMID: 22345702 DOI: 10.4049/jimmunol.1102439] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
Activating and inhibiting receptors of lymphocytes collect valuable information about their mikròs kósmos. This information is essential to initiate or to turn off complex signaling pathways. Irrespective of these advances, our knowledge on how these intracellular activation cascades are coordinated in a spatiotemporal manner is far from complete. Among multiple explanations, the scaffolding proteins have emerged as a critical piece of this evolutionary tangram. Among many, IQGAP1 is one of the essential scaffolding proteins that coordinate multiple signaling pathways. IQGAP1 possesses multiple protein interaction motifs to achieve its scaffolding functions. Using these domains, IQGAP1 has been shown to regulate a number of essential cellular events. This includes actin polymerization, tubulin multimerization, microtubule organizing center formation, calcium/calmodulin signaling, Pak/Raf/Mek1/2-mediated Erk1/2 activation, formation of maestrosome, E-cadherin, and CD44-mediated signaling and glycogen synthase kinase-3/adenomatous polyposis coli-mediated β-catenin activation. In this review, we summarize the recent developments and exciting new findings of cellular functions of IQGAP1.
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Affiliation(s)
- Subramaniam Malarkannan
- Laboratory of Molecular Immunology and Immunotherapy, Blood Research Institute, Milwaukee, WI 53226, USA.
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Krishnan S, Fernandez GE, Sacks DB, Prasadarao NV. IQGAP1 mediates the disruption of adherens junctions to promote Escherichia coli K1 invasion of brain endothelial cells. Cell Microbiol 2012; 14:1415-33. [PMID: 22519731 DOI: 10.1111/j.1462-5822.2012.01805.x] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/05/2011] [Revised: 04/09/2012] [Accepted: 04/16/2012] [Indexed: 12/17/2022]
Abstract
The transcellular entry of Escherichia coli K1 through human brain microvascular endothelial cells (HBMEC) is responsible for tight junction disruption, leading to brain oedema in neonatal meningitis. Previous studies demonstrated that outer membrane protein A (OmpA) of E. coli K1 interacts with its receptor, Ecgp96, to induce PKC-α phosphorylation, adherens junction (AJ) disassembly (by dislodging β-catenin from VE-cadherin), and remodelling of actin in HBMEC. We report here that IQGAP1 mediates β-catenin dissociation from AJs to promote actin polymerization required for E. coli K1 invasion of HBMEC. Overexpression of C-terminal truncated IQGAP1 (IQΔC) that cannot bind β-catenin prevents both AJ disruption and E. coli K1 entry. Of note, phospho-PKC-α interacts with the C-terminal portion of Ecgp96 as well as with VE-cadherin after IQGAP1-mediated AJ disassembly. HBMEC overexpressing either C-terminal truncated Ecgp96 (Ecgp96Δ200) or IQΔC upon infection with E. coli showed no interaction of phospho-PKC-α with Ecgp96. These data indicate that the binding of OmpA to Ecgp96 induces PKC-α phosphorylation and association of phospho-PKC-α with Ecgp96, and then signals IQGAP1 to detach β-catenin from AJs. Subsequently, IQGAP1/β-catenin bound actin translocates to the site of E. coli K1 attachment to promote invasion.
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Affiliation(s)
- Subramanian Krishnan
- Division of Infectious Diseases, Department of Pediatrics, The Saban Research Institute, Children's Hospital Los Angeles, University of Southern California School of Medicine, Los Angeles, CA 90027, USA
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