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Yang J, Wang P, Zhang Y, Zhang M, Sun Q, Chen H, Dong L, Chu Z, Xue B, Hoff WD, Zhao C, Wang W, Wei Q, Cao Y. Photo-tunable hydrogels reveal cellular sensing of rapid rigidity changes through the accumulation of mechanical signaling molecules. Cell Stem Cell 2025; 32:121-136.e6. [PMID: 39437791 DOI: 10.1016/j.stem.2024.09.016] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/25/2024] [Revised: 07/08/2024] [Accepted: 09/23/2024] [Indexed: 10/25/2024]
Abstract
Cells use traction forces to sense mechanical cues in their environment. While the molecular clutch model effectively explains how cells exert more forces on stiffer substrates, it falls short in addressing their adaptation to dynamic mechanical fluctuations prevalent in tissues and organs. Here, using hydrogel with photo-responsive rigidity, we show that cells' response to rigidity changes is frequency dependent. Strikingly, at certain frequencies, cellular traction forces exceed those on static substrates 4-fold stiffer, challenging the established molecular clutch model. We discover that the discrepancy between the rapid adaptation of traction forces and the slower deactivation of mechanotransduction signaling proteins results in their accumulation, thereby enhancing long-term cellular traction in dynamic settings. Consequently, we propose a new model that melds immediate mechanosensing with extended mechanical signaling. Our study underscores the significance of dynamic rigidity in the development of synthetic biomaterials, emphasizing the importance of considering both immediate and prolonged cellular responses.
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Affiliation(s)
- Jiapeng Yang
- Jinan Microecological Biomedicine Shandong Laboratory, Jinan 250021, China; College of Polymer Science and Engineering, State Key Laboratory of Polymer Materials and Engineering, Sichuan University, Chengdu 610065, China; Chemistry and Biomedicine Innovation Center (ChemBIC), ChemBioMed Interdisciplinary Research Center at Nanjing University, Department of Physics, Nanjing University, Nanjing 210093, China
| | - Peng Wang
- College of Polymer Science and Engineering, State Key Laboratory of Polymer Materials and Engineering, Sichuan University, Chengdu 610065, China
| | - Yu Zhang
- Jinan Microecological Biomedicine Shandong Laboratory, Jinan 250021, China; Chemistry and Biomedicine Innovation Center (ChemBIC), ChemBioMed Interdisciplinary Research Center at Nanjing University, Department of Physics, Nanjing University, Nanjing 210093, China
| | - Man Zhang
- College of Biomedical Engineering, Sichuan University, Chengdu 610065, China
| | - Qian Sun
- College of Polymer Science and Engineering, State Key Laboratory of Polymer Materials and Engineering, Sichuan University, Chengdu 610065, China
| | - Huiyan Chen
- Chemistry and Biomedicine Innovation Center (ChemBIC), ChemBioMed Interdisciplinary Research Center at Nanjing University, Department of Physics, Nanjing University, Nanjing 210093, China
| | - Liang Dong
- Chemistry and Biomedicine Innovation Center (ChemBIC), ChemBioMed Interdisciplinary Research Center at Nanjing University, Department of Physics, Nanjing University, Nanjing 210093, China
| | - Zhiqin Chu
- Department of Electrical and Electronic Engineering, The University of Hong Kong, Hong Kong 999077, China; Joint Appointment with School of Biomedical Sciences, The University of Hong Kong, Hong Kong 999077, China
| | - Bin Xue
- Chemistry and Biomedicine Innovation Center (ChemBIC), ChemBioMed Interdisciplinary Research Center at Nanjing University, Department of Physics, Nanjing University, Nanjing 210093, China
| | - Wouter David Hoff
- Department of Physics, Oklahoma State University, Stillwater, OK 74078, USA
| | - Changsheng Zhao
- College of Polymer Science and Engineering, State Key Laboratory of Polymer Materials and Engineering, Sichuan University, Chengdu 610065, China
| | - Wei Wang
- Chemistry and Biomedicine Innovation Center (ChemBIC), ChemBioMed Interdisciplinary Research Center at Nanjing University, Department of Physics, Nanjing University, Nanjing 210093, China
| | - Qiang Wei
- College of Polymer Science and Engineering, State Key Laboratory of Polymer Materials and Engineering, Sichuan University, Chengdu 610065, China.
| | - Yi Cao
- Jinan Microecological Biomedicine Shandong Laboratory, Jinan 250021, China; Chemistry and Biomedicine Innovation Center (ChemBIC), ChemBioMed Interdisciplinary Research Center at Nanjing University, Department of Physics, Nanjing University, Nanjing 210093, China; Wenzhou Institute, University of Chinese Academy of Sciences, Wenzhou 325001, China.
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2
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McDuffie EL, Panettieri RA, Scott CP. G 12/13 signaling in asthma. Respir Res 2024; 25:295. [PMID: 39095798 PMCID: PMC11297630 DOI: 10.1186/s12931-024-02920-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/14/2024] [Accepted: 07/19/2024] [Indexed: 08/04/2024] Open
Abstract
Shortening of airway smooth muscle and bronchoconstriction are pathognomonic for asthma. Airway shortening occurs through calcium-dependent activation of myosin light chain kinase, and RhoA-dependent calcium sensitization, which inhibits myosin light chain phosphatase. The mechanism through which pro-contractile stimuli activate calcium sensitization is poorly understood. Our review of the literature suggests that pro-contractile G protein coupled receptors likely signal through G12/13 to activate RhoA and mediate calcium sensitization. This hypothesis is consistent with the effects of pro-contractile agonists on RhoA and Rho kinase activation, actin polymerization and myosin light chain phosphorylation. Recognizing the likely role of G12/13 signaling in the pathophysiology of asthma rationalizes the effects of pro-contractile stimuli on airway hyperresponsiveness, immune activation and airway remodeling, and suggests new approaches for asthma treatment.
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Affiliation(s)
- Elizabeth L McDuffie
- Department of Biochemistry and Molecular Biology, Sidney Kimmel Medical College, Thomas Jefferson University, Philadelphia, PA, USA
| | - Reynold A Panettieri
- Rutgers Institute for Translational Medicine and Science, Child Health Institute, Rutgers University, New Brunswick, NJ, USA
| | - Charles P Scott
- Department of Biochemistry and Molecular Biology, Sidney Kimmel Medical College, Thomas Jefferson University, Philadelphia, PA, USA.
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3
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Zhang J, Wei K, Qu W, Wang M, Zhu Q, Dong X, Huang X, Yi W, Xu S, Li X. Ogt Deficiency Induces Abnormal Cerebellar Function and Behavioral Deficits of Adult Mice through Modulating RhoA/ROCK Signaling. J Neurosci 2023; 43:4559-4579. [PMID: 37225434 PMCID: PMC10286951 DOI: 10.1523/jneurosci.1962-22.2023] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/19/2022] [Revised: 04/10/2023] [Accepted: 04/13/2023] [Indexed: 05/26/2023] Open
Abstract
Previous studies have shown the essential roles of O-GlcNAc transferase (Ogt) and O-GlcNAcylation in neuronal development, function and neurologic diseases. However, the function of Ogt and O-GlcNAcylation in the adult cerebellum has not been well elucidated. Here, we have found that cerebellum has the highest level of O-GlcNAcylation relative to cortex and hippocampus of adult male mice. Specific deletion of Ogt in granule neuron precursors (GNPs) induces abnormal morphology and decreased size of the cerebellum in adult male Ogt deficient [conditional knock-out (cKO)] mice. Adult male cKO mice show the reduced density and aberrant distribution of cerebellar granule cells (CGCs), the disrupted arrangement of Bergman glia (BG) and Purkinje cells. In addition, adult male cKO mice exhibit aberrant synaptic connection, impaired motor coordination, and learning and memory abilities. Mechanistically, we have identified G-protein subunit α12 (Gα12) is modified by Ogt-mediated O-GlcNAcylation. O-GlcNAcylation of Gα12 facilitates its binding to Rho guanine nucleotide exchange factor 12 (Arhgef12) and consequently activates RhoA/ROCK signaling. RhoA/ROCK pathway activator LPA can rescue the developmental deficits of Ogt deficient CGCs. Therefore, our study has revealed the critical function and related mechanisms of Ogt and O-GlcNAcylation in the cerebellum of adult male mice.SIGNIFICANCE STATEMENT Cerebellar function are regulated by diverse mechanisms. To unveil novel mechanisms is critical for understanding the cerebellar function and the clinical therapy of cerebellum-related diseases. In the present study, we have shown that O-GlcNAc transferase gene (Ogt) deletion induces abnormal cerebellar morphology, synaptic connection, and behavioral deficits of adult male mice. Mechanistically, Ogt catalyzes O-GlcNAcylation of Gα12, which promotes the binding to Arhgef12, and regulates RhoA/ROCK signaling pathway. Our study has uncovered the important roles of Ogt and O-GlcNAcylation in regulating cerebellar function and cerebellum-related behavior. Our results suggest that Ogt and O-GlcNAcylation could be potential targets for some cerebellum-related diseases.
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Affiliation(s)
- Jinyu Zhang
- The Children's Hospital, National Clinical Research Center for Child Health, School of Medicine, Zhejiang University, Hangzhou 310052, China
- The Institute of Translational Medicine, School of Medicine, Zhejiang University, Hangzhou 310029, China
| | - Kaiyan Wei
- The Children's Hospital, National Clinical Research Center for Child Health, School of Medicine, Zhejiang University, Hangzhou 310052, China
| | - Wenzheng Qu
- The Children's Hospital, National Clinical Research Center for Child Health, School of Medicine, Zhejiang University, Hangzhou 310052, China
| | - Mengxuan Wang
- The Children's Hospital, National Clinical Research Center for Child Health, School of Medicine, Zhejiang University, Hangzhou 310052, China
- The Institute of Translational Medicine, School of Medicine, Zhejiang University, Hangzhou 310029, China
| | - Qiang Zhu
- MOE Key Laboratory of Biosystems Homeostasis and Protection, College of Life Sciences, Zhejiang University, Hangzhou 310058
- The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310002, China
| | - Xiaoxue Dong
- The Children's Hospital, National Clinical Research Center for Child Health, School of Medicine, Zhejiang University, Hangzhou 310052, China
- The Institute of Translational Medicine, School of Medicine, Zhejiang University, Hangzhou 310029, China
| | - Xiaoli Huang
- The Children's Hospital, National Clinical Research Center for Child Health, School of Medicine, Zhejiang University, Hangzhou 310052, China
| | - Wen Yi
- MOE Key Laboratory of Biosystems Homeostasis and Protection, College of Life Sciences, Zhejiang University, Hangzhou 310058
- The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310002, China
| | - Shunliang Xu
- Department of Neurology, The Second Hospital, Cheeloo College of Medicine, Shandong University, Jinan 250033, China
| | - Xuekun Li
- The Children's Hospital, National Clinical Research Center for Child Health, School of Medicine, Zhejiang University, Hangzhou 310052, China
- The Institute of Translational Medicine, School of Medicine, Zhejiang University, Hangzhou 310029, China
- Key Laboratory of Diagnosis and Treatment of Neonatal Diseases of Zhejiang Province, Hangzhou 310052, China
- Binjiang Institute of Zhejiang University, Hangzhou 310053, China
- Zhejiang University Cancer Center, Zhejiang University, Hangzhou 310029, China
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Abstract
The Ras homologous (Rho) protein family of GTPases (RhoA, RhoB and RhoC) are the members of the Ras superfamily and regulate cellular processes such as cell migration, proliferation, polarization, adhesion, gene transcription and cytoskeletal structure. Rho GTPases function as molecular switches that cycle between GTP-bound (active state) and GDP-bound (inactive state) forms. Leukaemia-associated RhoGEF (LARG) is a guanine nucleotide exchange factor (GEF) that activates RhoA subfamily GTPases by promoting the exchange of GDP for GTP. LARG is selective for RhoA subfamily GTPases and is an essential regulator of cell migration and invasion. Here, we describe the mechanisms by which LARG is regulated to facilitate the understanding of how LARG mediates functions like cell motility and to provide insight for better therapeutic targeting of these functions.
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Affiliation(s)
- Neda Z. Ghanem
- Cancer Biology Program, University of Hawaii Cancer Center, University of Hawaii at Mānoa, Honolulu, USA,Molecular Biosciences and BioEngineering Graduate Program, University of Hawaii at Mānoa, Honolulu, USA
| | - Michelle L. Matter
- Cancer Biology Program, University of Hawaii Cancer Center, University of Hawaii at Mānoa, Honolulu, USA,Molecular Biosciences and BioEngineering Graduate Program, University of Hawaii at Mānoa, Honolulu, USA
| | - Joe W. Ramos
- Cancer Biology Program, University of Hawaii Cancer Center, University of Hawaii at Mānoa, Honolulu, USA,Molecular Biosciences and BioEngineering Graduate Program, University of Hawaii at Mānoa, Honolulu, USA,CONTACT Joe W. Ramos Cancer Biology Program, University of Hawaii Cancer Center, University of Hawaii at Manoa, Honolulu, USA
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5
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Panagopoulos I, Andersen K, Eilert-Olsen M, Zeller B, Munthe-Kaas MC, Buechner J, Osnes LTN, Micci F, Heim S. Therapy-induced Deletion in 11q23 Leading to Fusion of KMT2A With ARHGEF12 and Development of B Lineage Acute Lymphoplastic Leukemia in a Child Treated for Acute Myeloid Leukemia Caused by t(9;11)(p21;q23)/ KMT2A-MLLT3. Cancer Genomics Proteomics 2021; 18:67-81. [PMID: 33419897 DOI: 10.21873/cgp.20242] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2020] [Revised: 10/25/2020] [Accepted: 10/27/2020] [Indexed: 11/10/2022] Open
Abstract
BACKGROUND/AIM Fusion of histone-lysine N-methyltransferase 2A gene (KMT2A) with the Rho guanine nucleotide exchange factor 12 gene (ARHGEF12), both located in 11q23, was reported in some leukemic patients. We report a KMT2A-ARHGEF12 fusion occurring during treatment of a pediatric acute myeloid leukemia (AML) with topoisomerase II inhibitors leading to a secondary acute lymphoblastic leukemia (ALL). MATERIALS AND METHODS Multiple genetic analyses were performed on bone marrow cells of a girl initially diagnosed with AML. RESULTS At the time of diagnosis with AML, the t(9;11)(p21;q23)/KMT2A-MLLT3 genetic abnormality was found. After chemotherapy resulting in AML clinical remission, a 2 Mb deletion in 11q23 was found generating a KMT2A-ARHGEF12 fusion gene. When the patient later developed B lineage ALL, a t(14;19)(q32;q13), loss of one chromosome 9, and KMT2A-ARHGEF12 were detected. CONCLUSION The patient sequentially developed AML and ALL with three leukemia-specific genomic abnormalities in her bone marrow cells, two of which were KMT2A-rearrangements.
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Affiliation(s)
- Ioannis Panagopoulos
- Section for Cancer Cytogenetics, Institute for Cancer Genetics and Informatics, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway;
| | - Kristin Andersen
- Section for Cancer Cytogenetics, Institute for Cancer Genetics and Informatics, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway
| | - Martine Eilert-Olsen
- Section for Cancer Cytogenetics, Institute for Cancer Genetics and Informatics, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway
| | - Bernward Zeller
- Department of Pediatric Hematology and Oncology, Oslo University Hospital, Rikshospitalet, Oslo, Norway
| | - Monica Cheng Munthe-Kaas
- Department of Pediatric Hematology and Oncology, Oslo University Hospital, Rikshospitalet, Oslo, Norway
| | - Jochen Buechner
- Department of Pediatric Hematology and Oncology, Oslo University Hospital, Rikshospitalet, Oslo, Norway
| | - Liv T N Osnes
- Department of Immunology, Oslo University Hospital, Rikshospitalet, Oslo, Norway
| | - Francesca Micci
- Section for Cancer Cytogenetics, Institute for Cancer Genetics and Informatics, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway
| | - Sverre Heim
- Section for Cancer Cytogenetics, Institute for Cancer Genetics and Informatics, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway.,Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway
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6
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Avet C, Sturino C, Grastilleur S, Gouill CL, Semache M, Gross F, Gendron L, Bennani Y, Mancini JA, Sayegh CE, Bouvier M. The PAR2 inhibitor I-287 selectively targets Gα q and Gα 12/13 signaling and has anti-inflammatory effects. Commun Biol 2020; 3:719. [PMID: 33247181 PMCID: PMC7695697 DOI: 10.1038/s42003-020-01453-8] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/05/2020] [Accepted: 10/29/2020] [Indexed: 01/01/2023] Open
Abstract
Protease-activated receptor-2 (PAR2) is involved in inflammatory responses and pain, therefore representing a promising therapeutic target for the treatment of immune-mediated inflammatory diseases. However, as for other GPCRs, PAR2 can activate multiple signaling pathways and those involved in inflammatory responses remain poorly defined. Here, we describe a new selective and potent PAR2 inhibitor (I-287) that shows functional selectivity by acting as a negative allosteric regulator on Gαq and Gα12/13 activity and their downstream effectors, while having no effect on Gi/o signaling and βarrestin2 engagement. Such selective inhibition of only a subset of the pathways engaged by PAR2 was found to be sufficient to block inflammation in vivo. In addition to unraveling the PAR2 signaling pathways involved in the pro-inflammatory response, our study opens the path toward the development of new functionally selective drugs with reduced liabilities that could arise from blocking all the signaling activities controlled by the receptor. Avet et al. characterize I-287, an inhibitor to protease-activated receptor 2 using BRET-assays. They find that I-287 selectively inhibits Gαq and Gα12/13 without affecting the activation of Gi/o or the recruitment of βarrestin2 and that it blocks inflammation in vitro and in vivo.
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Affiliation(s)
- Charlotte Avet
- Institute for Research in Immunology and Cancer, and Department of Biochemistry and Molecular Medicine, Université de Montréal, Montréal, QC, Canada, H3C 1J4
| | - Claudio Sturino
- Vertex Pharmaceuticals (Canada), Inc., Laval, QC, Canada, H7V 4A7.,Paraza Pharma, Inc., Saint-Laurent, QC, Canada, H4S 2E1
| | - Sébastien Grastilleur
- Département de Pharmacologie-Physiologie, Université de Sherbrooke, Centre de Recherche du CHU de Sherbrooke, Centre d'Excellence en Neurosciences de l'Université de Sherbrooke, Institut de Pharmacologie de Sherbrooke, Sherbrooke, QC, Canada, J1H 5N4
| | - Christian Le Gouill
- Institute for Research in Immunology and Cancer, and Department of Biochemistry and Molecular Medicine, Université de Montréal, Montréal, QC, Canada, H3C 1J4
| | - Meriem Semache
- Institute for Research in Immunology and Cancer, and Department of Biochemistry and Molecular Medicine, Université de Montréal, Montréal, QC, Canada, H3C 1J4.,Domain Therapeutics North America, Saint-Laurent, QC, Canada, H4S 1Z9
| | - Florence Gross
- Institute for Research in Immunology and Cancer, and Department of Biochemistry and Molecular Medicine, Université de Montréal, Montréal, QC, Canada, H3C 1J4.,Domain Therapeutics North America, Saint-Laurent, QC, Canada, H4S 1Z9
| | - Louis Gendron
- Département de Pharmacologie-Physiologie, Université de Sherbrooke, Centre de Recherche du CHU de Sherbrooke, Centre d'Excellence en Neurosciences de l'Université de Sherbrooke, Institut de Pharmacologie de Sherbrooke, Sherbrooke, QC, Canada, J1H 5N4
| | - Youssef Bennani
- Vertex Pharmaceuticals (Canada), Inc., Laval, QC, Canada, H7V 4A7.,AdMare BioInnovations, Saint-Laurent, QC, Canada, H4S 1Z9
| | - Joseph A Mancini
- Vertex Pharmaceuticals (Canada), Inc., Laval, QC, Canada, H7V 4A7.,Vertex Pharmaceuticals Inc., Boston, MA, 02210, USA
| | - Camil E Sayegh
- Vertex Pharmaceuticals (Canada), Inc., Laval, QC, Canada, H7V 4A7.,Ra Pharmaceuticals, Inc., Cambridge, MA, 02140, USA
| | - Michel Bouvier
- Institute for Research in Immunology and Cancer, and Department of Biochemistry and Molecular Medicine, Université de Montréal, Montréal, QC, Canada, H3C 1J4.
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7
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Garcia De Las Bayonas A, Philippe JM, Lellouch AC, Lecuit T. Distinct RhoGEFs Activate Apical and Junctional Contractility under Control of G Proteins during Epithelial Morphogenesis. Curr Biol 2019; 29:3370-3385.e7. [PMID: 31522942 PMCID: PMC6839405 DOI: 10.1016/j.cub.2019.08.017] [Citation(s) in RCA: 57] [Impact Index Per Article: 9.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/20/2019] [Revised: 07/15/2019] [Accepted: 08/07/2019] [Indexed: 01/08/2023]
Abstract
Small RhoGTPases direct cell shape changes and movements during tissue morphogenesis. Their activities are tightly regulated in space and time to specify the desired pattern of actomyosin contractility that supports tissue morphogenesis. This is expected to stem from polarized surface stimuli and from polarized signaling processing inside cells. We examined this general problem in the context of cell intercalation that drives extension of the Drosophila ectoderm. In the ectoderm, G protein-coupled receptors (GPCRs) and their downstream heterotrimeric G proteins (Gα and Gβγ) activate Rho1 both medial-apically, where it exhibits pulsed dynamics, and at junctions, where its activity is planar polarized. However, the mechanisms responsible for polarizing Rho1 activity are unclear. We report that distinct guanine exchange factors (GEFs) activate Rho1 in these two cellular compartments. RhoGEF2 acts uniquely to activate medial-apical Rho1 but is recruited both medial-apically and at junctions by Gα12/13-GTP, also called Concertina (Cta) in Drosophila. On the other hand, Dp114RhoGEF (Dp114), a newly characterized RhoGEF, is required for cell intercalation in the extending ectoderm, where it activates Rho1 specifically at junctions. Its localization is restricted to adherens junctions and is under Gβ13F/Gγ1 control. Furthermore, Gβ13F/Gγ1 activates junctional Rho1 and exerts quantitative control over planar polarization of Rho1. Finally, we found that Dp114RhoGEF is absent in the mesoderm, arguing for a tissue-specific control over junctional Rho1 activity. These results clarify the mechanisms of polarization of Rho1 activity in different cellular compartments and reveal that distinct GEFs are sensitive tuning parameters of cell contractility in remodeling epithelia.
Dp114RhoGEF activates junctional Rho1 and is involved in cell intercalation Gα/Cta and Gβγ subunits tune, respectively, RhoGEF2 and Dp114RhoGEF membrane levels Gβγ subunits control planar polarity of junctional Rho1 signaling via Dp114RhoGEF Tissue-specific RhoGEFs could diversify morphogenesis in different tissues
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Affiliation(s)
| | - Jean-Marc Philippe
- Aix Marseille Université, CNRS, IBDM-UMR7288, Turing Center for Living Systems, 13009 Marseille, France
| | - Annemarie C Lellouch
- Aix Marseille Université, CNRS, IBDM-UMR7288, Turing Center for Living Systems, 13009 Marseille, France
| | - Thomas Lecuit
- Aix Marseille Université, CNRS, IBDM-UMR7288, Turing Center for Living Systems, 13009 Marseille, France; Collège de France, 11 Place Marcelin Berthelot, 75005 Paris, France.
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8
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Feng X, Arang N, Rigiracciolo DC, Lee JS, Yeerna H, Wang Z, Lubrano S, Kishore A, Pachter JA, König GM, Maggiolini M, Kostenis E, Schlaepfer DD, Tamayo P, Chen Q, Ruppin E, Gutkind JS. A Platform of Synthetic Lethal Gene Interaction Networks Reveals that the GNAQ Uveal Melanoma Oncogene Controls the Hippo Pathway through FAK. Cancer Cell 2019; 35:457-472.e5. [PMID: 30773340 PMCID: PMC6737937 DOI: 10.1016/j.ccell.2019.01.009] [Citation(s) in RCA: 177] [Impact Index Per Article: 29.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/17/2018] [Revised: 12/03/2018] [Accepted: 01/15/2019] [Indexed: 02/05/2023]
Abstract
Activating mutations in GNAQ/GNA11, encoding Gαq G proteins, are initiating oncogenic events in uveal melanoma (UM). However, there are no effective therapies for UM. Using an integrated bioinformatics pipeline, we found that PTK2, encoding focal adhesion kinase (FAK), represents a candidate synthetic lethal gene with GNAQ activation. We show that Gαq activates FAK through TRIO-RhoA non-canonical Gαq-signaling, and genetic ablation or pharmacological inhibition of FAK inhibits UM growth. Analysis of the FAK-regulated transcriptome demonstrated that GNAQ stimulates YAP through FAK. Dissection of the underlying mechanism revealed that FAK regulates YAP by tyrosine phosphorylation of MOB1, inhibiting core Hippo signaling. Our findings establish FAK as a potential therapeutic target for UM and other Gαq-driven pathophysiologies that involve unrestrained YAP function.
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Affiliation(s)
- Xiaodong Feng
- Moores Cancer Center, University of California, San Diego, La Jolla, CA 92093, USA; State Key Laboratory of Oral Diseases, National Clinical Research Center for Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu 610041, China
| | - Nadia Arang
- Moores Cancer Center, University of California, San Diego, La Jolla, CA 92093, USA; Biomedical Sciences Graduate Program, University of California, San Diego, La Jolla, CA 92093, USA
| | - Damiano Cosimo Rigiracciolo
- Moores Cancer Center, University of California, San Diego, La Jolla, CA 92093, USA; Department of Pharmacy and Health and Nutritional Sciences, University of Calabria, Rende 87036, Italy
| | - Joo Sang Lee
- Cancer Data Science Laboratory, National Cancer Institute, National Institute of Health, Bethesda, MD 20892, USA; Center for Bioinformatics and Computational Biology & Department of Computer Sciences, University of Maryland, College Park, MD 20742, USA.
| | - Huwate Yeerna
- Moores Cancer Center, University of California, San Diego, La Jolla, CA 92093, USA
| | - Zhiyong Wang
- Moores Cancer Center, University of California, San Diego, La Jolla, CA 92093, USA; Biomedical Sciences Graduate Program, University of California, San Diego, La Jolla, CA 92093, USA
| | - Simone Lubrano
- Moores Cancer Center, University of California, San Diego, La Jolla, CA 92093, USA
| | - Ayush Kishore
- Moores Cancer Center, University of California, San Diego, La Jolla, CA 92093, USA
| | | | - Gabriele M König
- Molecular, Cellular and Pharmacobiology Section, Institute of Pharmaceutical Biology, University of Bonn, Bonn 53115, Germany
| | - Marcello Maggiolini
- Department of Pharmacy and Health and Nutritional Sciences, University of Calabria, Rende 87036, Italy
| | - Evi Kostenis
- Molecular, Cellular and Pharmacobiology Section, Institute of Pharmaceutical Biology, University of Bonn, Bonn 53115, Germany
| | - David D Schlaepfer
- Moores Cancer Center, University of California, San Diego, La Jolla, CA 92093, USA
| | - Pablo Tamayo
- Moores Cancer Center, University of California, San Diego, La Jolla, CA 92093, USA; Division of Medical Genetics, UC San Diego School of Medicine, La Jolla, CA 92093, USA
| | - Qianming Chen
- State Key Laboratory of Oral Diseases, National Clinical Research Center for Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu 610041, China.
| | - Eytan Ruppin
- Cancer Data Science Laboratory, National Cancer Institute, National Institute of Health, Bethesda, MD 20892, USA; Center for Bioinformatics and Computational Biology & Department of Computer Sciences, University of Maryland, College Park, MD 20742, USA.
| | - J Silvio Gutkind
- Moores Cancer Center, University of California, San Diego, La Jolla, CA 92093, USA.
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9
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Dee RA, Mangum KD, Bai X, Mack CP, Taylor JM. Druggable targets in the Rho pathway and their promise for therapeutic control of blood pressure. Pharmacol Ther 2019; 193:121-134. [PMID: 30189292 PMCID: PMC7235948 DOI: 10.1016/j.pharmthera.2018.09.001] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
The prevalence of high blood pressure (also known as hypertension) has steadily increased over the last few decades. Known as a silent killer, hypertension increases the risk for cardiovascular disease and can lead to stroke, heart attack, kidney failure and associated sequela. While numerous hypertensive therapies are currently available, it is estimated that only half of medicated patients exhibit blood pressure control. This signifies the need for a better understanding of the underlying cause of disease and for more effective therapies. While blood pressure homeostasis is very complex and involves the integrated control of multiple body systems, smooth muscle contractility and arterial resistance are important contributors. Strong evidence from pre-clinical animal models and genome-wide association studies indicate that smooth muscle contraction and BP homeostasis are governed by the small GTPase RhoA and its downstream target, Rho kinase. In this review, we summarize the signaling pathways and regulators that impart tight spatial-temporal control of RhoA activity in smooth muscle cells and discuss current therapeutic strategies to target these RhoA pathway components. We also discuss known allelic variations in the RhoA pathway and consider how these polymorphisms may affect genetic risk for hypertension and its clinical manifestations.
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Affiliation(s)
- Rachel A Dee
- Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, NC 27599, USA
| | - Kevin D Mangum
- Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, NC 27599, USA
| | - Xue Bai
- Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, NC 27599, USA
| | - Christopher P Mack
- Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, NC 27599, USA; McAllister Heart Institute, University of North Carolina, Chapel Hill, NC 27599, USA
| | - Joan M Taylor
- Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, NC 27599, USA; McAllister Heart Institute, University of North Carolina, Chapel Hill, NC 27599, USA.
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10
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Toro-Tapia G, Villaseca S, Beyer A, Roycroft A, Marcellini S, Mayor R, Torrejón M. The Ric-8A/Gα13/FAK signalling cascade controls focal adhesion formation during neural crest cell migration in Xenopus. Development 2018; 145:dev.164269. [PMID: 30297374 DOI: 10.1242/dev.164269] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/08/2018] [Accepted: 09/23/2018] [Indexed: 12/22/2022]
Abstract
Ric-8A is a pleiotropic guanine nucleotide exchange factor involved in the activation of various heterotrimeric G-protein pathways during adulthood and early development. Here, we sought to determine the downstream effectors of Ric-8A during the migration of the vertebrate cranial neural crest (NC) cells. We show that the Gα13 knockdown phenocopies the Ric-8A morphant condition, causing actin cytoskeleton alteration, protrusion instability, and a strong reduction in the number and dynamics of focal adhesions. In addition, the overexpression of Gα13 is sufficient to rescue Ric-8A-depleted cells. Ric-8A and Gα13 physically interact and colocalize in protrusions of the cells leading edge. The focal adhesion kinase FAK colocalizes and interacts with the endogenous Gα13, and a constitutively active form of Src efficiently rescues the Gα13 morphant phenotype in NC cells. We propose that Ric-8A-mediated Gα13 signalling is required for proper cranial NC cell migration by regulating focal adhesion dynamics and protrusion formation.
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Affiliation(s)
- Gabriela Toro-Tapia
- Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Biológicas, Universidad de Concepción, Casilla 160-C, Concepción 4030000, Chile
| | - Soraya Villaseca
- Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Biológicas, Universidad de Concepción, Casilla 160-C, Concepción 4030000, Chile
| | - Andrea Beyer
- Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Biológicas, Universidad de Concepción, Casilla 160-C, Concepción 4030000, Chile
| | - Alice Roycroft
- Department of Cell and Developmental Biology, University College London, London WC1E 6BT, UK
| | - Sylvain Marcellini
- Departamento de Biología Cellular, Facultad de Ciencias Biológicas, Universidad de Concepción, Casilla 160-C, Concepción 4030000, Chile
| | - Roberto Mayor
- Department of Cell and Developmental Biology, University College London, London WC1E 6BT, UK
| | - Marcela Torrejón
- Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Biológicas, Universidad de Concepción, Casilla 160-C, Concepción 4030000, Chile
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11
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PDZ-RhoGEF Is a Signaling Effector for TROY-Induced Glioblastoma Cell Invasion and Survival. Neoplasia 2018; 20:1045-1058. [PMID: 30219706 PMCID: PMC6140379 DOI: 10.1016/j.neo.2018.08.008] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/25/2018] [Revised: 08/17/2018] [Accepted: 08/20/2018] [Indexed: 11/24/2022] Open
Abstract
Glioblastoma multiforme (GBM) is the most common type of malignant brain tumors in adults and has a dismal prognosis. The highly aggressive invasion of malignant cells into the normal brain parenchyma renders complete surgical resection of GBM tumors impossible, increases resistance to therapeutic treatment, and leads to near-universal tumor recurrence. We have previously demonstrated that TROY (TNFRSF19) plays an important role in glioblastoma cell invasion and therapeutic resistance. However, the potential downstream effectors of TROY signaling have not been fully characterized. Here, we identified PDZ-RhoGEF as a binding partner for TROY that potentiated TROY-induced nuclear factor kappa B activation which is necessary for both cell invasion and survival. In addition, PDZ-RhoGEF also interacts with Pyk2, indicating that PDZ-RhoGEF is a component of a signalsome that includes TROY and Pyk2. PDZ-RhoGEF is overexpressed in glioblastoma tumors and stimulates glioma cell invasion via Rho activation. Increased PDZ-RhoGEF expression enhanced TROY-induced glioma cell migration. Conversely, silencing PDZ-RhoGEF expression inhibited TROY-induced glioma cell migration, increased sensitivity to temozolomide treatment, and extended survival of orthotopic xenograft mice. Furthermore, depletion of RhoC or RhoA inhibited TROY- and PDZ-RhoGEF-induced cell migration. Mechanistically, increased TROY expression stimulated Rho activation, and depletion of PDZ-RhoGEF expression reduced this activation. Taken together, these data suggest that PDZ-RhoGEF plays an important role in TROY signaling and provides insights into a potential node of vulnerability to limit GBM cell invasion and decrease therapeutic resistance.
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12
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Targeting Focal Adhesion Kinase Using Inhibitors of Protein-Protein Interactions. Cancers (Basel) 2018; 10:cancers10090278. [PMID: 30134553 PMCID: PMC6162372 DOI: 10.3390/cancers10090278] [Citation(s) in RCA: 24] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/18/2018] [Revised: 08/08/2018] [Accepted: 08/14/2018] [Indexed: 12/19/2022] Open
Abstract
Focal adhesion kinase (FAK) is a cytoplasmic non-receptor protein tyrosine kinase that is overexpressed and activated in many human cancers. FAK transmits signals to a wide range of targets through both kinase-dependant and independent mechanism thereby playing essential roles in cell survival, proliferation, migration and invasion. In the past years, small molecules that inhibit FAK kinase function have been developed and show reduced cancer progression and metastasis in several preclinical models. Clinical trials have been conducted and these molecules display limited adverse effect in patients. FAK contain multiple functional domains and thus exhibit both important scaffolding functions. In this review, we describe the major FAK interactions relevant in cancer signalling and discuss how such knowledge provide rational for the development of Protein-Protein Interactions (PPI) inhibitors.
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13
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Amado-Azevedo J, de Menezes RX, van Nieuw Amerongen GP, van Hinsbergh VWM, Hordijk PL. A functional siRNA screen identifies RhoGTPase-associated genes involved in thrombin-induced endothelial permeability. PLoS One 2018; 13:e0201231. [PMID: 30048510 PMCID: PMC6062096 DOI: 10.1371/journal.pone.0201231] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/01/2018] [Accepted: 07/11/2018] [Indexed: 12/18/2022] Open
Abstract
Thrombin and other inflammatory mediators may induce vascular permeability through the disruption of adherens junctions between adjacent endothelial cells. If uncontrolled, hyperpermeability leads to an impaired barrier, fluid leakage and edema, which can contribute to multi-organ failure and death. RhoGTPases control cytoskeletal dynamics, adhesion and migration and are known regulators of endothelial integrity. Knowledge of the precise role of each RhoGTPase, and their associated regulatory and effector genes, in endothelial integrity is incomplete. Using a combination of a RNAi screen with electrical impedance measurements, we quantified the effect of individually silencing 270 Rho-associated genes on the barrier function of thrombin-activated, primary endothelial cells. Known and novel RhoGTPase-associated regulators that modulate the response to thrombin were identified (RTKN, TIAM2, MLC1, ARPC1B, SEPT2, SLC9A3R1, RACGAP1, RAPGEF2, RHOD, PREX1, ARHGEF7, PLXNB2, ARHGAP45, SRGAP2, ARHGEF5). In conclusion, with this siRNA screen, we confirmed the roles of known regulators of endothelial integrity but also identified new, potential key players in thrombin-induced endothelial signaling.
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Affiliation(s)
- Joana Amado-Azevedo
- Department of Physiology, Amsterdam Cardiovascular Sciences, VU University Medical Center, Amsterdam, The Netherlands
| | - Renee X. de Menezes
- Department of Epidemiology and Biostatistics, VU University Medical Center, Amsterdam, The Netherlands
| | | | - Victor W. M. van Hinsbergh
- Department of Physiology, Amsterdam Cardiovascular Sciences, VU University Medical Center, Amsterdam, The Netherlands
| | - Peter L. Hordijk
- Department of Physiology, Amsterdam Cardiovascular Sciences, VU University Medical Center, Amsterdam, The Netherlands
- * E-mail:
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14
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Dada O, Gutowski S, Brautigam CA, Chen Z, Sternweis PC. Direct regulation of p190RhoGEF by activated Rho and Rac GTPases. J Struct Biol 2018; 202:13-24. [PMID: 29196061 PMCID: PMC5835413 DOI: 10.1016/j.jsb.2017.11.014] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/26/2017] [Revised: 11/21/2017] [Accepted: 11/27/2017] [Indexed: 12/16/2022]
Abstract
Rho family GTPases regulate a wide range of cellular processes. This includes cellular dynamics where three subfamilies, Rho, Rac, and Cdc42, are known to regulate cell shape and migration though coordinate action. Activation of Rho proteins largely depends on Rho Guanine nucleotide Exchange Factors (RhoGEFs) through a catalytic Dbl homology (DH) domain linked to a pleckstrin homology (PH) domain that subserves various functions. The PH domains from Lbc RhoGEFs, which specifically activate RhoA, have been shown to bind to activated RhoA. Here, p190RhoGEF is shown to also bind Rac1·GTP. Crystal structures reveal that activated Rac1 and RhoA use their effector-binding surfaces to associate with the same hydrophobic surface on the PH domain. Both activated RhoA and Rac1 can stimulate exchange of nucleotide on RhoA by localization of p190RhoGEF to its substrate, RhoA·GDP, in vitro. The binding of activated RhoA provides a mechanism for positive feedback regulation as previously proposed for the family of Lbc RhoGEFs. In contrast, the novel interaction between activated Rac1 and p190RhoGEF reveals a potential mechanism for cross-talk regulation where Rac can directly effect stimulation of RhoA. The greater capacity of Rac1 to stimulate p190RhoGEF among the Lbc RhoGEFs suggests functional specialization.
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Affiliation(s)
- Olugbenga Dada
- Department of Pharmacology, The University of Texas Southwestern Medical Center, 6001 Forest Park Road, Dallas, TX 75390, USA.
| | - Stephen Gutowski
- Department of Pharmacology, The University of Texas Southwestern Medical Center, 6001 Forest Park Road, Dallas, TX 75390, USA.
| | - Chad A Brautigam
- Department of Biophysics, The University of Texas Southwestern Medical Center, 6001 Forest Park Road, Dallas, TX 75390, USA; Department of Microbiology, The University of Texas Southwestern Medical Center, 6001 Forest Park Road, Dallas, TX 75390, USA.
| | - Zhe Chen
- Department of Biophysics, The University of Texas Southwestern Medical Center, 6001 Forest Park Road, Dallas, TX 75390, USA.
| | - Paul C Sternweis
- Department of Pharmacology, The University of Texas Southwestern Medical Center, 6001 Forest Park Road, Dallas, TX 75390, USA.
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15
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Chen R, Zhao H, Wu D, Zhao C, Zhao W, Zhou X. The role of SH3GL3 in myeloma cell migration/invasion, stemness and chemo-resistance. Oncotarget 2018; 7:73101-73113. [PMID: 27683032 PMCID: PMC5341966 DOI: 10.18632/oncotarget.12231] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2016] [Accepted: 09/12/2016] [Indexed: 11/25/2022] Open
Abstract
Multiple myeloma (MM) is an incurable cancer characterized by clonal expansion of malignant plasma cells in the bone marrow and their egress into peripheral blood. The mechanisms of myeloma cells migration/invasion have remained unclear. Herein, we found SH3GL3 was highly expressed in the CD138-negative (CD138−) myeloma cells. The migration/invasion capability of CD138− cells was significantly higher than that in the CD138-positive (CD138+) cells. Silencing SH3GL3 using shRNA reduced myeloma cells migration/invasion. Conversely, overexpression of SH3GL3 increased myeloma cells migration/invasion. Moreover, SH3GL3 is also associated with the stemness and chemo-resistance of CD138− myeloma cells. Elevated expression of stem cell and multi-drug resistant markers were seen in the myeloma cells with overexpressed SH3GL3; while knocking-down SH3GL3 reduced the expression of these markers. A marked increase in p-PI3K and p-FAK was observed in the cells with overexpressed SH3GL3. To test if FAK/PI3K signaling pathway was involved in the SH3GL3-mediated myeloma cells migration, the cells transfected w/wo SH3GL3 cDNA were treated with FAK inhibitor 14 and PI3K inhibitor LY294002. Inhibition of FAK and PI3K attenuated SH3GL3-mediated migration /invasion. Our findings indicate that SH3GL3 plays an important role in myeloma cell migration/invasion, stemness and chemo-resistance. The SH3GL3-mediated myeloma cell migration/invasion is mediated by FAK/PI3K signaling pathway.
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Affiliation(s)
- Ruoying Chen
- Department of Radiology, Wake Forest School of Medicine, Medical Center Boulevard, Winston-Salem, NC27157, USA
| | - Hong Zhao
- Department of Blood Transfusion, The Second Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang Province 150001, China
| | - Dan Wu
- Department of Radiology, Wake Forest School of Medicine, Medical Center Boulevard, Winston-Salem, NC27157, USA
| | - Chen Zhao
- Department of Radiology, Wake Forest School of Medicine, Medical Center Boulevard, Winston-Salem, NC27157, USA
| | - Weiling Zhao
- Department of Radiology, Wake Forest School of Medicine, Medical Center Boulevard, Winston-Salem, NC27157, USA
| | - Xiaobo Zhou
- Department of Radiology, Wake Forest School of Medicine, Medical Center Boulevard, Winston-Salem, NC27157, USA.,College of Computer Science and Software Engineering, Shenzhen University, Shenzhen, China
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16
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MacKay CE, Shaifta Y, Snetkov VV, Francois AA, Ward JPT, Knock GA. ROS-dependent activation of RhoA/Rho-kinase in pulmonary artery: Role of Src-family kinases and ARHGEF1. Free Radic Biol Med 2017; 110:316-331. [PMID: 28673614 PMCID: PMC5542024 DOI: 10.1016/j.freeradbiomed.2017.06.022] [Citation(s) in RCA: 39] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/01/2016] [Revised: 06/12/2017] [Accepted: 06/29/2017] [Indexed: 12/11/2022]
Abstract
The role of reactive oxygen species (ROS) in smooth muscle contraction is poorly understood. We hypothesised that G-protein coupled receptor (GPCR) activation and hypoxia induce Rho-kinase activity and contraction in rat intra-pulmonary artery (IPA) via stimulation of ROS production and subsequent Src-family kinase (SrcFK) activation. The T-type prostanoid receptor agonist U46619 induced ROS production in pulmonary artery smooth muscle cells (PASMC). U46619 also induced c-Src cysteine oxidation, SrcFK auto-phosphorylation, MYPT-1 and MLC20 phosphorylation and contraction in IPA, and all these responses were inhibited by antioxidants (ebselen, Tempol). Contraction and SrcFK/MYPT-1/MLC20 phosphorylations were also inhibited by combined superoxide dismutase and catalase, or by the SrcFK antagonist PP2, while contraction and MYPT-1/MLC20 phosphorylations were inhibited by the Rho guanine nucleotide exchange factor (RhoGEF) inhibitor Y16. H2O2 and the superoxide-generating quinoledione LY83583 both induced c-Src oxidation, SrcFK auto-phosphorylation and contraction in IPA. LY83583 and H2O2-induced contractions were inhibited by PP2, while LY83583-induced contraction was also inhibited by antioxidants and Y16. SrcFK auto-phosphorylation and MYPT-1/MLC20 phosphorylation was also induced by hypoxia in IPA and this was blocked by mitochondrial inhibitors rotenone and myxothiazol. In live PASMC, sub-cellular translocation of RhoA and the RhoGEF ARHGEF1 was triggered by both U46619 and LY83583 and this translocation was blocked by antioxidants and PP2. RhoA translocation was also inhibited by an ARHGEF1 siRNA. U46619 enhanced ROS-dependent co-immunoprecipitation of ARHGEF1 with c-Src. Our results demonstrate a link between GPCR-induced cytosolic ROS or hypoxia-induced mitochondrial ROS and SrcFK activity, Rho-kinase activity and contraction. ROS and SrcFK activate RhoA via ARHGEF1.
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Affiliation(s)
- Charles E MacKay
- Asthma, Allergy & Lung Biology, Faculty of Life Sciences & Medicine, King's College London, London, United Kingdom
| | - Yasin Shaifta
- Asthma, Allergy & Lung Biology, Faculty of Life Sciences & Medicine, King's College London, London, United Kingdom
| | - Vladimir V Snetkov
- Asthma, Allergy & Lung Biology, Faculty of Life Sciences & Medicine, King's College London, London, United Kingdom
| | - Asvi A Francois
- Cardiovascular Division, Faculty of Life Sciences & Medicine, King's College London, London, United Kingdom
| | - Jeremy P T Ward
- Asthma, Allergy & Lung Biology, Faculty of Life Sciences & Medicine, King's College London, London, United Kingdom
| | - Greg A Knock
- Asthma, Allergy & Lung Biology, Faculty of Life Sciences & Medicine, King's College London, London, United Kingdom.
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17
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Abstract
Rho GTPases regulate cytoskeletal and cell adhesion dynamics and thereby coordinate a wide range of cellular processes, including cell migration, cell polarity and cell cycle progression. Most Rho GTPases cycle between a GTP-bound active conformation and a GDP-bound inactive conformation to regulate their ability to activate effector proteins and to elicit cellular responses. However, it has become apparent that Rho GTPases are regulated by post-translational modifications and the formation of specific protein complexes, in addition to GTP-GDP cycling. The canonical regulators of Rho GTPases - guanine nucleotide exchange factors, GTPase-activating proteins and guanine nucleotide dissociation inhibitors - are regulated similarly, creating a complex network of interactions to determine the precise spatiotemporal activation of Rho GTPases.
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Affiliation(s)
- Richard G Hodge
- Randall Division of Cell and Molecular Biophysics, King's College London, New Hunt's House, Guy's Campus, London SE1 1UL, UK
| | - Anne J Ridley
- Randall Division of Cell and Molecular Biophysics, King's College London, New Hunt's House, Guy's Campus, London SE1 1UL, UK
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18
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The GNA13-RhoA signaling axis suppresses expression of tumor protective Kallikreins. Cell Signal 2016; 28:1479-88. [PMID: 27424208 DOI: 10.1016/j.cellsig.2016.07.001] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/16/2016] [Revised: 07/04/2016] [Accepted: 07/04/2016] [Indexed: 11/22/2022]
Abstract
Gα13 (encoded by GNA13 gene) is the alpha subunit of a heterotrimeric G-protein that mediates signaling through specific G-protein-coupled receptors (GPCRs). Increased GNA13 expression has been observed in metastatic breast cancer cells. Recently, we have shown that enhanced GNA13 signaling in MCF-10a cells, a benign breast cancer cell line increased its invasiveness. Previous studies have reported that Kallikrein-related peptidases (KLKs 1-15) are down-regulated in breast tumors and may have a tumor protective function. However, the mechanisms that lead to the down-regulation of KLK genes in breast cancer are yet to be elucidated. We found that enhanced GNA13 signaling represses KLK gene expression in breast cancer, and undertook examination of the mechanisms involved. A microarray analysis revealed down-regulation of several members of the Kallikrein-related peptidases (KLK) gene family, namely KLK5, KLK6, KLK7, KLK8 and KLK10, in MCF-10a lines with enhanced GNA13 protein expression. Using real-time PCR and promoter analysis, we identified that the mRNA expression and promoter activities of these KLKs are suppressed upon enforced expression of GNA13 in MCF-10a cells. Using Rhotekin pull-down assays, we identified that GNA13 suppressed Rho-A activation and protein levels in MCF-10a cells. Blocking Rho-A activation using C3-toxin or by inhibiting its down-stream effector, Rho-associated kinase (ROCK), reduced the above-mentioned KLK mRNAs in MCF-10A cells. Importantly, in a metastatic breast cancer cell line MDA-MB-157, knock down of GNA13 alone was sufficient to induce the expression KLK mRNAs. Taken together, our findings suggested that enhanced GNA13 signaling down-regulates KLK gene transcription. The ability of enhanced GNA13 signaling to suppress KLK gene expression appears at least in part due to the ability of enhanced GNA13 signaling to negatively impact Rho/ROCK-signaling.
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Abstract
Rho GTPases are crucial signaling molecules that regulate a plethora of biological functions. Traditional biochemical, cell biological, and genetic approaches have founded the basis of Rho GTPase biology. The development of biosensors then allowed measuring Rho GTPase activity with unprecedented spatio-temporal resolution. This revealed that Rho GTPase activity fluctuates on time and length scales of tens of seconds and micrometers, respectively. In this review, we describe Rho GTPase activity patterns observed in different cell systems. We then discuss the growing body of evidence that upstream regulators such as guanine nucleotide exchange factors and GTPase-activating proteins shape these patterns by precisely controlling the spatio-temporal flux of Rho GTPase activity. Finally, we comment on additional mechanisms that might feed into the regulation of these signaling patterns and on novel technologies required to dissect this spatio-temporal complexity.
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Affiliation(s)
| | - Olivier Pertz
- Department of Biomedicine, University of Basel, Basel, Switzerland; Institute of Cell Biology, University of Bern, Bern, Switzerland
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20
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Bai X, Dee R, Mangum KD, Mack CP, Taylor JM. RhoA signaling and blood pressure: The consequence of failing to “Tone it Down”. World J Hypertens 2016; 6:18-35. [DOI: 10.5494/wjh.v6.i1.18] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/30/2015] [Revised: 11/24/2015] [Accepted: 01/22/2016] [Indexed: 02/06/2023] Open
Abstract
Uncontrolled high blood pressure is a major risk factor for heart attack, stroke, and kidney failure and contributes to an estimated 25% of deaths worldwide. Despite numerous treatment options, estimates project that reasonable blood pressure (BP) control is achieved in only about half of hypertensive patients. Improvements in the detection and management of hypertension will undoubtedly be accomplished through a better understanding of the complex etiology of this disease and a more comprehensive inventory of the genes and genetic variants that influence BP regulation. Recent studies (primarily in pre-clinical models) indicate that the small GTPase RhoA and its downstream target, Rho kinase, play an important role in regulating BP homeostasis. Herein, we summarize the underlying mechanisms and highlight signaling pathways and regulators that impart tight spatial-temporal control of RhoA activity. We also discuss known allelic variations in the RhoA pathway and consider how these polymorphisms may affect genetic risk for hypertension and its clinical manifestations. Finally, we summarize the current (albeit limited) clinical data on the efficacy of targeting the RhoA pathway in hypertensive patients.
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21
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Zhang H, Cooper LF, Zhang X, Zhang Y, Deng F, Song J, Yang S. Titanium nanotubes induce osteogenic differentiation through the FAK/RhoA/YAP cascade. RSC Adv 2016. [DOI: 10.1039/c6ra04002k] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/19/2023] Open
Abstract
TNT topography restricts cell spreading, impairs the FAK recruitment in FAs, and thereby attenuates RhoA activity as well as cytoskeleton formation, which in turn expels YAP from that cell nucleus to the cytoplasm and initiates osteodifferentiation.
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Affiliation(s)
- He Zhang
- Chongqing Key Laboratory for Oral Diseases and Biomedical Sciences
- Chongqing Municipal Key Laboratory of Oral Biomedical Engineering of Higher Education
- College of Stomatology
- Chongqing Medical University
- Chongqing
| | - Lyndon F. Cooper
- Department Head
- Oral Biology
- University of Illinois at Chicago
- College of Dentistry
- Chicago
| | - Xiaonan Zhang
- Chongqing Key Laboratory for Oral Diseases and Biomedical Sciences
- Chongqing Municipal Key Laboratory of Oral Biomedical Engineering of Higher Education
- College of Stomatology
- Chongqing Medical University
- Chongqing
| | - Yi Zhang
- Chongqing Key Laboratory for Oral Diseases and Biomedical Sciences
- Chongqing Municipal Key Laboratory of Oral Biomedical Engineering of Higher Education
- College of Stomatology
- Chongqing Medical University
- Chongqing
| | - Feng Deng
- Chongqing Key Laboratory for Oral Diseases and Biomedical Sciences
- Chongqing Municipal Key Laboratory of Oral Biomedical Engineering of Higher Education
- College of Stomatology
- Chongqing Medical University
- Chongqing
| | - Jinlin Song
- Chongqing Key Laboratory for Oral Diseases and Biomedical Sciences
- Chongqing Municipal Key Laboratory of Oral Biomedical Engineering of Higher Education
- College of Stomatology
- Chongqing Medical University
- Chongqing
| | - Sheng Yang
- Chongqing Key Laboratory for Oral Diseases and Biomedical Sciences
- Chongqing Municipal Key Laboratory of Oral Biomedical Engineering of Higher Education
- College of Stomatology
- Chongqing Medical University
- Chongqing
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22
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Shaifta Y, Irechukwu N, Prieto-Lloret J, MacKay CE, Marchon KA, Ward JPT, Knock GA. Divergent modulation of Rho-kinase and Ca(2+) influx pathways by Src family kinases and focal adhesion kinase in airway smooth muscle. Br J Pharmacol 2015; 172:5265-80. [PMID: 26294392 PMCID: PMC4864488 DOI: 10.1111/bph.13313] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/12/2015] [Revised: 08/02/2015] [Accepted: 08/19/2015] [Indexed: 02/06/2023] Open
Abstract
Background and Purpose The importance of tyrosine kinases in airway smooth muscle (ASM) contraction is not fully understood. The aim of this study was to investigate the role of Src‐family kinases (SrcFK) and focal adhesion kinase (FAK) in GPCR‐mediated ASM contraction and associated signalling events. Experimental Approach Contraction was recorded in intact or α‐toxin permeabilized rat bronchioles. Phosphorylation of SrcFK, FAK, myosin light‐chain‐20 (MLC20) and myosin phosphatase targeting subunit‐1 (MYPT‐1) was evaluated in cultured human ASM cells (hASMC). [Ca2+]i was evaluated in Fura‐2 loaded hASMC. Responses to carbachol (CCh) and bradykinin (BK) and the contribution of SrcFK and FAK to these responses were determined. Key Results Contractile responses in intact bronchioles were inhibited by antagonists of SrcFK, FAK and Rho‐kinase, while after α‐toxin permeabilization, they were sensitive to inhibition of SrcFK and Rho‐kinase, but not FAK. CCh and BK increased phosphorylation of MYPT‐1 and MLC20 and auto‐phosphorylation of SrcFK and FAK. MYPT‐1 phosphorylation was sensitive to inhibition of Rho‐kinase and SrcFK, but not FAK. Contraction induced by SR Ca2+ depletion and equivalent [Ca2+]i responses in hASMC were sensitive to inhibition of both SrcFK and FAK, while depolarization‐induced contraction was sensitive to FAK inhibition only. SrcFK auto‐phosphorylation was partially FAK‐dependent, while FAK auto‐phosphorylation was SrcFK‐independent. Conclusions and Implications SrcFK mediates Ca2+‐sensitization in ASM, while SrcFK and FAK together and individually influence multiple Ca2+ influx pathways. Tyrosine phosphorylation is therefore a key upstream signalling event in ASM contraction and may be a viable target for modulating ASM tone in respiratory disease.
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Affiliation(s)
- Yasin Shaifta
- Division of Asthma, Allergy and Lung Biology, Faculty of Life Sciences and Medicine, King's College London, London, UK
| | - Nneka Irechukwu
- Division of Asthma, Allergy and Lung Biology, Faculty of Life Sciences and Medicine, King's College London, London, UK
| | - Jesus Prieto-Lloret
- Division of Asthma, Allergy and Lung Biology, Faculty of Life Sciences and Medicine, King's College London, London, UK
| | - Charles E MacKay
- Division of Asthma, Allergy and Lung Biology, Faculty of Life Sciences and Medicine, King's College London, London, UK
| | - Keisha A Marchon
- Division of Asthma, Allergy and Lung Biology, Faculty of Life Sciences and Medicine, King's College London, London, UK
| | - Jeremy P T Ward
- Division of Asthma, Allergy and Lung Biology, Faculty of Life Sciences and Medicine, King's College London, London, UK
| | - Greg A Knock
- Division of Asthma, Allergy and Lung Biology, Faculty of Life Sciences and Medicine, King's College London, London, UK
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Helms MC, Grabocka E, Martz MK, Fischer CC, Suzuki N, Wedegaertner PB. Mitotic-dependent phosphorylation of leukemia-associated RhoGEF (LARG) by Cdk1. Cell Signal 2015; 28:43-52. [PMID: 26483157 DOI: 10.1016/j.cellsig.2015.10.004] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/08/2015] [Accepted: 10/15/2015] [Indexed: 01/14/2023]
Abstract
Rho GTPases are integral to the regulation of actin cytoskeleton-dependent processes, including mitosis. Rho and leukemia-associated Rho guanine-nucleotide exchange factor (LARG), also known as ARHGEF12, are involved in mitosis as well as diseases such as cancer and heart disease. Since LARG has a role in mitosis and diverse signaling functions beyond mitosis, it is important to understand the regulation of the protein through modifications such as phosphorylation. Here we report that LARG undergoes a mitotic-dependent and cyclin-dependent kinase 1 (Cdk1) inhibitor-sensitive phosphorylation. Additionally, LARG is phosphorylated at the onset of mitosis and dephosphorylated as cells exit mitosis, concomitant with Cdk1 activity. Furthermore, using an in vitro kinase assay, we show that LARG can be directly phosphorylated by Cdk1. Through expression of phosphonull mutants that contain non-phosphorylatable alanine mutations at potential Cdk1 S/TP sites, we demonstrate that LARG phosphorylation occurs in both termini. Using phosphospecific antibodies, we confirm that two sites, serine 190 and serine 1176, are phosphorylated during mitosis in a Cdk1-dependent manner. In addition, these phosphospecific antibodies show phosphorylated LARG at specific mitotic locations, namely the mitotic organizing centers and flanking the midbody. Lastly, RhoA activity assays reveal that phosphonull LARG is more active in cells than phosphomimetic LARG. Our data thus identifies LARG as a phosphoregulated RhoGEF during mitosis.
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Affiliation(s)
- Michelle C Helms
- Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA, USA
| | - Elda Grabocka
- Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA, USA
| | - Matthew K Martz
- Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA, USA
| | - Christopher C Fischer
- Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA, USA
| | - Nobuchika Suzuki
- Department of Signal Dynamics, University of Tokyo, Meguro, Tokyo, Japan
| | - Philip B Wedegaertner
- Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA, USA.
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Dunn HA, Ferguson SSG. PDZ Protein Regulation of G Protein-Coupled Receptor Trafficking and Signaling Pathways. Mol Pharmacol 2015; 88:624-39. [PMID: 25808930 DOI: 10.1124/mol.115.098509] [Citation(s) in RCA: 89] [Impact Index Per Article: 8.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/18/2015] [Accepted: 03/25/2015] [Indexed: 02/14/2025] Open
Abstract
G protein-coupled receptors (GPCRs) contribute to the regulation of every aspect of human physiology and are therapeutic targets for the treatment of numerous diseases. As a consequence, understanding the myriad of mechanisms controlling GPCR signaling and trafficking is essential for the development of new pharmacological strategies for the treatment of human pathologies. Of the many GPCR-interacting proteins, postsynaptic density protein of 95 kilodaltons, disc large, zona occludens-1 (PDZ) domain-containing proteins appear most abundant and have similarly been implicated in disease mechanisms. PDZ proteins play an important role in regulating receptor and channel protein localization within synapses and tight junctions and function to scaffold intracellular signaling protein complexes. In the current study, we review the known functional interactions between PDZ domain-containing proteins and GPCRs and provide insight into the potential mechanisms of action. These PDZ domain-containing proteins include the membrane-associated guanylate-like kinases [postsynaptic density protein of 95 kilodaltons; synapse-associated protein of 97 kilodaltons; postsynaptic density protein of 93 kilodaltons; synapse-associated protein of 102 kilodaltons; discs, large homolog 5; caspase activation and recruitment domain and membrane-associated guanylate-like kinase domain-containing protein 3; membrane protein, palmitoylated 3; calcium/calmodulin-dependent serine protein kinase; membrane-associated guanylate kinase protein (MAGI)-1, MAGI-2, and MAGI-3], Na(+)/H(+) exchanger regulatory factor proteins (NHERFs) (NHERF1, NHERF2, PDZ domain-containing kidney protein 1, and PDZ domain-containing kidney protein 2), Golgi-associated PDZ proteins (Gα-binding protein interacting protein, C-terminus and CFTR-associated ligand), PDZ domain-containing guanine nucleotide exchange factors (GEFs) 1 and 2, regulator of G protein signaling (RGS)-homology-RhoGEFs (PDZ domain-containing RhoGEF and leukemia-associated RhoGEF), RGS3 and RGS12, spinophilin and neurabin-1, SRC homology 3 domain and multiple ankyrin repeat domain (Shank) proteins (Shank1, Shank2, and Shank3), partitioning defective proteins 3 and 6, multiple PDZ protein 1, Tamalin, neuronal nitric oxide synthase, syntrophins, protein interacting with protein kinase C α 1, syntenin-1, and sorting nexin 27.
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Affiliation(s)
- Henry A Dunn
- J. Allyn Taylor Centre for Cell Biology, Robarts Research Institute, and the Department of Physiology and Pharmacology, University of Western Ontario, London, Ontario, Canada
| | - Stephen S G Ferguson
- J. Allyn Taylor Centre for Cell Biology, Robarts Research Institute, and the Department of Physiology and Pharmacology, University of Western Ontario, London, Ontario, Canada
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25
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Roche PL, Filomeno KL, Bagchi RA, Czubryt MP. Intracellular Signaling of Cardiac Fibroblasts. Compr Physiol 2015; 5:721-60. [DOI: 10.1002/cphy.c140044] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023]
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Goicoechea SM, Awadia S, Garcia-Mata R. I'm coming to GEF you: Regulation of RhoGEFs during cell migration. Cell Adh Migr 2014; 8:535-49. [PMID: 25482524 PMCID: PMC4594598 DOI: 10.4161/cam.28721] [Citation(s) in RCA: 67] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
Cell migration is a highly regulated multistep process that requires the coordinated regulation of cell adhesion, protrusion, and contraction. These processes require numerous protein–protein interactions and the activation of specific signaling pathways. The Rho family of GTPases plays a key role in virtually every aspect of the cell migration cycle. The activation of Rho GTPases is mediated by a large and diverse family of proteins; the guanine nucleotide exchange factors (RhoGEFs). GEFs work immediately upstream of Rho proteins to provide a direct link between Rho activation and cell–surface receptors for various cytokines, growth factors, adhesion molecules, and G protein-coupled receptors. The regulated targeting and activation of RhoGEFs is essential to coordinate the migratory process. In this review, we summarize the recent advances in our understanding of the role of RhoGEFs in the regulation of cell migration.
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Key Words
- DH, Dbl-homology
- DHR, DOCK homology region
- DOCK, dedicator of cytokinesis
- ECM, extracellular matrix
- EGF, epidermal growth factor
- FA, focal adhesion
- FN, fibronectin
- GAP, GTPase activating protein
- GDI, guanine nucleotide dissociation inhibitor
- GEF, guanine nucleotide exchange factor
- GPCR, G protein-coupled receptor
- HGF, hepatocyte growth factor
- LPA, lysophosphatidic acid
- MII, myosin II
- PA, phosphatidic acid
- PDGF, platelet-derived growth factor
- PH, pleckstrin-homology
- PIP2, phosphatidylinositol 4, 5-bisphosphate
- PIP3, phosphatidylinositol (3, 4, 5)-trisphosphate.
- Rho GEFs
- Rho GTPases
- bFGF, basic fibroblast growth factor
- cell migration
- cell polarization
- focal adhesions
- guanine nucleotide exchange factors
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Affiliation(s)
- Silvia M Goicoechea
- a Department of Biological Sciences ; University of Toledo ; Toledo , OH USA
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Datla SR, McGrail DJ, Vukelic S, Huff LP, Lyle AN, Pounkova L, Lee M, Seidel-Rogol B, Khalil MK, Hilenski LL, Terada LS, Dawson MR, Lassègue B, Griendling KK. Poldip2 controls vascular smooth muscle cell migration by regulating focal adhesion turnover and force polarization. Am J Physiol Heart Circ Physiol 2014; 307:H945-57. [PMID: 25063792 DOI: 10.1152/ajpheart.00918.2013] [Citation(s) in RCA: 49] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
Abstract
Polymerase-δ-interacting protein 2 (Poldip2) interacts with NADPH oxidase 4 (Nox4) and regulates migration; however, the precise underlying mechanisms are unclear. Here, we investigated the role of Poldip2 in focal adhesion turnover, as well as traction force generation and polarization. Poldip2 overexpression (AdPoldip2) in vascular smooth muscle cells (VSMCs) impairs PDGF-induced migration and induces a characteristic phenotype of long cytoplasmic extensions. AdPoldip2 also prevents the decrease in spreading and increased aspect ratio observed in response to PDGF and slightly impairs cell contraction. Moreover, AdPoldip2 blocks focal adhesion dissolution and sustains H2O2 levels in focal adhesions, whereas Poldip2 knockdown (siPoldip2) significantly decreases the number of focal adhesions. RhoA activity is unchanged when focal adhesion dissolution is stimulated in control cells but increases in AdPoldip2-treated cells. Inhibition of RhoA blocks Poldip2-mediated attenuation of focal adhesion dissolution, and overexpression of RhoA or focal adhesion kinase (FAK) reverses the loss of focal adhesions induced by siPoldip2, indicating that RhoA and FAK mediate the effect of Poldip2 on focal adhesions. Nox4 silencing prevents focal adhesion stabilization by AdPoldip2 and induces a phenotype similar to siPoldip2, suggesting a role for Nox4 in Poldip2-induced focal adhesion stability. As a consequence of impaired focal adhesion turnover, PDGF-treated AdPoldip2 cells are unable to reduce and polarize traction forces, a necessary first step in migration. These results implicate Poldip2 in VSMC migration via regulation of focal adhesion turnover and traction force generation in a Nox4/RhoA/FAK-dependent manner.
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Affiliation(s)
- Srinivasa Raju Datla
- Department of Medicine, Division of Cardiology, Emory University School of Medicine, Atlanta
| | | | - Sasa Vukelic
- Department of Medicine, Division of Cardiology, Emory University School of Medicine, Atlanta
| | - Lauren P Huff
- Department of Medicine, Division of Cardiology, Emory University School of Medicine, Atlanta
| | - Alicia N Lyle
- Department of Medicine, Division of Cardiology, Emory University School of Medicine, Atlanta
| | - Lily Pounkova
- Department of Medicine, Division of Cardiology, Emory University School of Medicine, Atlanta
| | - Minyoung Lee
- Department of Medicine, Division of Cardiology, Emory University School of Medicine, Atlanta
| | - Bonnie Seidel-Rogol
- Department of Medicine, Division of Cardiology, Emory University School of Medicine, Atlanta
| | - Mazen K Khalil
- Department of Medicine, Division of Cardiology, Emory University School of Medicine, Atlanta
| | - Lula L Hilenski
- Department of Medicine, Division of Cardiology, Emory University School of Medicine, Atlanta
| | - Lance S Terada
- Department of Internal Medicine, Division of Pulmonary and Critical Care, University of Texas Southwestern Medical Center, Dallas, Texas
| | - Michelle R Dawson
- Department of Chemical and Biomolecular Engineering and The Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, Georgia
| | - Bernard Lassègue
- Department of Medicine, Division of Cardiology, Emory University School of Medicine, Atlanta
| | - Kathy K Griendling
- Department of Medicine, Division of Cardiology, Emory University School of Medicine, Atlanta;
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Bkca opener, NS1619 pretreatment protects against shock-induced vascular hyporeactivity through PDZ-Rho GEF-RhoA-Rho kinase pathway in rats. J Trauma Acute Care Surg 2014; 76:394-401. [PMID: 24398773 DOI: 10.1097/ta.0b013e3182aa2d98] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
Abstract
BACKGROUND Our previous study showed that the ischemic preconditioning and pretreatment of adenosine triphosphate-sensitive potassium channel (KATP) opener, pinacidil, may induce a good protective effect on shock-induced vascular hyporeactivity. Whether the pretreatment of opener/activator of the large-conductance calcium-activated potassium channel (Bkca), NS1619, can also induce a protective effect on vascular reactivity and play a beneficial effect on subsequent hemorrhagic shock is not clear. METHODS With Sprague-Dawley rats subjected to hemorrhagic shock and their isolated superior mesenteric artery, the protective effect of NS1619 (0.5, 1, 2, and 4 mg/kg) pretreatment (30 minutes before hemorrhage shock) on vascular reactivity and the underlying mechanisms were observed. RESULTS NS1619 pretreatment significantly improved the 72-hour survival of hemorrhagic shock rats, alleviated shock-induced decrease of vascular reactivity and calcium sensitivity, and increased the cardiac output and oxygen delivery. NS1619 2 mg/kg had the best effect. These protective effects of NS1619 pretreatment on vascular reactivity and calcium sensitivity were antagonized by RhoA inhibitor, C3 transferase, and Rho kinase antagonist, Y-27632. NS1619 pretreatment up-regulated the activities of RhoA, Rho-kinase, and PDZ-Rho GEF (guanine nucleotide exchange factor). These effects of NS1619 pretreatment were eliminated by RhoA inhibitor, C3 transferase. CONCLUSION Bkca opener, NS1619 pretreatment has good protective effect on vascular reactivity and calcium sensitivity, which plays a good beneficial effect on hemorrhagic shock. The mechanism may be mainly through PDZ-Rho GEF-RhoA-Rho kinase pathway. Bkca channel may be a potential target for the treatment of shock-induced vascular hyporeactivity.
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Lessey-Morillon EC, Osborne LD, Monaghan-Benson E, Guilluy C, O'Brien ET, Superfine R, Burridge K. The RhoA guanine nucleotide exchange factor, LARG, mediates ICAM-1-dependent mechanotransduction in endothelial cells to stimulate transendothelial migration. THE JOURNAL OF IMMUNOLOGY 2014; 192:3390-8. [PMID: 24585879 DOI: 10.4049/jimmunol.1302525] [Citation(s) in RCA: 46] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/13/2023]
Abstract
RhoA-mediated cytoskeletal rearrangements in endothelial cells (ECs) play an active role in leukocyte transendothelial cell migration (TEM), a normal physiological process in which leukocytes cross the endothelium to enter the underlying tissue. Although much has been learned about RhoA signaling pathways downstream from ICAM-1 in ECs, little is known about the consequences of the tractional forces that leukocytes generate on ECs as they migrate over the surface before TEM. We have found that after applying mechanical forces to ICAM-1 clusters, there is an increase in cellular stiffening and enhanced RhoA signaling compared with ICAM-1 clustering alone. We have identified that leukemia-associated Rho guanine nucleotide exchange factor (LARG), also known as Rho GEF 12 (ARHGEF12) acts downstream of clustered ICAM-1 to increase RhoA activity, and that this pathway is further enhanced by mechanical force on ICAM-1. Depletion of LARG decreases leukocyte crawling and inhibits TEM. To our knowledge, this is the first report of endothelial LARG regulating leukocyte behavior and EC stiffening in response to tractional forces generated by leukocytes.
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Affiliation(s)
- Elizabeth C Lessey-Morillon
- Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599
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30
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Sato K, Sugiyama T, Nagase T, Kitade Y, Ueda H. Threonine 680 phosphorylation of FLJ00018/PLEKHG2, a Rho family-specific guanine nucleotide exchange factor, by epidermal growth factor receptor signaling regulates cell morphology of Neuro-2a cells. J Biol Chem 2014; 289:10045-56. [PMID: 24554703 DOI: 10.1074/jbc.m113.521880] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
FLJ00018/PLEKHG2 is a guanine nucleotide exchange factor for the small GTPases Rac and Cdc42 and has been shown to mediate the signaling pathways leading to actin cytoskeleton reorganization. The function of FLJ00018 is regulated by the interaction of heterotrimeric GTP-binding protein Gβγ subunits or cytosolic actin. However, the details underlying the molecular mechanisms of FLJ00018 activation have yet to be elucidated. In the present study we show that FLJ00018 is phosphorylated and activated by β1-adrenergic receptor stimulation-induced EGF receptor (EGFR) transactivation in addition to Gβγ signaling. FLJ00018 is also phosphorylated and activated by direct EGFR stimulation. The phosphorylation of FLJ00018 by EGFR stimulation is mediated by the Ras/mitogen-activated protein kinase (MAPK) pathway. Through deletion and site-directed mutagenesis studies, we have identified Thr-680 as the major site of phosphorylation by EGFR stimulation. FLJ00018 T680A, in which the phosphorylation site is replaced by alanine, showed a limited response of the Neuro-2a cell morphology to EGF stimulation. Our results provide evidence that stimulation of the Ras/MAPK pathway by EGFR results in FLJ00018 phosphorylation at Thr-680, which in turn controls changes in cell shape.
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Affiliation(s)
- Katsuya Sato
- From the United Graduate School of Drug Discovery and Medical Information Sciences and
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31
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Kwon MS, Park BO, Kim HM, Kim S. Leucine-rich repeat-containing G-protein coupled receptor 5/GPR49 activates G12/13-Rho GTPase pathway. Mol Cells 2013; 36:267-72. [PMID: 23912594 PMCID: PMC3887977 DOI: 10.1007/s10059-013-0173-z] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/05/2013] [Revised: 06/27/2013] [Accepted: 06/27/2013] [Indexed: 10/26/2022] Open
Abstract
Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5/GPR49) is highly expressed in adult stem cells of various tissues, such as intestine, hair follicles, and stomach. LGR5 is also overexpressed in some colon and ovarian tumors. Recent reports show that R-spondin (RSPO) family ligands bind to and activate LGR5, enhancing canonical Wnt signaling via the interaction with LRP5/6 and Frizzled. The identity of heterotrimeric G-proteins coupled to LGR5, however, remains unclear. Here, we show that Rho GTPase is a downstream target of LGR5. Overexpression of LGR5 induced SRF-RE luciferase activity, a reporter of Rho signaling. RSPOs, ligands for LGR4, LGR5, and LGR6, however, did not induce SRF-RE reporter activity in the presence of LGR5. Consistently, LGR5-induced activity of the SRF-RE reporter was inhibited by Rho inhibitor C3 transferase and RhoA N19 mutant, and knockdown of Gα12/13 genes blocked the reporter activity induced by LGR5. In addition, focal adhesion kinase, NF-κB and c-fos, targets of Rho GTPase, were shown to be regulated by LGR5. Here, we have demonstrated, for the first time, that LGR5 is coupled to the Rho pathway through G12/13 signaling.
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Affiliation(s)
- Mi So Kwon
- Targeted Medicine Research Center, Korea Research Institute of Bioscience and Biotechnology, Cheongwon 363-883, Korea
- Biomolecular Science Major, University of Science and Technology, Daejeon 305-350, Korea
| | - Bi-oh Park
- Targeted Medicine Research Center, Korea Research Institute of Bioscience and Biotechnology, Cheongwon 363-883, Korea
| | - Ho Min Kim
- Graduate School of Medical Science and Engineering, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Korea
| | - Sunhong Kim
- Targeted Medicine Research Center, Korea Research Institute of Bioscience and Biotechnology, Cheongwon 363-883, Korea
- Biomolecular Science Major, University of Science and Technology, Daejeon 305-350, Korea
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Struckhoff AP, Rana MK, Kher SS, Burow ME, Hagan JL, Del Valle L, Worthylake RA. PDZ-RhoGEF is essential for CXCR4-driven breast tumor cell motility through spatial regulation of RhoA. J Cell Sci 2013; 126:4514-26. [PMID: 23868972 DOI: 10.1242/jcs.132381] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022] Open
Abstract
The CXCL12-CXCR4 chemokine signaling pathway is a well-established driver of cancer progression. One key process promoted by CXCR4 stimulation is tumor cell motility; however, the specific signaling pathways leading to migration remain poorly understood. Previously, we have shown that CXCL12 stimulation of migration depends on temporal regulation of RhoA. However, the specific RhoGEF that translates CXCR4 signaling into RhoA activity and cell motility is unknown. We screened the three regulator of G-protein signaling RhoGEFs (LSC, LARG and PRG) and found that PRG selectively regulated the migration and invasion of CXCR4-overexpressing breast tumor cells. Interestingly, we found that PDZ-RhoGEF (PRG) was required for spatial organization of F-actin structures in the center, but not periphery of the cells. The effects on the cytoskeleton were mirrored by the spatial effects on RhoA activity that were dependent upon PRG. Loss of PRG also enhanced adherens junctions in the epithelial-like MCF7-CXCR4 cell line, and inhibited directional persistence and polarity in the more mesenchymal MDA-MB-231 cell line. Thus, PRG is essential for CXCR4-driven tumor cell migration through spatial regulation of RhoA and the subsequent organization of the cytoskeletal structures that support motility. Furthermore, immunohistochemical analysis of human breast tumor tissues shows a significant increase of PRG expression in the invasive areas of the tumors, suggesting that this RhoGEF is associated with breast tumor invasion in vivo.
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Affiliation(s)
- Amanda P Struckhoff
- Stanley S. Scott Cancer Center, Louisiana State University Health, New Orleans, Louisiana, USA
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33
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Schmidt TT, Tauseef M, Yue L, Bonini MG, Gothert J, Shen TL, Guan JL, Predescu S, Sadikot R, Mehta D. Conditional deletion of FAK in mice endothelium disrupts lung vascular barrier function due to destabilization of RhoA and Rac1 activities. Am J Physiol Lung Cell Mol Physiol 2013; 305:L291-300. [PMID: 23771883 DOI: 10.1152/ajplung.00094.2013] [Citation(s) in RCA: 49] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022] Open
Abstract
Loss of lung-fluid homeostasis is the hallmark of acute lung injury (ALI). Association of catenins and actin cytoskeleton with vascular endothelial (VE)-cadherin is generally considered the main mechanism for stabilizing adherens junctions (AJs), thereby preventing disruption of lung vascular barrier function. The present study identifies endothelial focal adhesion kinase (FAK), a nonreceptor tyrosine kinase that canonically regulates focal adhesion turnover, as a novel AJ-stabilizing mechanism. In wild-type mice, induction of ALI by intraperitoneal administration of lipopolysaccharide or cecal ligation and puncture markedly decreased FAK expression in lungs. Using a mouse model in which FAK was conditionally deleted only in endothelial cells (ECs), we show that loss of EC-FAK mimicked key features of ALI (diffuse lung hemorrhage, increased transvascular albumin influx, edema, and neutrophil accumulation in the lung). EC-FAK deletion disrupted AJs due to impairment of the fine balance between the activities of RhoA and Rac1 GTPases. Deletion of EC-FAK facilitated RhoA's interaction with p115-RhoA guanine exchange factor, leading to activation of RhoA. Activated RhoA antagonized Rac1 activity, destabilizing AJs. Inhibition of Rho kinase, a downstream effector of RhoA, reinstated normal endothelial barrier function in FAK-/- ECs and lung vascular integrity in EC-FAK-/- mice. Our findings demonstrate that EC-FAK plays an essential role in maintaining AJs and thereby lung vascular barrier function by establishing the normal balance between RhoA and Rac1 activities.
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Affiliation(s)
- Tracy Thennes Schmidt
- Dept. of Pharmacology, The Univ. of Illinois, College of Medicine, 835 S. Wolcott Ave., Chicago, IL 60612.
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Mikelis CM, Palmby TR, Simaan M, Li W, Szabo R, Lyons R, Martin D, Yagi H, Fukuhara S, Chikumi H, Galisteo R, Mukouyama YS, Bugge TH, Gutkind JS. PDZ-RhoGEF and LARG are essential for embryonic development and provide a link between thrombin and LPA receptors and Rho activation. J Biol Chem 2013; 288:12232-43. [PMID: 23467409 DOI: 10.1074/jbc.m112.428599] [Citation(s) in RCA: 50] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
G protein-coupled receptors (GPCRs) linked to both members of the Gα12 family of heterotrimeric G proteins α subunits, Gα12 and Gα13, regulate the activation of Rho GTPases, thereby contributing to many key biological processes. Multiple Rho GEFs have been proposed to link Gα12/13 GPCRs to Rho activation, including PDZ-RhoGEF (PRG), leukemia-associated Rho GEF (LARG), p115-RhoGEF (p115), lymphoid blast crisis (Lbc), and Dbl. PRG, LARG, and p115 share the presence of a regulator of G protein signaling homology (RGS) domain. There is limited information on the biological roles of this RGS-containing family of RhoGEFs in vivo. p115-deficient mice are viable with some defects in the immune system and gastrointestinal motor dysfunctions, whereas in an initial study we showed that mice deficient for Larg are viable and resistant to salt-induced hypertension. Here, we generated knock-out mice for Prg and observed that these mice do not display any overt phenotype. However, deficiency in Prg and Larg leads to complex developmental defects and early embryonic lethality. Signaling from Gα11/q-linked GPCRs to Rho was not impaired in mouse embryonic fibroblasts defective in all three RGS-containing RhoGEFs. However, a combined lack of Prg, Larg, and p115 expression abolished signaling through Gα12/13 to Rho and thrombin-induced cell proliferation, directional migration, and nuclear signaling through JNK and p38. These findings provide evidence of an essential role for the RGS-containing RhoGEF family in signaling to Rho by Gα12/13-coupled GPCRs, which may likely play a critical role during embryonic development.
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Affiliation(s)
- Constantinos M Mikelis
- Oral and Pharyngeal Cancer Branch, NIDCR, Genetics and Developmental Biology Center, NHLBI, National Institutes of Health, Bethesda, Maryland 20892, USA
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Boulter E, Estrach S, Errante A, Pons C, Cailleteau L, Tissot F, Meneguzzi G, Féral CC. CD98hc (SLC3A2) regulation of skin homeostasis wanes with age. ACTA ACUST UNITED AC 2013; 210:173-90. [PMID: 23296466 PMCID: PMC3549711 DOI: 10.1084/jem.20121651] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
Loss of CD98hc expression in young adult skin induces changes similar to those associated with aging, including improper skin homeostasis and epidermal wound healing. Skin aging is linked to reduced epidermal proliferation and general extracellular matrix atrophy. This involves factors such as the cell adhesion receptors integrins and amino acid transporters. CD98hc (SLC3A2), a heterodimeric amino acid transporter, modulates integrin signaling in vitro. We unravel CD98hc functions in vivo in skin. We report that CD98hc invalidation has no appreciable effect on cell adhesion, clearly showing that CD98hc disruption phenocopies neither CD98hc knockdown in cultured keratinocytes nor epidermal β1 integrin loss in vivo. Instead, we show that CD98hc deletion in murine epidermis results in improper skin homeostasis and epidermal wound healing. These defects resemble aged skin alterations and correlate with reduction of CD98hc expression observed in elderly mice. We also demonstrate that CD98hc absence in vivo induces defects as early as integrin-dependent Src activation. We decipher the molecular mechanisms involved in vivo by revealing a crucial role of the CD98hc/integrins/Rho guanine nucleotide exchange factor (GEF) leukemia-associated RhoGEF (LARG)/RhoA pathway in skin homeostasis. Finally, we demonstrate that the deregulation of RhoA activation in the absence of CD98hc is also a result of impaired CD98hc-dependent amino acid transports.
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Affiliation(s)
- Etienne Boulter
- Institute for Research on Cancer and Aging, Nice, AVENIR Team, University of Nice Sophia-Antipolis, Institut National de la Santé et de la Recherche Médicale U1081, Centre National de la Recherche Scientifique UMR 7284, Centre Antoine Lacassagne, Nice 06107, France
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Abstract
Spatio-temporal control of RhoA GTPase is critical for regulation of cell migration, attachment to extracellular matrix, and cell-cell adhesions. Activation of RhoA is mediated by guanine nucleotide exchange factors (GEFs), a diverse family of enzymes that are controlled by multiple signaling pathways regulating actin cytoskeleton and cell migration. GEFs can be regulated by different mechanisms. Growing evidence demonstrates that phosphorylation serves as one of the predominant signals controlling activity, interactions, and localization of RhoGEFs. It acts as a positive and a negative regulator, and allows for regulation of RhoGEFs by multiple signaling cascades. Although there are common trends in phosphorylation-mediated regulation of some RhoGEF homologs, the majority of GEFs utilize distinct mechanisms that are dictated by their unique structure and interaction networks. This diversity enables multiple signaling pathways to use different RhoGEFs for regulation of a single central-RhoA. Here, we review current examples of phosphorylation-mediated regulation of GEFs for RhoA and its role in cell migration, discuss mechanisms, and provide insights into potential future directions.
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Affiliation(s)
- Maulik Patel
- Department of Pharmacology; University of Illinois at Chicago; Chicago, IL USA
| | - Andrei V Karginov
- Department of Pharmacology; University of Illinois at Chicago; Chicago, IL USA
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Wu X, Chen H, Gao Q, Bai J, Wang X, Zhou J, Qiu S, Xu Y, Shi Y, Wang X, Zhou J, Fan J. Downregulation of JWA promotes tumor invasion and predicts poor prognosis in human hepatocellular carcinoma. Mol Carcinog 2012; 53:325-36. [PMID: 23169062 DOI: 10.1002/mc.21981] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/05/2012] [Revised: 10/17/2012] [Accepted: 10/22/2012] [Indexed: 12/13/2022]
Abstract
We previously identified JWA as a novel microtubule-associated protein (MAP), which is implicated in carcinogenesis and tumor progression. The aims of the present study were to investigate the biological action and the prognostic significance of JWA in hepatocellular carcinoma (HCC). Quantitative real-time PCR and Western blot were used to detect JWA mRNA and protein expression, respectively, in stepwise metastatic HCC cell lines and HCC tissues. Short hairpin RNA was used to inhibit JWA expression in HCC cells. The effects of JWA depletion on cell migration, invasion, adhesion and in vivo metastasis were investigated. Immunohistochemistry of JWA was conducted in microarrays with tissue from 314 HCC patients who had undergone surgical resection. Prognostic significance was assessed using the Kaplan-Meier method and log-rank tests. The result showed JWA expression was decreased in the highly metastatic HCC cell lines and HCC tissues. Depletion of JWA caused a notable increase in cell migration, invasion and adhesion in vitro and metastasis in vivo. Furthermore, there was an inverse correlation between JWA expression and FAK expression and phosphorylation, RhoA activation and matrix metalloproteinase-2 (MMP-2) activity in HCC cells. More notably, multivariate analysis revealed that a low level of JWA expression was an independent prognosticator for both recurrence-free and overall survival for HCC patients after surgical resection, especially for AFP-normal HCC patients. Taken together, our data demonstrate that JWA plays a crucial role in HCC progression and suggest JWA may be a potential prognostic biomarker and therapeutic target for HCC.
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Affiliation(s)
- Xiaofeng Wu
- Liver Transplantation Center, First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu Province, People's Republic of, China
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Kach J, Sethakorn N, Dulin NO. A finer tuning of G-protein signaling through regulated control of RGS proteins. Am J Physiol Heart Circ Physiol 2012; 303:H19-35. [PMID: 22542620 DOI: 10.1152/ajpheart.00764.2011] [Citation(s) in RCA: 40] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
Regulators of G-protein signaling (RGS) proteins are GTPase-activating proteins (GAP) for various Gα subunits of heterotrimeric G proteins. Through this mechanism, RGS proteins regulate the magnitude and duration of G-protein-coupled receptor signaling and are often referred to as fine tuners of G-protein signaling. Increasing evidence suggests that RGS proteins themselves are regulated through multiple mechanisms, which may provide an even finer tuning of G-protein signaling and crosstalk between G-protein-coupled receptors and other signaling pathways. This review summarizes the current data on the control of RGS function through regulated expression, intracellular localization, and covalent modification of RGS proteins, as related to cell function and the pathogenesis of diseases.
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Affiliation(s)
- Jacob Kach
- Department of Medicine, University of Chicago, Illinois, 60637, USA
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39
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Sun T, Krishnan R, Swiercz JM. Grb2 mediates semaphorin-4D-dependent RhoA inactivation. J Cell Sci 2012; 125:3557-67. [PMID: 22505611 DOI: 10.1242/jcs.101063] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Signaling through the semaphorin 4D (Sema4D) receptor plexin-B1 is modulated by its interaction with tyrosine kinases ErbB-2 and Met. In cells expressing the plexin-B1-ErbB-2 receptor complex, ligand stimulation results in the activation of small GTPase RhoA and stimulation of cellular migration. By contrast, in cells expressing plexin-B1 and Met, ligand stimulation results in an association with the RhoGTPase-activating protein p190 RhoGAP and subsequent RhoA inactivation--a process that involves the tyrosine phosphorylation of plexin-B1 by Met. Inactivation of RhoA is necessary for Sema4D-mediated inhibition of cellular migration. It is, however, unknown how plexin-B1 phosphorylation regulates RhoGAP interaction and activity. Here we show that the activation of plexin-B1 by Sema4D and its subsequent tyrosine phosphorylation by Met creates a docking site for the SH2 domain of growth factor receptor bound-2 (Grb2). Grb2 is thereby recruited into the plexin-B1 receptor complex and, through its SH3 domain, interacts with p190 RhoGAP and mediates RhoA deactivation. Phosphorylation of plexin-B1 by Met and the recruitment of Grb2 have no effect on the R-RasGAP activity of plexin-B1, but are required for Sema4D-induced, RhoA-dependent antimigratory effects of Sema4D on breast cancer cells. These data show Grb2 as a direct link between plexin and p190-RhoGAP-mediated downstream signaling.
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Affiliation(s)
- Tianliang Sun
- Department of Pharmacology, Max-Planck Institute for Heart and Lung Research, Ludwigstr 43, 61231 Bad Nauheim, Germany
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40
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Lock FE, Ryan KR, Poulter NS, Parsons M, Hotchin NA. Differential regulation of adhesion complex turnover by ROCK1 and ROCK2. PLoS One 2012; 7:e31423. [PMID: 22348083 PMCID: PMC3278444 DOI: 10.1371/journal.pone.0031423] [Citation(s) in RCA: 67] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/03/2011] [Accepted: 01/07/2012] [Indexed: 01/09/2023] Open
Abstract
BACKGROUND ROCK1 and ROCK2 are serine/threonine kinases that function downstream of the small GTP-binding protein RhoA. Rho signalling via ROCK regulates a number of cellular functions including organisation of the actin cytoskeleton, cell adhesion and cell migration. METHODOLOGY/PRINCIPAL FINDINGS In this study we use RNAi to specifically knockdown ROCK1 and ROCK2 and analyse their role in assembly of adhesion complexes in human epidermal keratinocytes. We observe that loss of ROCK1 inhibits signalling via focal adhesion kinase resulting in a failure of immature adhesion complexes to form mature stable focal adhesions. In contrast, loss of ROCK2 expression results in a significant reduction in adhesion complex turnover leading to formation of large, stable focal adhesions. Interestingly, loss of either ROCK1 or ROCK2 expression significantly impairs cell migration indicating both ROCK isoforms are required for normal keratinocyte migration. CONCLUSIONS ROCK1 and ROCK2 have distinct and separate roles in adhesion complex assembly and turnover in human epidermal keratinocytes.
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Affiliation(s)
- Frances E. Lock
- School of Biosciences, University of Birmingham, Edgbaston, United Kingdom
| | - Katie R. Ryan
- School of Biosciences, University of Birmingham, Edgbaston, United Kingdom
| | - Natalie S. Poulter
- School of Biosciences, University of Birmingham, Edgbaston, United Kingdom
| | - Maddy Parsons
- Randall Division of Cell and Molecular Biophysics, King's College London, London, United Kingdom
| | - Neil A. Hotchin
- School of Biosciences, University of Birmingham, Edgbaston, United Kingdom
- * E-mail:
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Badgwell DB, Lu Z, Le K, Gao F, Yang M, Suh GK, Bao JJ, Das P, Andreeff M, Chen W, Yu Y, Ahmed AA, Liao WSL, Bast RC. The tumor-suppressor gene ARHI (DIRAS3) suppresses ovarian cancer cell migration through inhibition of the Stat3 and FAK/Rho signaling pathways. Oncogene 2012; 31:68-79. [PMID: 21643014 PMCID: PMC3170676 DOI: 10.1038/onc.2011.213] [Citation(s) in RCA: 72] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/23/2010] [Revised: 03/13/2011] [Accepted: 04/11/2011] [Indexed: 01/01/2023]
Abstract
Ovarian cancers migrate and metastasize over the surface of the peritoneal cavity. Consequently, dysregulation of mechanisms that limit cell migration may be particularly important in the pathogenesis of the disease. ARHI is an imprinted tumor-suppressor gene that is downregulated in >60% of ovarian cancers, and its loss is associated with decreased progression-free survival. ARHI encodes a 26-kDa GTPase with homology to Ras. In contrast to Ras, ARHI inhibits cell growth, but whether it also regulates cell motility has not been studied previously. Here we report that re-expression of ARHI decreases the motility of IL-6- and epidermal growth factor (EGF)-stimulated SKOv3 and Hey ovarian cancer cells, inhibiting both chemotaxis and haptotaxis. ARHI binds to and sequesters Stat3 in the cytoplasm, preventing its translocation to the nucleus and localization in focal adhesion complexes. Stat3 siRNA or the JAK2 inhibitor AG490 produced similar inhibition of motility. However, the combination of ARHI expression with Stat3 knockdown or inhibition produced greatest inhibition in ovarian cancer cell migration, consistent with Stat3-dependent and Stat3-independent mechanisms. Consistent with two distinct signaling pathways, knockdown of Stat3 selectively inhibited IL-6-stimulated migration, whereas knockdown of focal adhesion kinase (FAK) preferentially inhibited EGF-stimulated migration. In EGF-stimulated ovarian cancer cells, re-expression of ARHI inhibited FAK(Y397) and Src(Y416) phosphorylation, disrupted focal adhesions, and blocked FAK-mediated RhoA signaling, resulting in decreased levels of GTP-RhoA. Re-expression of ARHI also disrupted the formation of actin stress fibers in a FAK- and RhoA-dependent manner. Thus, ARHI has a critical and previously uncharacterized role in the regulation of ovarian cancer cell migration, exerting inhibitory effects on two distinct signaling pathways.
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Affiliation(s)
- Donna B. Badgwell
- Department of Experimental Therapeutics, The University of Texas M. D. Anderson Cancer Center, Houston, Texas, 77030
| | - Zhen Lu
- Department of Experimental Therapeutics, The University of Texas M. D. Anderson Cancer Center, Houston, Texas, 77030
| | - Kim Le
- Department of Experimental Therapeutics, The University of Texas M. D. Anderson Cancer Center, Houston, Texas, 77030
| | - Fengqin Gao
- Department of Experimental Therapeutics, The University of Texas M. D. Anderson Cancer Center, Houston, Texas, 77030
| | - Maojie Yang
- Department of Experimental Therapeutics, The University of Texas M. D. Anderson Cancer Center, Houston, Texas, 77030
| | - Grace K. Suh
- Department of Experimental Therapeutics, The University of Texas M. D. Anderson Cancer Center, Houston, Texas, 77030
| | - Jia-Ju Bao
- Department of Experimental Therapeutics, The University of Texas M. D. Anderson Cancer Center, Houston, Texas, 77030
| | - Partha Das
- Department of Experimental Therapeutics, The University of Texas M. D. Anderson Cancer Center, Houston, Texas, 77030
| | - Michael Andreeff
- Department of Blood and Marrow Transplantation, The University of Texas M. D. Anderson Cancer Center, Houston, Texas, 77030
| | - Wenting Chen
- Department of Experimental Therapeutics, The University of Texas M. D. Anderson Cancer Center, Houston, Texas, 77030
| | - Yinhua Yu
- Department of Experimental Therapeutics, The University of Texas M. D. Anderson Cancer Center, Houston, Texas, 77030
| | - Ahmed Ashour Ahmed
- Department of Experimental Therapeutics, The University of Texas M. D. Anderson Cancer Center, Houston, Texas, 77030
| | - Warren S.-L. Liao
- Department of Experimental Therapeutics, The University of Texas M. D. Anderson Cancer Center, Houston, Texas, 77030
| | - Robert C. Bast
- Department of Experimental Therapeutics, The University of Texas M. D. Anderson Cancer Center, Houston, Texas, 77030
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Thennes T, Mehta D. Heterotrimeric G proteins, focal adhesion kinase, and endothelial barrier function. Microvasc Res 2012; 83:31-44. [PMID: 21640127 PMCID: PMC3214251 DOI: 10.1016/j.mvr.2011.05.004] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/10/2011] [Revised: 05/04/2011] [Accepted: 05/12/2011] [Indexed: 12/18/2022]
Abstract
Ligands by binding to G protein coupled receptors (GPCRs) stimulate dissociation of heterotrimeric G proteins into Gα and Gβγ subunits. Released Gα and Gβγ subunits induce discrete signaling cues that differentially regulate focal adhesion kinase (FAK) activity and endothelial barrier function. Activation of G proteins downstream of receptors such as protease activated receptor 1 (PAR1) and histamine receptors rapidly increases endothelial permeability which reverses naturally within the following 1-2 h. However, activation of G proteins coupled to the sphingosine-1-phosphate receptor 1 (S1P1) signal cues that enhance basal barrier endothelial function and restore endothelial barrier function following the increase in endothelial permeability by edemagenic agents. Intriguingly, both PAR1 and S1P1 activation stimulates FAK activity, which associates with alteration in endothelial barrier function by these agonists. In this review, we focus on the role of the G protein subunits downstream of PAR1 and S1P1 in regulating FAK activity and endothelial barrier function.
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Affiliation(s)
- Tracy Thennes
- Department of Pharmacology, University of Illinois at Chicago, Chicago, IL 60612, USA
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43
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Asada M, Yoshida M, Hatachi Y, Sasaki T, Yasuda H, Deng X, Nishimura H, Kubo H, Nagatomi R, Yamaya M. l-carbocisteine inhibits respiratory syncytial virus infection in human tracheal epithelial cells. Respir Physiol Neurobiol 2011; 180:112-8. [PMID: 22080978 DOI: 10.1016/j.resp.2011.10.017] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/20/2011] [Revised: 10/18/2011] [Accepted: 10/27/2011] [Indexed: 11/25/2022]
Abstract
To examine the effects of l-carbocisteine on airway infection with respiratory syncytial (RS) virus, human tracheal epithelial cells were pretreated with l-carbocisteine and infected with RS virus. Viral titer, virus RNA, and pro-inflammatory cytokine secretion, including interleukin (IL)-1 and IL-6, increased with time after infection. l-carbocisteine reduced the viral titer in the supernatant fluids, the amount of RS virus RNA, RS virus infection susceptibility, and the concentration of pro-inflammatory cytokines induced by virus infection. l-carbocisteine reduced the expression of intercellular adhesion molecule (ICAM)-1, an RS virus receptor, on the cells. However, l-carbocisteine had no effects on the expression of heparan sulfate, a glycosaminoglycan that binds to the RS virus attachment protein, or on the amount of intracellular activated-RhoA, isoform A of the Ras-homologous family, that binds to the RS virus fusion protein. These findings suggest that l-carbocisteine may inhibit RS virus infection by reducing the expression of ICAM-1. It may also modulate airway inflammation during RS virus infection.
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Affiliation(s)
- Masanori Asada
- Department of Infectious Disease, Sendai City Hospital, Sendai, Japan
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Bielnicki JA, Shkumatov AV, Derewenda U, Somlyo AV, Svergun DI, Derewenda ZS. Insights into the molecular activation mechanism of the RhoA-specific guanine nucleotide exchange factor, PDZRhoGEF. J Biol Chem 2011; 286:35163-75. [PMID: 21816819 PMCID: PMC3186380 DOI: 10.1074/jbc.m111.270918] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/11/2011] [Revised: 07/07/2011] [Indexed: 11/06/2022] Open
Abstract
PDZRhoGEF (PRG) belongs to a small family of RhoA-specific nucleotide exchange factors that mediates signaling through select G-protein-coupled receptors via Gα(12/13) and activates RhoA by catalyzing the exchange of GDP to GTP. PRG is a multidomain protein composed of PDZ, regulators of G-protein signaling-like (RGSL), Dbl-homology (DH), and pleckstrin-homology (PH) domains. It is autoinhibited in cytosol and is believed to undergo a conformational rearrangement and translocation to the membrane for full activation, although the molecular details of the regulation mechanism are not clear. It has been shown recently that the main autoregulatory elements of PDZRhoGEF, the autoinhibitory "activation box" and the "GEF switch," which is required for full activation, are located directly upstream of the catalytic DH domain and its RhoA binding surface, emphasizing the functional role of the RGSL-DH linker. Here, using a combination of biophysical and biochemical methods, we show that the mechanism of PRG regulation is yet more complex and may involve an additional autoinhibitory element in the form of a molten globule region within the linker between RGSL and DH domains. We propose a novel, two-tier model of autoinhibition where the activation box and the molten globule region act synergistically to impair the ability of RhoA to bind to the catalytic DH-PH tandem. The molten globule region and the activation box become less ordered in the PRG-RhoA complex and dissociate from the RhoA-binding site, which may constitute a critical step leading to PRG activation.
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Affiliation(s)
- Jakub A. Bielnicki
- From the Department of Molecular Physiology and Biological Physics University of Virginia, Charlottesville, Virginia 22908 and
| | - Alexander V. Shkumatov
- the European Molecular Biology Laboratory, Hamburg Outstation, EMBL c/o DESY, Notkestrasse 85, D-22603 Hamburg, Germany
| | - Urszula Derewenda
- From the Department of Molecular Physiology and Biological Physics University of Virginia, Charlottesville, Virginia 22908 and
| | - Avril V. Somlyo
- From the Department of Molecular Physiology and Biological Physics University of Virginia, Charlottesville, Virginia 22908 and
| | - Dmitri I. Svergun
- the European Molecular Biology Laboratory, Hamburg Outstation, EMBL c/o DESY, Notkestrasse 85, D-22603 Hamburg, Germany
| | - Zygmunt S. Derewenda
- From the Department of Molecular Physiology and Biological Physics University of Virginia, Charlottesville, Virginia 22908 and
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Kozasa T, Hajicek N, Chow CR, Suzuki N. Signalling mechanisms of RhoGTPase regulation by the heterotrimeric G proteins G12 and G13. J Biochem 2011; 150:357-69. [PMID: 21873336 DOI: 10.1093/jb/mvr105] [Citation(s) in RCA: 58] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022] Open
Abstract
G protein-mediated signal transduction can transduce signals from a large variety of extracellular stimuli into cells and is the most widely used mechanism for cell communication at the membrane. The RhoGTPase family has been well established as key regulators of cell growth, differentiation and cell shape changes. Among G protein-mediated signal transduction, G12/13-mediated signalling is one mechanism to regulate RhoGTPase activity in response to extracellular stimuli. The alpha subunits of G12 or G13 have been shown to interact with members of the RH domain containing guanine nucleotide exchange factors for Rho (RH-RhoGEF) family of proteins to directly connect G protein-mediated signalling and RhoGTPase signalling. The G12/13-RH-RhoGEF signalling mechanism is well conserved over species and is involved in critical steps for cell physiology and disease conditions, including embryonic development, oncogenesis and cancer metastasis. In this review, we will summarize current progress on this important signalling mechanism.
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Affiliation(s)
- Tohru Kozasa
- Laboratory of Systems Biology and Medicine, Research Center for Advanced Science and Technology, University of Tokyo, Tokyo 153-8904, Japan.
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46
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Role of myosin light chain kinase and myosin light chain phosphatase in the resistance arterial myogenic response to intravascular pressure. Arch Biochem Biophys 2011; 510:160-73. [DOI: 10.1016/j.abb.2011.02.024] [Citation(s) in RCA: 87] [Impact Index Per Article: 6.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/16/2010] [Revised: 02/24/2011] [Accepted: 02/28/2011] [Indexed: 12/19/2022]
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Guilluy C, Swaminathan V, Garcia-Mata R, O’Brien ET, Superfine R, Burridge K. The Rho GEFs LARG and GEF-H1 regulate the mechanical response to force on integrins. Nat Cell Biol 2011; 13:722-7. [PMID: 21572419 PMCID: PMC3107386 DOI: 10.1038/ncb2254] [Citation(s) in RCA: 303] [Impact Index Per Article: 21.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/01/2011] [Accepted: 04/06/2011] [Indexed: 12/16/2022]
Abstract
How individual cells respond to mechanical forces is of considerable interest to biologists as force affects many aspects of cell behaviour. The application of force on integrins triggers cytoskeletal rearrangements and growth of the associated adhesion complex, resulting in increased cellular stiffness, also known as reinforcement. Although RhoA has been shown to play a role during reinforcement, the molecular mechanisms that regulate its activity are unknown. By combining biochemical and biophysical approaches, we identified two guanine nucleotide exchange factors (GEFs), LARG and GEF-H1, as key molecules that regulate the cellular adaptation to force. We show that stimulation of integrins with tensional force triggers activation of these two GEFs and their recruitment to adhesion complexes. Surprisingly, activation of LARG and GEF-H1 involves distinct signalling pathways. Our results reveal that LARG is activated by the Src family tyrosine kinase Fyn, whereas GEF-H1 catalytic activity is enhanced by ERK downstream of a signalling cascade that includes FAK and Ras.
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Affiliation(s)
- Christophe Guilluy
- Department of Cell and Developmental Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA
| | - Vinay Swaminathan
- Curriculum in Applied Sciences and Engineering, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
| | - Rafael Garcia-Mata
- Department of Cell and Developmental Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA
| | - E. Timothy O’Brien
- Department of Physics and Astronomy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA
| | - Richard Superfine
- Department of Physics and Astronomy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA
| | - Keith Burridge
- Department of Cell and Developmental Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA
- Lineberger Comprehensive Cancer Center, and UNC McAllister Heart Institute, University of North Carolina, Chapel Hill, North Carolina 27599, USA
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48
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Association of ARHGEF11 R1467H polymorphism with risk for type 2 diabetes mellitus and insulin resistance in Chinese population. Mol Biol Rep 2011; 38:2499-505. [DOI: 10.1007/s11033-010-0387-5] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/23/2010] [Accepted: 11/08/2010] [Indexed: 01/25/2023]
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49
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HGAL, a germinal center specific protein, decreases lymphoma cell motility by modulation of the RhoA signaling pathway. Blood 2010; 116:5217-27. [PMID: 20844236 DOI: 10.1182/blood-2010-04-281568] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023] Open
Abstract
HGAL is a germinal center (GC)-specific gene that negatively regulates lymphocyte motility and whose expression predicts improved survival of patients with diffuse large B-cell lymphoma (DLBCL) and classical Hodgkin lymphoma (cHL). We demonstrate that HGAL serves as a regulator of the RhoA signaling pathway. HGAL enhances activation of RhoA and its down-stream effectors by a novel mechanism - direct binding to the catalytic DH-domain of the RhoA-specific guanine nucleotide exchange factors (RhoGEFs) PDZ-RhoGEF and LARG that stimulate the GDP-GTP exchange rate of RhoA. We delineate the structural domain of HGAL that mediates its interaction with the PDZ-RhoGEF protein. These observations reveal a novel molecular mechanism underlying the inhibitory effects of GC-specific HGAL protein on the motility of GC-derived lymphoma cells. This mechanism may underlie the limited dissemination and better outcome of patients with HGAL-expressing DLBCL and cHL.
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50
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Lim ST, Chen XL, Tomar A, Miller NLG, Yoo J, Schlaepfer DD. Knock-in mutation reveals an essential role for focal adhesion kinase activity in blood vessel morphogenesis and cell motility-polarity but not cell proliferation. J Biol Chem 2010; 285:21526-36. [PMID: 20442405 PMCID: PMC2898428 DOI: 10.1074/jbc.m110.129999] [Citation(s) in RCA: 94] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/01/2010] [Revised: 05/03/2010] [Indexed: 12/27/2022] Open
Abstract
Focal adhesion kinase (FAK) associates with both integrins and growth factor receptors in the control of cell motility and survival. Loss of FAK during mouse development results in lethality at embryonic day 8.5 (E8.5) and a block in cell proliferation. Because FAK serves as both a scaffold and signaling protein, gene knock-outs do not provide mechanistic insights in distinguishing between these modes of FAK function. To determine the role of FAK activity during development, a knock-in point mutation (lysine 454 to arginine (R454)) within the catalytic domain was introduced by homologous recombination. Homozygous FAK(R454/R454) mutation was lethal at E9.5 with defects in blood vessel formation as determined by lack of yolk sac primary capillary plexus formation and disorganized endothelial cell patterning in FAK(R454/R454) embryos. In contrast to the inability of embryonic FAK(-/-) cells to proliferate ex vivo, primary FAK(R454/R454) mouse embryo fibroblasts (MEFs) were established from E8.5 embryos. R454 MEFs exhibited no difference in cell growth compared with normal MEFs, and R454 FAK localized to focal adhesions but was not phosphorylated at Tyr-397. In E8.5 embryos and primary MEFs, FAK R454 mutation resulted in decreased c-Src Tyr-416 phosphorylation. R454 MEFs exhibited enhanced focal adhesion formation, decreased migration, and defects in cell polarity. Within immortalized MEFs, FAK activity was required for fibronectin-stimulated FAK-p190RhoGAP association and p190RhoGAP tyrosine phosphorylation linked to decreased RhoA GTPase activity, focal adhesion turnover, and directional motility. Our results establish that intrinsic FAK activity is essential for developmental processes controlling blood vessel formation and cell motility-polarity but not cell proliferation. This work supports the use of FAK inhibitors to disrupt neovascularization.
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Affiliation(s)
- Ssang-Taek Lim
- From the Department of Reproductive Medicine, Moores Cancer Center, University of California San Diego, La Jolla, California 92093
| | - Xiao Lei Chen
- From the Department of Reproductive Medicine, Moores Cancer Center, University of California San Diego, La Jolla, California 92093
| | - Alok Tomar
- From the Department of Reproductive Medicine, Moores Cancer Center, University of California San Diego, La Jolla, California 92093
| | - Nichol L. G. Miller
- From the Department of Reproductive Medicine, Moores Cancer Center, University of California San Diego, La Jolla, California 92093
| | - Jiyeon Yoo
- From the Department of Reproductive Medicine, Moores Cancer Center, University of California San Diego, La Jolla, California 92093
| | - David D. Schlaepfer
- From the Department of Reproductive Medicine, Moores Cancer Center, University of California San Diego, La Jolla, California 92093
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