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1
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Steenkiste EM, Berndt JD, Pilling C, Simpkins C, Cooper JA. A Cas-BCAR3 co-regulatory circuit controls lamellipodia dynamics. eLife 2021; 10:67078. [PMID: 34169835 PMCID: PMC8266394 DOI: 10.7554/elife.67078] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/30/2021] [Accepted: 06/21/2021] [Indexed: 11/13/2022] Open
Abstract
Integrin adhesion complexes regulate cytoskeletal dynamics during cell migration. Adhesion activates phosphorylation of integrin-associated signaling proteins, including Cas (p130Cas, BCAR1), by Src-family kinases. Cas regulates leading-edge protrusion and migration in cooperation with its binding partner, BCAR3. However, it has been unclear how Cas and BCAR3 cooperate. Here, using normal epithelial cells, we find that BCAR3 localization to integrin adhesions requires Cas. In return, Cas phosphorylation, as well as lamellipodia dynamics and cell migration, requires BCAR3. These functions require the BCAR3 SH2 domain and a specific phosphorylation site, Tyr 117, that is also required for BCAR3 downregulation by the ubiquitin-proteasome system. These findings place BCAR3 in a co-regulatory positive-feedback circuit with Cas, with BCAR3 requiring Cas for localization and Cas requiring BCAR3 for activation and downstream signaling. The use of a single phosphorylation site in BCAR3 for activation and degradation ensures reliable negative feedback by the ubiquitin-proteasome system.
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Affiliation(s)
- Elizabeth M Steenkiste
- Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, United States.,Molecular and Cellular Biology Program, University of Washington, Seattle, United States
| | - Jason D Berndt
- Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, United States
| | - Carissa Pilling
- Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, United States.,Molecular and Cellular Biology Program, University of Washington, Seattle, United States
| | - Christopher Simpkins
- Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, United States
| | - Jonathan A Cooper
- Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, United States.,Molecular and Cellular Biology Program, University of Washington, Seattle, United States
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2
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Katoh K. FAK-Dependent Cell Motility and Cell Elongation. Cells 2020; 9:cells9010192. [PMID: 31940873 PMCID: PMC7017285 DOI: 10.3390/cells9010192] [Citation(s) in RCA: 70] [Impact Index Per Article: 14.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/10/2019] [Revised: 01/02/2020] [Accepted: 01/08/2020] [Indexed: 12/20/2022] Open
Abstract
Fibroblastic cells show specific substrate selectivity for typical cell–substrate adhesion. However, focal adhesion kinase (FAK) contributes to controlling the regulation of orientation and polarity. When fibroblasts attach to micropatterns, tyrosine-phosphorylated proteins and FAK are both detected along the inner border between the adhesive micropatterns and the nonadhesive glass surface. FAK likely plays important roles in regulation of cell adhesion to the substrate, as FAK is a tyrosine-phosphorylated protein that acts as a signal transduction molecule at sites of cell–substrate attachment, called focal adhesions. FAK has been suggested to play a role in the attachment of cells at adhesive micropatterns by affecting cell polarity. Therefore, the localization of FAK might play a key role in recognition of the border of the cell with the adhesive micropattern, thus regulating cell polarity and the cell axis. This review discusses the regulation and molecular mechanism of cell proliferation and cell elongation by FAK and its associated signal transduction proteins.
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Affiliation(s)
- Kazuo Katoh
- Laboratory of Human Anatomy and Cell Biology, Faculty of Health Sciences, Tsukuba University of Technology Tsukuba-city, Ibaraki, Japan
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3
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Palanisamy AP, Suryakumar G, Panneerselvam K, Willey CD, Kuppuswamy D. A Kinase-Independent Function of c-Src Mediates p130Cas Phosphorylation at the Serine-639 Site in Pressure Overloaded Myocardium. J Cell Biochem 2016; 116:2793-803. [PMID: 25976166 DOI: 10.1002/jcb.25224] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/11/2015] [Accepted: 05/08/2015] [Indexed: 12/11/2022]
Abstract
Early work in pressure overloaded (PO) myocardium shows that integrins mediate focal adhesion complex formation by recruiting the adaptor protein p130Cas (Cas) and nonreceptor tyrosine kinase c-Src. To explore c-Src role in Cas-associated changes during PO, we used a feline right ventricular in vivo PO model and a three-dimensional (3D) collagen-embedded adult cardiomyocyte in vitro model that utilizes a Gly-Arg-Gly-Asp-Ser (RGD) peptide for integrin stimulation. Cas showed slow electrophoretic mobility (band-shifting), recruitment to the cytoskeleton, and tyrosine phosphorylation at 165, 249, and 410 sites in both 48 h PO myocardium and 1 h RGD-stimulated cardiomyocytes. Adenoviral mediated expression of kinase inactive (negative) c-Src mutant with intact scaffold domains (KN-Src) in cardiomyocytes did not block the RGD stimulated changes in Cas. Furthermore, expression of KN-Src or kinase active c-Src mutant with intact scaffold function (A-Src) in two-dimensionally (2D) cultured cardiomyocytes was sufficient to cause Cas band-shifting, although tyrosine phosphorylation required A-Src. These data indicate that c-Src's adaptor function, but not its kinase function, is required for a serine/threonine specific phosphorylation(s) responsible for Cas band-shifting. To explore this possibility, Chinese hamster ovary cells that stably express Cas were infected with either β-gal or KN-Src adenoviruses and used for Cas immunoprecipitation combined with mass spectrometry analysis. In the KN-Src expressing cells, Cas showed phosphorylation at the serine-639 (human numbering) site. A polyclonal antibody raised against phospho-serine-639 detected Cas phosphorylation in 24-48 h PO myocardium. Our studies indicate that c-Src's adaptor function mediates serine-639 phosphorylation of Cas during integrin activation in PO myocardium.
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Affiliation(s)
- Arun P Palanisamy
- Division of Cardiology, Department of Medicine, Gazes Cardiac Research Institute, Medical University of South Carolina, Charleston, South Carolina, 29425-2221
| | - Geetha Suryakumar
- Division of Cardiology, Department of Medicine, Gazes Cardiac Research Institute, Medical University of South Carolina, Charleston, South Carolina, 29425-2221
| | - Kavin Panneerselvam
- Division of Cardiology, Department of Medicine, Gazes Cardiac Research Institute, Medical University of South Carolina, Charleston, South Carolina, 29425-2221
| | - Christopher D Willey
- Division of Cardiology, Department of Medicine, Gazes Cardiac Research Institute, Medical University of South Carolina, Charleston, South Carolina, 29425-2221
| | - Dhandapani Kuppuswamy
- Division of Cardiology, Department of Medicine, Gazes Cardiac Research Institute, Medical University of South Carolina, Charleston, South Carolina, 29425-2221
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4
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Luo LY, Hahn WC. Oncogenic Signaling Adaptor Proteins. J Genet Genomics 2015; 42:521-529. [PMID: 26554907 PMCID: PMC4643408 DOI: 10.1016/j.jgg.2015.09.001] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/28/2015] [Revised: 08/31/2015] [Accepted: 09/02/2015] [Indexed: 02/08/2023]
Abstract
Signal transduction pathways activated by receptor tyrosine kinases (RTK) play a critical role in many aspects of cell function. Adaptor proteins serve an important scaffolding function that facilitates key signaling transduction events downstream of RTKs. Recent work integrating both structural and functional genomic approaches has identified several adaptor proteins as new oncogenes. In this review, we focus on the discovery, structure and function, and therapeutic implication of three of these adaptor oncogenes, CRKL, GAB2, and FRS2. Each of the three genes is recurrently amplified in lung adenocarcinoma or ovarian cancer, and is essential to cancer cell lines that harbor such amplification. Overexpression of each gene is able to transform immortalized human cell lines in in vitro or in vivo models. These observations identify adaptor protein as a distinct class of oncogenes and potential therapeutic targets.
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Affiliation(s)
- Leo Y Luo
- Health Sciences and Technology Program, Harvard Medical School, Boston, MA 02115, USA
| | - William C Hahn
- Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02215, USA; Broad Institute of Harvard and Massachusetts Institute of Technology, Cambridge, MA 02142, USA; Center for Cancer Genome Discovery, Dana-Farber Cancer Institute, Boston, MA 02215, USA; Department of Medicine, Brigham and Women's Hospital, Boston, MA 02215, USA.
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5
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Gonsior C, Binamé F, Frühbeis C, Bauer NM, Hoch-Kraft P, Luhmann HJ, Trotter J, White R. Oligodendroglial p130Cas is a target of Fyn kinase involved in process formation, cell migration and survival. PLoS One 2014; 9:e89423. [PMID: 24586768 PMCID: PMC3931761 DOI: 10.1371/journal.pone.0089423] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/08/2013] [Accepted: 01/21/2014] [Indexed: 01/06/2023] Open
Abstract
Oligodendrocytes are the myelinating glial cells of the central nervous system. In the course of brain development, oligodendrocyte precursor cells migrate, scan the environment and differentiate into mature oligodendrocytes with multiple cellular processes which recognize and ensheath neuronal axons. During differentiation, oligodendrocytes undergo dramatic morphological changes requiring cytoskeletal rearrangements which need to be tightly regulated. The non-receptor tyrosine kinase Fyn plays a central role in oligodendrocyte differentiation and myelination. In order to improve our understanding of the role of oligodendroglial Fyn kinase, we have identified Fyn targets in these cells. Purification and mass-spectrometric analysis of tyrosine-phosphorylated proteins in response to overexpressed active Fyn in the oligodendrocyte precursor cell line Oli-neu, yielded the adaptor molecule p130Cas. We analyzed the function of this Fyn target in oligodendroglial cells and observed that reduction of p130Cas levels by siRNA affects process outgrowth, the thickness of cellular processes and migration behavior of Oli-neu cells. Furthermore, long term p130Cas reduction results in decreased cell numbers as a result of increased apoptosis in cultured primary oligodendrocytes. Our data contribute to understanding the molecular events taking place during oligodendrocyte migration and morphological differentiation and have implications for myelin formation.
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Affiliation(s)
- Constantin Gonsior
- Department of Biology, Molecular Cell Biology, Johannes Gutenberg-University of Mainz, Mainz, Germany
| | - Fabien Binamé
- Department of Biology, Molecular Cell Biology, Johannes Gutenberg-University of Mainz, Mainz, Germany
| | - Carsten Frühbeis
- Department of Biology, Molecular Cell Biology, Johannes Gutenberg-University of Mainz, Mainz, Germany
| | - Nina M. Bauer
- Institute of Physiology, University Medical Center of the Johannes Gutenberg-University, Mainz, Germany
| | - Peter Hoch-Kraft
- Department of Biology, Molecular Cell Biology, Johannes Gutenberg-University of Mainz, Mainz, Germany
| | - Heiko J. Luhmann
- Institute of Physiology, University Medical Center of the Johannes Gutenberg-University, Mainz, Germany
| | - Jacqueline Trotter
- Department of Biology, Molecular Cell Biology, Johannes Gutenberg-University of Mainz, Mainz, Germany
| | - Robin White
- Institute of Physiology, University Medical Center of the Johannes Gutenberg-University, Mainz, Germany
- * E-mail:
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6
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Zhang X, Moore SW, Iskratsch T, Sheetz MP. N-WASP-directed actin polymerization activates Cas phosphorylation and lamellipodium spreading. J Cell Sci 2014; 127:1394-405. [PMID: 24481817 DOI: 10.1242/jcs.134692] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/26/2023] Open
Abstract
Tyrosine phosphorylation of the substrate domain of Cas (CasSD) correlates with increased cell migration in healthy and diseased cells. Here, we address the mechanism leading to the phosphorylation of CasSD in the context of fibronectin-induced early spreading of fibroblasts. We have previously demonstrated that mechanical stretching of CasSD exposes phosphorylation sites for Src family kinases (SFKs). Surprisingly, phosphorylation of CasSD was independent of myosin contractile activity but dependent on actin polymerization. Furthermore, we found that CasSD phosphorylation in the early stages of cell spreading required: (1) integrin anchorage and integrin-mediated activation of SFKs, (2) association of Cas with focal adhesion kinase (FAK), and (3) N-WASP-driven actin-assembly activity. These findings, and analyses of the interactions of the Cas domains, indicate that the N-terminus of Cas associates with the FAK-N-WASP complex at the protrusive edge of the cell and that the C-terminus of Cas associates with the immobilized integrin-SFK cluster. Thus, extension of the leading edge mediated by actin polymerization could stretch Cas during early cell spreading, priming it for phosphorylation.
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Affiliation(s)
- Xian Zhang
- Department of Biological Sciences, Columbia University, 1212 Amsterdam Avenue, New York, NY 10027, USA
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7
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Burdisso JE, González Á, Arregui CO. PTP1B promotes focal complex maturation, lamellar persistence and directional migration. J Cell Sci 2013; 126:1820-31. [DOI: 10.1242/jcs.118828] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023] Open
Abstract
Previous findings established that ER-bound PTP1B targets peripheral cell-matrix adhesions and regulates positively cell adhesion to fibronectin. Here we show that PTP1B enhances focal complex lifetime at the lamellipodium base, delaying their turnover and facilitating α-actinin incorporation. We demonstrate the presence of catalytic PTP1BD181A-α-actinin complexes at focal complexes. Kymograph analysis reveals that PTP1B contributes to lamellar protrusion persistence and directional cell migration. Pull down and FRET analysis also shows that PTP1B is required for efficient integrin-dependent downregulation of RhoA and upregulation of Rac1 during spreading. A substrate trap strategy revealed that FAK/Src recruitment and Src activity were essential for the generation of PTP1B substrates in adhesions. PTP1B targets the negative regulatory site of Src (phosphotyrosine 529), paxillin and p130Cas at peripheral cell-matrix adhesions. We postulate that PTP1B modulates more than one pathway required for focal complex maturation and membrane protrusion, including α-actinin-mediated cytoskeletal anchorage, integrin-dependent activation of the FAK/Src signaling pathway, and RhoA and Rac1 GTPase activity. By doing so, PTP1B contributes to coordinate adhesion turnover, lamellar stability and directional cell migration.
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8
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Cabodi S, del Pilar Camacho-Leal M, Di Stefano P, Defilippi P. Integrin signalling adaptors: not only figurants in the cancer story. Nat Rev Cancer 2010; 10:858-70. [PMID: 21102636 DOI: 10.1038/nrc2967] [Citation(s) in RCA: 241] [Impact Index Per Article: 16.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Current evidence highlights the ability of adaptor (or scaffold) proteins to create signalling platforms that drive cellular transformation upon integrin-dependent adhesion and growth factor receptor activation. The understanding of the biological effects that are regulated by these adaptors in tumours might be crucial for the identification of new targets and the development of innovative therapeutic strategies for human cancer. In this Review we discuss the relevance of adaptor proteins in signalling that originates from integrin-mediated cell-extracellular matrix (ECM) adhesion and growth factor stimulation in the context of cell transformation and tumour progression. We specifically underline the contribution of p130 Crk-associated substrate (p130CAS; also known as BCAR1), neural precursor cell expressed, developmentally down-regulated 9 (NEDD9; also known as HEF1), CRK and the integrin-linked kinase (ILK)-pinch-parvin (IPP) complex to cancer, along with the more recently identified p140 Cas-associated protein (p140CAP; also known as SRCIN1).
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Affiliation(s)
- Sara Cabodi
- Molecular Biotechnology Centre and Department of Genetics, Biology and Biochemistry, University of Torino, Via Nizza 52, Torino 10126, Italy
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9
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Donato DM, Ryzhova LM, Meenderink LM, Kaverina I, Hanks SK. Dynamics and mechanism of p130Cas localization to focal adhesions. J Biol Chem 2010; 285:20769-79. [PMID: 20430882 PMCID: PMC2898362 DOI: 10.1074/jbc.m109.091207] [Citation(s) in RCA: 45] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/02/2009] [Revised: 03/10/2010] [Indexed: 01/09/2023] Open
Abstract
The docking protein p130Cas is a major Src substrate involved in integrin signaling and mechanotransduction. Tyrosine phosphorylation of p130Cas in focal adhesions (FAs) has been linked to enhanced cell migration, invasion, proliferation, and survival. However, the mechanism of p130Cas targeting to FAs is uncertain, and dynamic aspects of its localization have not been explored. Using live cell microscopy, we show that fluorophore-tagged p130Cas is a component of FAs throughout the FA assembly and disassembly stages, although it resides transiently in FAs with a high mobile fraction. Deletion of either the N-terminal Src homology 3 (SH3) domain or the Cas-family C-terminal homology (CCH) domain significantly impaired p130Cas FA localization, and deletion of both domains resulted in full exclusion. Focal adhesion kinase was implicated in the FA targeting function of the p130Cas SH3 domain. Consistent with their roles in FA targeting, both the SH3 and CCH domains were found necessary for p130Cas to fully undergo tyrosine phosphorylation and promote cell migration. By revealing the capacity of p130Cas to function in FAs throughout their lifetime, clarifying FA targeting mechanism, and demonstrating the functional importance of the highly conserved CCH domain, our results advance the understanding of an important aspect of integrin signaling.
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Affiliation(s)
- Dominique M. Donato
- From the Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232
| | - Larisa M. Ryzhova
- From the Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232
| | - Leslie M. Meenderink
- From the Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232
| | - Irina Kaverina
- From the Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232
| | - Steven K. Hanks
- From the Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232
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10
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Tikhmyanova N, Little JL, Golemis EA. CAS proteins in normal and pathological cell growth control. Cell Mol Life Sci 2010; 67:1025-48. [PMID: 19937461 PMCID: PMC2836406 DOI: 10.1007/s00018-009-0213-1] [Citation(s) in RCA: 150] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/06/2009] [Revised: 11/03/2009] [Accepted: 11/09/2009] [Indexed: 12/20/2022]
Abstract
Proteins of the CAS (Crk-associated substrate) family (BCAR1/p130Cas, NEDD9/HEF1/Cas-L, EFS/SIN and CASS4/HEPL) are integral players in normal and pathological cell biology. CAS proteins act as scaffolds to regulate protein complexes controlling migration and chemotaxis, apoptosis, cell cycle, and differentiation, and have more recently been linked to a role in progenitor cell function. Reflecting these complex functions, over-expression of CAS proteins has now been strongly linked to poor prognosis and increased metastasis in cancer, as well as resistance to first-line chemotherapeutics in multiple tumor types including breast and lung cancers, glioblastoma, and melanoma. Further, CAS proteins have also been linked to additional pathological conditions including inflammatory disorders, Alzheimer's and Parkinson's disease, as well as developmental defects. This review will explore the roles of the CAS proteins in normal and pathological states in the context of the many mechanistic insights into CAS protein function that have emerged in the past decade.
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Affiliation(s)
- Nadezhda Tikhmyanova
- Fox Chase Cancer Center, 333 Cottman Ave., Philadelphia, PA 19111 USA
- Department of Biochemistry, Drexel University Medical School, Philadelphia, PA 19102 USA
| | - Joy L. Little
- Fox Chase Cancer Center, 333 Cottman Ave., Philadelphia, PA 19111 USA
| | - Erica A. Golemis
- Fox Chase Cancer Center, 333 Cottman Ave., Philadelphia, PA 19111 USA
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11
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Patwardhan P, Shiba K, Gordon C, Craddock BP, Tamiko M, Miller WT. Synthesis of functional signaling domains by combinatorial polymerization of phosphorylation motifs. ACS Chem Biol 2009; 4:751-8. [PMID: 19627099 DOI: 10.1021/cb900059f] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
The adaptor protein Cas contains a core substrate domain with multiple YXXP motifs that are phosphorylated by Src and other tyrosine kinases. Here, we used a synthetic strategy to determine the importance of the arrangement, spacing, and identity of the YXXP motifs. By polymerizing short DNA sequences encoding two phosphorylation motifs, we created a panel of Cas mutants in which the entire substrate domain was replaced by synthetic domains containing random numbers and arrangements of the motifs. Most of these synthetic Cas variants were recognized and phosphorylated by Src in vitro and in intact mammalian cells. The random polymer mutants also restored migration activity to Cas knockout cells; even artificial proteins containing a single motif retained some biological function. Our results suggest that the arrangement of Cas motifs is not critical for signaling. This method could be used to identify the minimal functional units in other signaling proteins.
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Affiliation(s)
- Parag Patwardhan
- Department of Physiology and Biophysics, School of Medicine, State University of New York at Stony Brook, Stony Brook, New York 11794
| | - Kiyotaka Shiba
- Department of Protein Engineering, Cancer Institute, Japanese Foundation for Cancer Research, Tokyo 135-8550, Japan
| | - Chris Gordon
- Department of Physiology and Biophysics, School of Medicine, State University of New York at Stony Brook, Stony Brook, New York 11794
| | - Barbara P. Craddock
- Department of Physiology and Biophysics, School of Medicine, State University of New York at Stony Brook, Stony Brook, New York 11794
| | - Minamisawa Tamiko
- Department of Protein Engineering, Cancer Institute, Japanese Foundation for Cancer Research, Tokyo 135-8550, Japan
| | - W. Todd Miller
- Department of Physiology and Biophysics, School of Medicine, State University of New York at Stony Brook, Stony Brook, New York 11794
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12
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Birge RB, Kalodimos C, Inagaki F, Tanaka S. Crk and CrkL adaptor proteins: networks for physiological and pathological signaling. Cell Commun Signal 2009; 7:13. [PMID: 19426560 PMCID: PMC2689226 DOI: 10.1186/1478-811x-7-13] [Citation(s) in RCA: 214] [Impact Index Per Article: 13.4] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/02/2009] [Accepted: 05/10/2009] [Indexed: 01/24/2023] Open
Abstract
The Crk adaptor proteins (Crk and CrkL) constitute an integral part of a network of essential signal transduction pathways in humans and other organisms that act as major convergence points in tyrosine kinase signaling. Crk proteins integrate signals from a wide variety of sources, including growth factors, extracellular matrix molecules, bacterial pathogens, and apoptotic cells. Mounting evidence indicates that dysregulation of Crk proteins is associated with human diseases, including cancer and susceptibility to pathogen infections. Recent structural work has identified new and unusual insights into the regulation of Crk proteins, providing a rationale for how Crk can sense diverse signals and produce a myriad of biological responses.
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Affiliation(s)
- Raymond B Birge
- Department of Biochemistry & Molecular Biology, UMDNJ-New Jersey Medical School, 185 South Orange Ave, Newark, NJ 07103, USA.
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13
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Structural and functional basis of a role for CRKL in a fibroblast growth factor 8-induced feed-forward loop. Mol Cell Biol 2009; 29:3076-87. [PMID: 19307307 DOI: 10.1128/mcb.01686-08] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/13/2023] Open
Abstract
The adapter protein CRKL is required for the normal development of multiple tissues that rely on fibroblast growth factor 8 (FGF8). The precise role of CRKL in receptor signaling has been unclear, however. To address this issue, we first modeled the three-dimensional structure of CRKL by molecular dynamics. By taking advantage of structural simulations, we performed in silico analysis of the interactions of the autophosphorylation sites of FGR receptor 1 (FGFR1) with the SH2 domain of CRKL or a highly related protein, CRK. As predicted by simulations, we confirm the specific physical interaction of phosphorylated Y463 (pY463) in FGFR1 with the CRKL SH2 domain at an affinity approximately 30-fold stronger than that of CRK. We also provide evidence that interactions outside of the core YXXP motif have a significant impact on physical association, which is consistent with predictions from molecular-dynamics simulations. Furthermore, we identify CRKL as an essential component of an FGF8-induced feed-forward loop permissive for efficient activation of the mitogen-activated protein kinase Erk1/2, as well as FGF8-induced anchorage-independent cell growth, using Crkl-deficient cells or a pY463 synthetic peptide. Although many cells generally require cell-matrix adhesion, our results demonstrate that CRKL permits cells to bypass the strict need for adhesion in response to FGF8 through direct interaction with receptor.
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14
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Jia L, Uekita T, Sakai R. Hyperphosphorylated cortactin in cancer cells plays an inhibitory role in cell motility. Mol Cancer Res 2008; 6:654-62. [PMID: 18403644 DOI: 10.1158/1541-7786.mcr-07-0220] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
Cortactin is frequently overexpressed in cancer cells, and changes of the levels of its tyrosine phosphorylation have been observed in several cancer cells. However, how the expression level and phosphorylation state of cortactin would influence the ultimate cellular function of cancer cells is unknown. In this study, we analyzed the role of cortactin in gastric and breast cancer cell lines using RNA interference technique and found that knockdown of cortactin inhibited cell migration in a subset of gastric cancer cells with a lower level of its tyrosine phosphorylation, whereas it greatly enhanced cell migration and increased tyrosine phosphorylation of p130Cas in other subsets of cells with hyperphosphorylated cortactin. Consistent results were obtained when hyperphosphorylation of cortactin was induced in MCF7 breast cancer cells by expressing Fyn tyrosine kinase. Additionally, immunostaining analysis showed that knockdown of hyperphosphorylated cortactin resulted in the recruitment of p130Cas to focal adhesions. These results suggest that cortactin hyperphosphorylation suppresses cell migration possibly through the inhibition of membrane localization and tyrosine phosphorylation of p130Cas.
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Affiliation(s)
- Lin Jia
- National Cancer Center Research Institute, Chuo-ku, Tokyo 104-0045, Japan
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15
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Payne SL, Hendrix MJC, Kirschmann DA. Lysyl oxidase regulates actin filament formation through the p130(Cas)/Crk/DOCK180 signaling complex. J Cell Biochem 2006; 98:827-37. [PMID: 16440329 DOI: 10.1002/jcb.20792] [Citation(s) in RCA: 43] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/10/2022]
Abstract
We have previously demonstrated that lysyl oxidase (LOX) is expressed in invasive breast cancer cells compared to poorly invasive cells. Additionally, we have recently shown that LOX regulates cell migration, a key step in the invasion process, through a hydrogen peroxide-dependent mechanism involving the focal adhesion kinase (FAK)/Src signaling complex. Here we further elucidate the role of LOX in cell motility/migration by examining the role of LOX in actin filament polymerization. We demonstrate that inhibition of LOX leads to an increase in phalloidin staining, directly associated with an increase in actin stress fiber formation. This increase in staining was confirmed by activity assays showing an increase in Rho activity with decreased LOX activity. Additionally, Rac and Cdc42 activity decreased with the reduction in LOX activity. Taken together, these data demonstrate a loss of a motogenic phenotype with decreased LOX activity. Finally, in order to elucidate the mechanism by which LOX regulates actin polymerization, we have demonstrated that LOX facilitates p130(Cas) phosphorylation, which allows for the binding to CAS related kinase (Crk) and formation of the p130(Cas)/Crk/DOCK180 signaling complex. Formation of this complex leads to an increase in Rac-GTP, which decreases actin stress fiber formation and increases formation of lamellipodium. These data demonstrate that LOX regulates cell motility/migration through changes in actin filament polymerization, which involve the regulation of the p130(Cas)/Crk/DOCK180 signaling pathway. Elucidating the role of LOX in the regulation of cell motility will allow the development of more effective therapeutic strategies to treat invasive/metastatic breast cancer.
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Affiliation(s)
- Stacey L Payne
- Children's Memorial Research Center, Cancer Biology and Epigenomics Program, Robert H. Lurie Comprehensive Cancer Center, Feinberg School of Medicine at Northwestern University, Chicago, Illinois 60614, USA
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Dadke D, Jarnik M, Pugacheva EN, Singh MK, Golemis EA. Deregulation of HEF1 impairs M-phase progression by disrupting the RhoA activation cycle. Mol Biol Cell 2006; 17:1204-17. [PMID: 16394104 PMCID: PMC1382310 DOI: 10.1091/mbc.e05-03-0237] [Citation(s) in RCA: 48] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023] Open
Abstract
The focal adhesion-associated signaling protein HEF1 undergoes a striking relocalization to the spindle at mitosis, but a function for HEF1 in mitotic signaling has not been demonstrated. We here report that overexpression of HEF1 leads to failure of cells to progress through cytokinesis, whereas depletion of HEF1 by small interfering RNA (siRNA) leads to defects earlier in M phase before cleavage furrow formation. These defects can be explained mechanistically by our determination that HEF1 regulates the activation cycle of RhoA. Inactivation of RhoA has long been known to be required for cytokinesis, whereas it has recently been determined that activation of RhoA at the entry to M phase is required for cellular rounding. We find that increased HEF1 sustains RhoA activation, whereas depleted HEF1 by siRNA reduces RhoA activation. Furthermore, we demonstrate that chemical inhibition of RhoA is sufficient to reverse HEF1-dependent cellular arrest at cytokinesis. Finally, we demonstrate that HEF1 associates with the RhoA-GTP exchange factor ECT2, an orthologue of the Drosophila cytokinetic regulator Pebble, providing a direct means for HEF1 control of RhoA. We conclude that HEF1 is a novel component of the cell division control machinery and that HEF1 activity impacts division as well as cell attachment signaling events.
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Affiliation(s)
- Disha Dadke
- Division of Basic Science, Fox Chase Cancer Center, Philadelphia, PA 19111, USA
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Brábek J, Constancio SS, Siesser PF, Shin NY, Pozzi A, Hanks SK. Crk-associated substrate tyrosine phosphorylation sites are critical for invasion and metastasis of SRC-transformed cells. Mol Cancer Res 2005; 3:307-15. [PMID: 15972849 DOI: 10.1158/1541-7786.mcr-05-0015] [Citation(s) in RCA: 94] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
Crk-associated substrate (CAS, p130Cas) is a major tyrosine phosphorylated protein in cells transformed by v-crk and v-src oncogenes. We recently reported that reexpression of CAS in CAS-deficient mouse embryo fibroblasts transformed by oncogenic Src promoted an invasive phenotype associated with enhanced cell migration through Matrigel, organization of actin into large podosome ring and belt structures, activation of matrix metalloproteinase-2, and elevated tyrosine phosphorylation of the focal adhesion proteins FAK and paxillin. We have now extended these studies to examine the mechanism by which CAS achieves these changes and to evaluate the potential role for CAS in promoting in vivo tumor growth and metastasis. Whereas the presence or absence of CAS did not alter the primary growth of subcutaneous-injected Src-transformed mouse embryo fibroblasts, CAS expression was required to promote lung metastasis following removal of the primary tumor. The substrate domain YxxP tyrosines, the major sites of CAS phosphorylation by Src that mediate interactions with Crk, were found to be critical for promoting both invasive and metastatic properties of the cells. The ability of CAS to promote Matrigel invasion, formation of large podosome structures, and tyrosine phosphorylation of Src substrates, including FAK, paxillin, and cortactin, was also strictly dependent on the YxxP tyrosines. In contrast, matrix metalloproteinase-2 activation was most dependent on the CAS SH3 domain, whereas the substrate domain YxxP sites also contributed to this property. Thus multiple CAS-mediated signaling events are implicated in promoting invasive and metastatic properties of Src-transformed cells.
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Affiliation(s)
- Jan Brábek
- Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37212, USA
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18
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Li X, Hyink DP, Polgar K, Gusella GL, Wilson PD, Burrow CR. Protein Kinase X Activates Ureteric Bud Branching Morphogenesis in Developing Mouse Metanephric Kidney. J Am Soc Nephrol 2005; 16:3543-52. [PMID: 16236808 DOI: 10.1681/asn.2005030240] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/03/2022] Open
Abstract
The human protein kinase X (PRKX) gene was identified previously as a cAMP-dependent serine/threonine kinase that is aberrantly expressed in autosomal dominant polycystic disease kidneys and normally expressed in fetal kidneys. The PRKX kinase belongs to a serine/threonine kinase family that is phylogenetically and functionally distinct from classical protein kinase A kinases. Expression of PRKX activates cAMP-dependent renal epithelial cell migration and tubular morphogenesis in cell culture, suggesting that it might regulate branching growth of the collecting duct system in the fetal kidney. With the use of a mouse embryonic kidney organ culture system that recapitulates early kidney development in vitro, it is demonstrated that lentiviral vector-driven expression of a constitutively active, cAMP-independent PRKX in the ureteric bud epithelium stimulates branching morphogenesis and results in a 2.5-fold increase in glomerular number. These results suggest that PRKX stimulates epithelial branching morphogenesis by activating cell migration and support a role for this kinase in the regulation of nephrogenesis and of collecting system development in the fetal kidney.
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Affiliation(s)
- Xiaohong Li
- Department of Medicine, Division of Nephrology, Mount Sinai School of Medicine, New York, NY 10029, USA
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19
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Nasertorabi F, Tars K, Becherer K, Kodandapani R, Liljas L, Vuori K, Ely KR. Molecular basis for regulation of Src by the docking protein p130Cas. J Mol Recognit 2005; 19:30-8. [PMID: 16245368 DOI: 10.1002/jmr.755] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/10/2022]
Abstract
The docking protein p130Cas (Cas) becomes tyrosine-phosphorylated in its central substrate domain in response to extracellular stimuli such as integrin-mediated cell adhesion, and transmits signals through interactions with various intracellular signaling molecules such as the adaptor protein Crk. Src-family kinases (SFKs) bind a specific site in the carboxyl-terminal region of Cas and subsequently SFKs phosphorylate progressively the substrate domain in Cas. In this study crystallography, mutagenesis and binding assays were used to understand the molecular basis for Cas interactions with SFKs. Tyrosine phosphorylation regulates binding of Cas to SFKs, and the primary site for this phosphorylation, Y762, has been proposed. A phosphorylated peptide corresponding to Cas residues 759MEDpYDYVHL767 containing the key phosphotyrosine was crystallized in complex with the SH3-SH2 domain of the SFK Lck. The results provide the first structural data for this protein-protein interaction. The motif in Cas 762pYDYV binds to the SH2 domain in a mode that mimics high-affinity ligands, involving dual contacts of Y762 and V765 with conserved residues in SFK SH2 domains. In addition, Y764 is in position to make an electrostatic contact after phosphorylation with a conserved SFK arginine that mediates interactions with other high-affinity SH2 binders. These new molecular data suggest that Cas may regulate activity of Src as a competing ligand to displace intramolecular interactions that occur in SFKs (between the C-terminal tail and the SH2 domain) and restrain and down-regulate the kinase in an inactive form.
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Affiliation(s)
- Fariborz Nasertorabi
- Cancer Center, The Burnham Institute for Medical Research, La Jolla, California 92037, USA
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20
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Chodniewicz D, Klemke RL. Regulation of integrin-mediated cellular responses through assembly of a CAS/Crk scaffold. BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH 2004; 1692:63-76. [PMID: 15246680 DOI: 10.1016/j.bbamcr.2004.03.006] [Citation(s) in RCA: 141] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Subscribe] [Scholar Register] [Received: 11/03/2003] [Accepted: 03/16/2004] [Indexed: 01/09/2023]
Abstract
The molecular coupling of CAS and Crk in response to integrin activation is an evolutionary conserved signaling module that controls cell proliferation, survival and migration. However, when deregulated, CAS/Crk signaling also contributes to cancer progression and developmental defects in humans. Here we highlight recent advances in our understanding of how CAS/Crk complexes assemble in cells to modulate the actin cytoskeleton, and the molecular mechanisms that regulate this process. We discuss in detail the spatiotemporal dynamics of CAS/Crk assembly and how this scaffold recruits specific effector proteins that couple integrin signaling networks to the migration machinery of cells. We also highlight the importance of CAS/Crk signaling in the dual regulation of cell migration and survival mechanisms that operate in invasive cells during development and pathological conditions associated with cancer metastasis.
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Affiliation(s)
- David Chodniewicz
- Department of Immunology, The Scripps Research Institute, 10550 North Torrey Pines Road, SP231, La Jolla, CA 92037, USA
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21
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Shin NY, Dise RS, Schneider-Mergener J, Ritchie MD, Kilkenny DM, Hanks SK. Subsets of the Major Tyrosine Phosphorylation Sites in Crk-associated Substrate (CAS) Are Sufficient to Promote Cell Migration. J Biol Chem 2004; 279:38331-7. [PMID: 15247284 DOI: 10.1074/jbc.m404675200] [Citation(s) in RCA: 85] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
Crk-associated substrate (p130(CAS) or CAS) is a major integrin-associated Src substrate that undergoes tyrosine phosphorylation at multiple YXXP motifs in its substrate domain (SD) to create docking sites for SH2-containing signaling effectors. Notably, recruitment of Crk adaptor proteins to the CAS SD sites is implicated in promoting cell migration. However, it is unclear which or how many of the 15 CAS SD YXXP tyrosines are critically involved. To gain a better understanding of CAS SD function, we assessed the signaling capacity of individual YXXP motifs. Using site-directed mutagenesis combined with tryptic phosphopeptide mapping, we determined that the ten tyrosines in YXXP motifs 6-15 are the major sites of CAS SD phosphorylation by Src. Phosphopeptide binding assays showed that all of these sites are capable of binding the Crk SH2 domain. To evaluate the requirement for CAS YXXP sites in stimulating cell migration, a series of phenylalanine substitution variants were expressed in CAS -/- mouse embryo fibroblasts. CAS expression enhanced the rate of cell migration into a monolayer wound in a manner dependent on the major sites of Src phosphorylation. Effective wound healing was achieved by CAS variants containing as few as four of the major sites, indicating sufficiency of partial SD signaling function in this cell migration response.
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Affiliation(s)
- Nah-Young Shin
- Department of Cell and Developmental Biology, Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA
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22
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Brábek J, Constancio SS, Shin NY, Pozzi A, Weaver AM, Hanks SK. CAS promotes invasiveness of Src-transformed cells. Oncogene 2004; 23:7406-15. [PMID: 15273716 DOI: 10.1038/sj.onc.1207965] [Citation(s) in RCA: 73] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/11/2023]
Abstract
CAS ('Crk-associated substrate') is an Src substrate found at sites of integrin-mediated cell adhesion and linked to cell motility and survival. In this study, the involvement of CAS in oncogenic transformation was evaluated through analysis of mouse embryo fibroblast populations expressing an activated Src mutant, either in the presence or absence of CAS expression. CAS was not found to be a critical determinant of either Src-mediated morphologic transformation or anchorage-independent growth. However, CAS had a profound effect on other aspects of oncogenic Src function. CAS expression led to a substantial increase in the phosphotyrosine content of FAK and paxillin, supporting a role for CAS as a positive regulator of Src activity at integrin adhesion sites. Importantly, CAS expression resulted in a striking enhancement of the capacity of Src-transformed cells to invade through Matrigel. The increased invasiveness was associated with increased activation of matrix metalloproteinase MMP-2 and formation of large actin-rich podosomal aggregates appearing as ring and belt structures. Thus, elevated CAS-associated tyrosine phosphorylation signaling events occurring at sites of integrin-mediated cell adhesion can have a major role in the development of an invasive cell phenotype.
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Affiliation(s)
- Jan Brábek
- Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37212, USA
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23
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Kuwabara K, Nakaoka T, Sato K, Nishishita T, Sasaki T, Yamashita N. Differential regulation of cell migration and proliferation through proline-rich tyrosine kinase 2 in endothelial cells. Endocrinology 2004; 145:3324-30. [PMID: 15070849 DOI: 10.1210/en.2003-1433] [Citation(s) in RCA: 18] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/30/2023]
Abstract
Proline-rich tyrosine kinase 2 (Pyk2), a member of the focal adhesion kinase family, is thought to act as a key component in vasculogenesis and angiogenesis. Therefore, we studied the effect of mutant Pyk2 expression on the migration and proliferation in endothelial cells (ECs). Two types of mutant Pyk2 were examined by adenovirus vectors AxCA-Pyk2K457A, expressing a kinase inactive mutant, and AxCA-Pyk2Y402F, expressing a tyrosine autophosphorylation site mutant, in addition to AxCA-Pyk2, expressing wild-type Pyk2. Migration of ECs infected with AxCA-Pyk2Y402F increased to a level similar to that of ECs infected with AxCA-Pyk2. The size of effect was dependent on the amount of applied adenoviruses within the range of 3-30 multiplicity of infection. In contrast, AxCA-Pyk2K457A infection did not show any significant effect on cell migration. Western blotting showed that both phosphorylation of Pyk2 Y(881) and association of p130(Cas) with Pyk2 were enhanced in ECs infected with AxCA-Pyk2Y402F as well as with AxCA-Pyk2, but not in ECs infected with AxCA-Pyk2K457A. Therefore, signaling mediated by Pyk2 Y(881) and p130(Cas) may be involved in the migration of ECs infected either with AxCA-Pyk2Y402F or with AxCA-Pyk2. In proliferation assay, AxCA-Pyk2 infection suppressed EC proliferation significantly; however, neither AxCA-Pyk2Y402F nor AxCA-Pyk2K457A showed such an inhibitory effect. Thus, the two Pyk2 mutants revealed that Pyk2 signaling differentially regulates cell migration and proliferation pathways.
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Affiliation(s)
- Koichiro Kuwabara
- Department of Advanced Medical Science, The Institute of Medical Science, The University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo 108-8639, Japan
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24
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Huang J, Asawa T, Takato T, Sakai R. Cooperative roles of Fyn and cortactin in cell migration of metastatic murine melanoma. J Biol Chem 2003; 278:48367-76. [PMID: 13129922 DOI: 10.1074/jbc.m308213200] [Citation(s) in RCA: 54] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022] Open
Abstract
Src family kinases are major regulators of various integrin-mediated biological processes, although their functional roles and substrates in cancer metastasis are unknown. We explored the roles of Src family tyrosine kinases in cell migration and the spread of K-1735 murine melanoma cell lines with low or high metastatic potential. Corresponding to elevated cell motility and spreading ability, Fyn was selectively activated among Src family kinases, and the cell motility was blocked by an inhibitor of Src family kinases. Significant tyrosine phosphorylation of cortactin, stable complex formation between activated Fyn and cortactin, and co-localization of cortactin with Fyn at cell membranes were all observed only in cells with high metastatic potential. Both integrin-mediated Fyn activation and hyperphosphorylation of cortactin were observed 2-5 h after stimulation in highly metastatic cells, and they required de novo protein synthesis. We demonstrate that cortactin is a specific substrate and cooperative effector of Fyn in integrin-mediated signaling processes regulating metastatic potential.
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Affiliation(s)
- Jinhong Huang
- Growth Factor Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan
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25
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Vial D, Oliver C, Jamur MC, Pastor MVD, da Silva Trindade E, Berenstein E, Zhang J, Siraganian RP. Alterations in Granule Matrix and Cell Surface of Focal Adhesion Kinase-Deficient Mast Cells. THE JOURNAL OF IMMUNOLOGY 2003; 171:6178-86. [PMID: 14634134 DOI: 10.4049/jimmunol.171.11.6178] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/03/2023]
Abstract
Focal adhesion kinase (FAK) is a nonreceptor protein tyrosine kinase that plays an important role in many cellular processes and is tyrosine phosphorylated after FcepsilonRI aggregation in mast cells. In mice, null mutation of the fak gene results in a lethal phenotype in which the embryos fail to develop past day 8.5 of gestation. To study the role of FAK in these mast cells, 8.5-day embryos were isolated and placed in culture with IL-3 and stem cell factor (SCF). Although FAK was not required for the development of mast cells in culture, the FAK(-/-) embryo-derived mast cells had several distinct characteristics. Compared with the controls, the mast cells that lack FAK were less metachromatic and by electron microscopy had granules that appeared largely electron lucid, although their histamine content was unchanged. The FAK-deficient mast cells had a reduction in the content of chondroitin/dermatan sulfate, the major glycosaminoglycan component of the granular matrix. The FAK-deficient cells had fewer microvilli that were fused with each other, giving the cell surface a ruffled appearance. There was also a 3-fold increase in the number of cells highly expressing beta(7) integrin. However, signal transduction from the high affinity IgE receptor for the secretion of histamine was similar in the wild-type, heterozygote, and the FAK-deficient cells. The FcepsilonRI-induced tyrosine phosphorylation of paxillin, Crk-associated tyrosine kinase substrate (CAS), and mitogen-activated protein kinase proteins was independent of FAK. These results indicate that FAK plays a role in regulating the glycosaminoglycan content of the secretory granules and influences the cell surface morphology of mast cells.
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Affiliation(s)
- Daniel Vial
- Receptors and Signal Transduction Section, Oral Infection and Immunity Branch, National Institute of Dental Research, National Institutes of Health, Bethesda, MD 20892, USA
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26
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Goldberg GS, Alexander DB, Pellicena P, Zhang ZY, Tsudal H, Miller WT. Src phosphorylates Cas on tyrosine 253 to promote migration of transformed cells. J Biol Chem 2003; 278:46533-40. [PMID: 12972425 PMCID: PMC2441571 DOI: 10.1074/jbc.m307526200] [Citation(s) in RCA: 78] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022] Open
Abstract
Cas is a member of the focal adhesion complex. Phosphorylation of Cas by Src is an important event leading to cell transformation. Using mass spectrometry, we have mapped 11 sites in Cas that are phosphorylated by Src. These sites are all located between residues 132 and 414 of Cas, in a region that is required for binding to a number of other proteins including Crk. We tested synthetic peptides modeled on Cas phosphorylation sites, and found that the sequence containing tyrosine 253 was phosphorylated by Src most efficiently. Using cells derived from Cas-deficient mice, we confirmed that Cas greatly enhanced the ability of Src to transform cells. Phosphorylation of Cas on tyrosine 253 was not required for Src to increase growth rate, suppress contact inhibition, or suppress anchorage dependence. Yet, in contrast to these growth characteristics, phosphorylation of Cas on tyrosine 253 was required for Src to promote cell migration. Thus, a single phosphorylation site on this focal adhesion adaptor protein can effectively separate cell migration from other transformed growth characteristics.
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Affiliation(s)
- Gary S. Goldberg
- Department of Physiology and Biophysics, School of Medicine, State University of New York at Stony Brook, Stony Brook, New York 11794-8661
- To whom correspondence may be addressed: Dept. of Physiology and Biophysics, School of Medicine, Basic Science Tower T6, Health Science Complex, State University of New York at Stony Brook, Stony Brook, NY 11794-8661. Tel.: 631-444-3533; Fax: 631-444-3432; E-mail: or
| | - David B. Alexander
- Department of Physiology and Biophysics, School of Medicine, State University of New York at Stony Brook, Stony Brook, New York 11794-8661
| | - Patricia Pellicena
- Department of Physiology and Biophysics, School of Medicine, State University of New York at Stony Brook, Stony Brook, New York 11794-8661
| | - Zhong-Yin Zhang
- Department of Molecular Pharmacology, College of Medicine, Albert Einstein University, Bronx, New York 10461
| | - Hiroyuki Tsudal
- Division of Experimental Pathology and Chemotherapy, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan
| | - W. Todd Miller
- Department of Physiology and Biophysics, School of Medicine, State University of New York at Stony Brook, Stony Brook, New York 11794-8661
- To whom correspondence may be addressed: Dept. of Physiology and Biophysics, School of Medicine, Basic Science Tower T6, Health Science Complex, State University of New York at Stony Brook, Stony Brook, NY 11794-8661. Tel.: 631-444-3533; Fax: 631-444-3432; E-mail: or
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27
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Abassi YA, Rehn M, Ekman N, Alitalo K, Vuori K. p130Cas Couples the tyrosine kinase Bmx/Etk with regulation of the actin cytoskeleton and cell migration. J Biol Chem 2003; 278:35636-43. [PMID: 12832404 DOI: 10.1074/jbc.m306438200] [Citation(s) in RCA: 51] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022] Open
Abstract
Bmx/Etk, a member of the Tec/Btk family of nonreceptor kinases, has recently been shown to mediate cell motility in signaling pathways that become activated upon integrin-mediated cell adhesion (Chen, R., Kim, O., Li, M., Xiong, X., Guan, J. L., Kung, H. J., Chen, H., Shimizu, Y., and Qiu, Y. (2001) Nat Cell Biol. 3, 439-444). The molecular mechanisms of Bmx-induced cell motility have so far remained unknown. Previous studies by us and others have demonstrated that a complex formation between the docking protein p130Cas (Cas) and the adapter protein Crk is instrumental in connecting several stimuli to the regulation of actin cytoskeleton and cell motility. We demonstrate here that expression of Bmx leads to an interaction between Bmx and Cas at membrane ruffles, which are sites of active actin remodeling in motile cells. Expression of Bmx also enhances tyrosine phosphorylation of Cas and Cas.Crk complex formation, and coexpression of Bmx with Cas results in an enhanced membrane ruffling and haptotactic cell migration. Importantly, a mutant form of Bmx that fails to interact with Cas also fails to induce cell migration. Furthermore, expression of a dominant-negative form of Cas that is incapable of interacting with Crk inhibits Bmx-induced membrane ruffling and cell migration. These studies suggest that Bmx-Cas interaction, phosphorylation of Cas by Bmx, and subsequent Cas.Crk complex formation functionally couple Bmx to the regulation of actin cytoskeleton and cell motility.
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Affiliation(s)
- Yama A Abassi
- Cancer Research Center, The Burnham Institute, La Jolla, California 92037, USA
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28
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Wei L, Yang Y, Zhang X, Yu Q. Anchorage-independent phosphorylation of p130(Cas) protects lung adenocarcinoma cells from anoikis. J Cell Biochem 2003; 87:439-49. [PMID: 12397603 DOI: 10.1002/jcb.10322] [Citation(s) in RCA: 44] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
The regulation and function of the signaling adaptor protein p130(Cas) in tumor cell anchorage-independent survival, or anoikis resistance, were investigated in human lung adenocarcinoma cells. The tyrosine phosphorylation and function of p130(Cas) during cell detachment were analyzed in tumor cells and compared with that of normal epithelial cells. Cell detachment trigged rapid dephosphorylation of p130(Cas) in the nontumorigenic and anoikis-sensitive normal epithelial cells, but had no effect on the tyrosine phosphorylation of p130(Cas) in the anoikis-resistant lung adenocarcinoma cells. Further analysis revealed that the total tyrosine kinase activities associated with p130(Cas) in the lung tumor cells are anchorage-independent and are significantly higher than that in the normal cells, in which the p130(Cas)-associated tyrosine kinase activities are anchorage-dependent. Analysis of two known p130(Cas)-associated tyrosine kinases FAK and Src indicated that the regulation of tyrosine phosphorylation of FAK and Src are altered in the tumor cells. Inhibition of Src specifically abolished phosphorylation of p130(Cas) and induced anoikis. Furthermore, overexpression of dominant-negative forms of p130(Cas) also induced apoptosis. Taken together, these data suggest that p130(Cas) mediates a cell survival signal from cell-matrix interaction. Alterations in tumor cells that lead to constitutive phosphorylation of p130(Cas) can prevent cells from anoikis, hence contribute to tumor cell anchorage independence and metastasis.
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Affiliation(s)
- Lin Wei
- Pulmonary Center, Department of Biochemistry, Boston University Medical Center, Boston, Massachusetts 02118, USA
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29
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Li L, Guris DL, Okura M, Imamoto A. Translocation of CrkL to focal adhesions mediates integrin-induced migration downstream of Src family kinases. Mol Cell Biol 2003; 23:2883-92. [PMID: 12665586 PMCID: PMC152569 DOI: 10.1128/mcb.23.8.2883-2892.2003] [Citation(s) in RCA: 59] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022] Open
Abstract
The adapter protein Crk-Like (CrkL) can associate with the Src substrate p130(Cas) (Cas). The biological role of CrkL downstream of Cas, however, has been largely obscure. Consistent with the ability of CrkL to biochemically associate with Cas, we found that Src triggers translocation of CrkL to focal adhesions (FAs) in a manner dependent on Cas. Forced localization of CRKL to FAs (FA-CRKL) by itself was sufficient to induce activation of Rac1 and Cdc42 and rescued haptotaxis defects of mouse embryonic fibroblasts (MEFs) lacking Src, Yes, and Fyn, three broadly expressed Src family members required for integrin-induced migration. Consistent with Rac1 activation, FA-CRKL induced cotranslocation of a Rac1 activator, Dock1, to focal adhesions. These results therefore indicate a role for CrkL in mediating Src signaling by activating small G proteins at focal adhesions. Furthermore, MEFs lacking CrkL show impaired integrin-induced migration despite expression of a closely related protein, Crk-II, in these cells. These results therefore provide formal evidence that CrkL plays a specific role in integrin-induced migration as a downstream mediator of Src.
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Affiliation(s)
- Leiming Li
- Committee on Cell Physiology and The Ben May Institute for Cancer Research and Center for Molecular Oncology, The University of Chicago, Chicago, Illinois 60637, USA
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30
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Hoon Kim D, Jeon Choi S, Kook S, Kim W, Keun Song W. Phosphorylation-dependent cleavage of p130cas in apoptotic rat-1 cells. Biochem Biophys Res Commun 2003; 300:141-8. [PMID: 12480533 DOI: 10.1016/s0006-291x(02)02786-9] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/17/2023]
Abstract
We previously demonstrated caspase-mediated cleavage of p130cas during apoptosis and identified two caspase-3 cleavage sites [1]. In this study, we investigated the phosphorylation-dependent cleavage of p130cas in apoptotic Rat-1 fibroblast cells. Lysophosphatidic acid and fibronectin induced p130cas phosphorylation, which in turn resulted in resistance to caspase-mediated cleavage. Alternatively, dephosphorylation by calf intestinal alkaline phosphatase, PP1, and LAR stimulated cleavage of p130cas by caspase-3, generating a 31-kDa fragment. During apoptosis, p130cas dephosphorylation seems to precede its cleavage. The phosphorylation of tyrosine and serine residues immediately adjacent to the two cleavage sites (DVPD(416) and DSPD(748)) strongly affected p130cas cleavage by caspase-3, both in vitro and in vivo. Furthermore, the generation of the 31-kDa cleavage fragment was strongly regulated by phosphorylation of a tyrosine residue at position 751 (DSPD(748) and GQY(751)). Our results collectively suggest that degradation of p130cas during apoptosis is modulated in a phosphorylation-dependent manner.
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Affiliation(s)
- Do Hoon Kim
- Department of Life Science, Kwangju Institute of Science and Technology, 1 Oryong-dong, Puk-gu, South Korea
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Miyake I, Hakomori Y, Shinohara A, Gamou T, Saito M, Iwamatsu A, Sakai R. Activation of anaplastic lymphoma kinase is responsible for hyperphosphorylation of ShcC in neuroblastoma cell lines. Oncogene 2002; 21:5823-34. [PMID: 12185581 DOI: 10.1038/sj.onc.1205735] [Citation(s) in RCA: 88] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/18/2002] [Revised: 05/14/2002] [Accepted: 06/07/2002] [Indexed: 01/26/2023]
Abstract
Shc family of docking proteins, ShcA, ShcB and ShcC, play roles in cellular signal transduction by binding to phosphotyrosine residues of various activated receptor tyrosine kinases. Both ShcB and ShcC proteins are selectively expressed in the neural system of adult mouse tissues. In most of neuroblastoma cells, obvious tyrosine phosphorylation of ShcC was observed, whereas expression of ShcB was considerably low. Phosphoproteins associated with hyperphosphorylated ShcC were purified from neuroblastoma cell lines, and identified by mass-spectrometry. Anaplastic lymphoma kinase (ALK), which turned out to be one of these phosphoproteins, was constitutively activated and associated with the PTB domain of ShcC in three neuroblastoma cells. In vitro kinase assay revealed that ShcC is a potent substrate of the activated ALK kinase. The ALK gene locus was significantly amplified in both of these cell lines, suggesting that gene amplification leads to constitutive activation of the ALK kinase, which results in hyperphosphorylation of ShcC. Constitutive activation of ALK appeared to interfere with signals from other receptor tyrosine kinases. ALK-ShcC signal activation, possibly caused by co-amplification with the N-myc gene, might give additional effects on malignant tumor progression of neuroblastoma.
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Affiliation(s)
- Izumi Miyake
- Cancer Signal Transduction Project, National Cancer Center Research Institute, 5-1-1 Tsukuji, Chuo-ku, Tokyo 104-0045, Japan
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Su JM, Gui L, Zhou YP, Zha XL. Expression of focal adhesion kinase and α5 and β1 integrins in carcinomas and its clinical significance. World J Gastroenterol 2002; 8:613-8. [PMID: 12174366 PMCID: PMC4656308 DOI: 10.3748/wjg.v8.i4.613] [Citation(s) in RCA: 44] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To detect the expression pattern of FAK (focal adhesion kinase) and integrin α5 and β1 subunits in different kinds of cancerous tissues and to study their correlation with clinicopathological data including tumor type, grade and lymph node status.
METHODS: Using an immunohistochemical technique, we examined the expression of FAK and integrin and 1 subunits in cancerous and noncancerous tissues obtained from 75 patients with gastric carcinomas, 21 colorectal carcinomas, 16 hepatocellular carcinomas, 20 uterocervical carcinomas, and 20 breast carcinomas.
RESULTS: The staining of FAK was stronger in cancerous than in noncancerous areas. Enhanced expression of FAKwas detected in poor-differentiated carcinoma of the stomach and colorectum. Tumors with lymph node metastases had more FAK protein than those without metastases. In addition, the deeper the extent of tumor infiltration, the higher the FAK expression. The expression of integrin α5 and β1 subunits was lower in cancerous areas than in noncancerous areas, but it was higher in well-differentiated cancerous tissues than in poor differentiated tissues. The relationship between the expression of integrin α5 and β1 subunits and infiltration or metastasis was not significant. Cancerous tissues with stronger FAK expression (++ or +++) also had a higher expression of integrin α5 and β1 subunits in the tumor and its unaffected margins.
CONCLUSION: FAK is a better marker for carcinogenesis and the progression of cancer than integrin α5 or β1 subunit, and it may be not only a transformation-linked enzyme but also a progression-linked enzyme.
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Affiliation(s)
- Jian-Min Su
- Department of Biochemistry, FuDan University Medical Center, 138 Yixueyuan Road, Shanghai 200032, China
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Sarkar S, Svoboda M, de Beaumont R, Freedman AS. The role of Aktand RAFTK in beta1 integrin mediated survival of precursor B-acute lymphoblastic leukemia cells. Leuk Lymphoma 2002; 43:1663-71. [PMID: 12400610 DOI: 10.1080/1042819021000003009] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/28/2022]
Abstract
In this study, we report that the related adhesion focal tyrosine kinase RAFTK, is an upstream kinase in beta1 integrin mediated activation of Akt. Stimulation through beta1 integrins by fibronectin reversed apoptosis induced by adriamycin. Inhibitors of phosphatidylinositol 3-kinase (PI3 kinase)/Akt (LY 294002), tyrosine kinases (Herbimycin-A) and the cytotoxic agent adriamycin induced apoptosis of REH cells. beta1 integrin ligation induced activation of Akt, and tyrosine phosphorylation of RAFTK and FAK, but not SYK in REH cells. This suggested that RAFTK and FAK activation might be linked to Akt activation. Evidence that RAFTK is a modulator of Akt came from phorbol myristic acetate (PMA) stimulation. RAFTK and Akt were activated but FAK was not. Using fibroblasts from FAK -/- mice, which express high levels of RAFTK, fibronectin plating enhanced Akt activation. Pretreatment of REH cells with a P13 kinase/Akt inhibitor LY 294002 did not inhibit RAFTK tyrosine phosphorylation showing that RAFTK is upstream of P13k/Akt. Further evidence for a link between RAFTK tyrosine phosphorylation and Akt activation was the observation that the p85 subunit of P13 kinase associated with RAFTK following integrin ligation in REH cells. These results suggest that RAFTK plays an anti-apoptotic role through the activation of Akt.
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Affiliation(s)
- Sibaji Sarkar
- Department of Adult Oncology, Dana-Farber Cancer Institute, Boston, MA 02115 USA
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Huang J, Hamasaki H, Nakamoto T, Honda H, Hirai H, Saito M, Takato T, Sakai R. Differential regulation of cell migration, actin stress fiber organization, and cell transformation by functional domains of Crk-associated substrate. J Biol Chem 2002; 277:27265-72. [PMID: 12011056 DOI: 10.1074/jbc.m203063200] [Citation(s) in RCA: 60] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023] Open
Abstract
The Crk-associated substrate (Cas) is a unique docking protein that possesses a repetitive stretch of tyrosine-containing motifs and an Src homology 3 (SH3) domain. Embryonic fibroblasts lacking Cas demonstrated resistance to Src-induced transformation along with impaired actin bundling and cell motility, indicating critical roles of Cas in actin cytoskeleton organization, cell migration, and oncogenesis. To gain further insight into roles of each domain of Cas in these processes, a compensation assay was performed by expressing a series of Cas mutants in Cas-deficient fibroblasts. The results showed that motifs containing YDxP were indispensable for actin cytoskeleton organization and cell migration, suggesting that CrkII-mediated signaling regulates these biological processes. The C-terminal Src-binding domain played essential roles in cell migration and membrane localization of Cas, although it was dispensable in the organization of actin stress fibers. Furthermore, the Src-binding domain was also a prerequisite for Src transformation possibly, because of its crucial role in the phosphorylation of Cas during transformation. Overall, differential uses of the Cas domains in individual biological processes were demonstrated.
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Affiliation(s)
- Jinhong Huang
- Cancer Signal Transduction Project, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045
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Kirsch KH, Kensinger M, Hanafusa H, August A. A p130Cas tyrosine phosphorylated substrate domain decoy disrupts v-crk signaling. BMC Cell Biol 2002; 3:18. [PMID: 12119061 PMCID: PMC117778 DOI: 10.1186/1471-2121-3-18] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/17/2002] [Accepted: 07/15/2002] [Indexed: 01/22/2023] Open
Abstract
BACKGROUND The adaptor protein p130Cas (Cas) has been shown to be involved in different cellular processes including cell adhesion, migration and transformation. This protein has a substrate domain with up to 15 tyrosines that are potential kinase substrates, able to serve as docking sites for proteins with SH2 or PTB domains. Cas interacts with focal adhesion plaques and is phosphorylated by the tyrosine kinases FAK and Src. A number of effector molecules have been shown to interact with Cas and play a role in its function, including c-crk and v-crk, two adaptor proteins involved in intracellular signaling. Cas function is dependent on tyrosine phosphorylation of its substrate domain, suggesting that tyrosine phosphorylation of Cas in part regulates its control of adhesion and migration. To determine whether the substrate domain alone when tyrosine phosphorylated could signal, we have constructed a chimeric Cas molecule that is phosphorylated independently of upstream signals. RESULTS We found that a tyrosine phosphorylated Cas substrate domain acts as a dominant negative mutant by blocking Cas-mediated signaling events, including JNK activation by the oncogene v-crk in transient and stable lines and v-crk transformation. This block was the result of competition for binding partners as the chimera competed for binding to endogenous c-crk and exogenously expressed v-crk. CONCLUSION Our approach suggests a novel method to study adaptor proteins that require phosphorylation, and indicates that mere tyrosine phosphorylation of the substrate domain of Cas is not sufficient for its function.
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Affiliation(s)
- Kathrin H Kirsch
- Laboratory of Molecular Oncology, The Rockefeller University, NY, NY, 10021, USA
- Department of Biochemistry, Boston University School of Medicine, 715 Albany Street, K-225, Boston, MA 02118, USA
| | - Margaret Kensinger
- Immunology Research Laboratories, Department of Veterinary Science, Penn State University, 115 Henning Building, University Park, PA 16802, USA
| | - Hidesaburo Hanafusa
- Laboratory of Molecular Oncology, The Rockefeller University, NY, NY, 10021, USA
- Osaka Bioscience Institute, 6-2-4 Furuedai, Suita, Osaka 565-0874, Japan
| | - Avery August
- Immunology Research Laboratories, Department of Veterinary Science, Penn State University, 115 Henning Building, University Park, PA 16802, USA
- Laboratory of Molecular Oncology, The Rockefeller University, NY, NY, 10021, USA
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Ruest PJ, Shin NY, Polte TR, Zhang X, Hanks SK. Mechanisms of CAS substrate domain tyrosine phosphorylation by FAK and Src. Mol Cell Biol 2001; 21:7641-52. [PMID: 11604500 PMCID: PMC99935 DOI: 10.1128/mcb.21.22.7641-7652.2001] [Citation(s) in RCA: 130] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022] Open
Abstract
Tyrosine phosphorylation of CAS (Crk-associated substrate, p130(Cas)) has been implicated as a key signaling step in integrin control of normal cellular behaviors, including motility, proliferation, and survival. Aberrant CAS tyrosine phosphorylation may contribute to cell transformation by certain oncoproteins, including v-Crk and v-Src, and to tumor growth and metastasis. The CAS substrate domain (SD) contains 15 Tyr-X-X-Pro motifs, which are thought to represent the major tyrosine phosphorylation sites and to function by recruiting downstream signaling effectors, including c-Crk and Nck. CAS makes multiple interactions, direct and indirect, with the tyrosine kinases Src and focal adhesion kinase (FAK), and as a result of this complexity, several plausible models have been proposed for the mechanism of CAS-SD phosphorylation. The objective of this study was to provide experimental tests of these models in order to determine the most likely mechanism(s) of CAS-SD tyrosine phosphorylation by FAK and Src. In vitro kinase assays indicated that FAK has a very poor capacity to phosphorylate CAS-SD, relative to Src. However, FAK expression along with Src was found to be important for achieving high levels of CAS tyrosine phosphorylation in COS-7 cells, as well as recovery of CAS-associated Src activity toward the SD. Structure-functional studies for both FAK and CAS further indicated that FAK plays a major role in regulating CAS-SD phosphorylation by acting as a docking or scaffolding protein to recruit Src to phosphorylate CAS, while a secondary FAK-independent mechanism involves Src directly bound to the CAS Src-binding domain (SBD). Our results do not support models in which FAK either phosphorylates CAS-SD directly or phosphorylates CAS-SBD to promote Src binding to this site.
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Affiliation(s)
- P J Ruest
- Department of Cell Biology, Vanderbilt University School of Medicine, Nahville, Tennessee 37232, USA
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37
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Abstract
Crk family adaptors are widely expressed and mediate the timely formation of signal transduction protein complexes upon a variety of extracellular stimuli, including various growth and differentiation factors. Selective formation of multi-protein complexes by the Crk and Crk-like (CRKL) proteins depends on specific motifs recognized by their SH2 and SH3 domains. In the case of the first SH3 domains [SH3(1)] a P-x-x-P-x-K motif is crucial for highly selective binding, while the SH2 domains prefer motifs which conform to the consensus pY-x-x-P. Crk family proteins are involved in the relocalization and activation of several different effector proteins which include guanine nucleotide releasing proteins like C3G, protein kinases of the Abl- and GCK-families and small GTPases like Rap1 and Rac. Crk-type proteins have been found not only in vertebrates but also in flies and nematodes. Major insight into the function of Crk within organisms came from the genetic model organism C. elegans, where the Crk-homologue CED-2 regulates cell engulfment and phagocytosis. Other biological outcomes of the Crk-activated signal transduction cascades include the modulation of cell adhesion, cell migration and immune cell responses. Crk family adaptors also appear to play a role in mediating the action of human oncogenes like the leukaemia-inducing Bcr-Abl protein. This review summarizes some key findings and highlights recent insights and open questions.
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Affiliation(s)
- S M Feller
- Cell Signalling Laboratory, Imperial Cancer Research Fund, University of Oxford, Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DS, UK.
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Pellicena P, Miller WT. Processive phosphorylation of p130Cas by Src depends on SH3-polyproline interactions. J Biol Chem 2001; 276:28190-6. [PMID: 11389136 DOI: 10.1074/jbc.m100055200] [Citation(s) in RCA: 95] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Many in vivo substrates of Src family tyrosine kinases possess sequences conforming to Src homology 2 and 3 (SH2 and SH3) domain-binding motifs. One such substrate is p130Cas, a protein that is hyperphosphorylated in v-Src transformed cells. Cas contains a substrate domain consisting of 15 potential tyrosine phosphorylation sites, C- and N-terminal polyproline regions fitting the consensus sequence for SH3 domain ligands, and a YDYV motif that binds the Src SH2 domain when phosphorylated. In an effort to understand the mechanisms of processive phosphorylation, we have explored the regions of Cas necessary for interaction with Src using the yeast two-hybrid system. Mutations in the SH2 domain-binding region of Cas or the Src SH2 domain have little effect in Cas-Src complex formation or phosphorylation. However, disruption of the C-terminal polyproline region of Cas completely abolishes interaction between the two proteins and results in impaired phosphorylation of Cas. Kinetic analyses using purified proteins indicated that multisite phosphorylation of Cas by Src follows a processive rather than a distributive mechanism. Furthermore, the kinetic studies show that there are two properties of the polyproline region of Cas that are important in enhancing substrate phosphorylation. First, the C-terminal polyproline serves to activate Src kinases through the process of SH3 domain displacement. Second, this region aids in anchoring the kinase to Cas to facilitate processive phosphorylation of the substrate domain. The two processes combine to ensure phosphorylation of Cas with high efficiency.
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Affiliation(s)
- P Pellicena
- Department of Physiology and Biophysics, School of Medicine, State University of New York at Stony Brook, Stony Brook, New York 11794-8661, USA
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Sinnett-Smith J, Lunn JA, Leopoldt D, Rozengurt E. Y-27632, an inhibitor of Rho-associated kinases, prevents tyrosine phosphorylation of focal adhesion kinase and paxillin induced by bombesin: dissociation from tyrosine phosphorylation of p130(CAS). Exp Cell Res 2001; 266:292-302. [PMID: 11399057 DOI: 10.1006/excr.2001.5219] [Citation(s) in RCA: 64] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
A rapid increase in tyrosine phosphorylation of focal adhesion kinase (FAK), paxillin, and Crk-associated substrate (CAS) are prominent early events triggered by many G protein-coupled receptors (GPCRs), but the mechanisms involved remain unclear. Here, we examined whether the Rho-associated protein serine/threonine kinase family (ROCK) is a critical Rho effector in the pathway that links GPCR activation to the tyrosine phosphorylation of FAK, CAS, and paxillin. Treatment of Swiss 3T3 cells with Y-27632, a preferential inhibitor of ROCK, dramatically inhibited the formation of actin stress fibers, the assembly of focal contacts, and the increase in tyrosine phosphorylation of FAK and paxillin induced by bombesin in these cells. Surprisingly, we found that treatment with Y-27632 did not produce any detectable effect on bombesin-elicited CAS tyrosine phosphorylation even at the highest concentrations of Y-27632 tested. HA-1077, a preferential inhibitor of ROCK activity structurally unrelated to Y-27632, also attenuated the increase in the tyrosine phosphorylation of FAK and paxillin but did not affect the tyrosine phosphorylation of CAS induced by bombesin in Swiss 3T3 cells. The results demonstrate that ROCK-dependent tyrosine phosphorylation of FAK and paxillin can be dissociated from a ROCK-independent pathway leading to tyrosine phosphorylation of CAS.
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Affiliation(s)
- J Sinnett-Smith
- Department of Medicine, School of Medicine and Molecular Biology Institute, University of California, Los Angeles, 90095-1786, USA
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40
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Reiske HR, Zhao J, Han DC, Cooper LA, Guan JL. Analysis of FAK-associated signaling pathways in the regulation of cell cycle progression. FEBS Lett 2000; 486:275-80. [PMID: 11119718 DOI: 10.1016/s0014-5793(00)02295-x] [Citation(s) in RCA: 41] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
Focal adhesion kinase (FAK) is an important mediator of signal transduction pathways initiated by integrins in cell migration, survival and cell cycle regulation. The ability of FAK to mediate integrin signaling in the regulation of cell cycle progression depends on the phosphorylation of Tyr397, which implies a functional significance for the formation of FAK signaling complexes with Src, phosphatidylinositol-3-kinase (PI3K) and Grb7. We have previously described a FAK mutant, D395A, that selectively disrupts FAK binding to PI3K, but allows FAK association with Src. Using this mutation in a mislocalized FAK mutant background, we show here that formation of a FAK/PI3K complex is not sufficient for cell cycle progression but the formation of a FAK/Src complex plays an essential role. We also show that mutation of D395 to A disrupted FAK association with Grb7. This suggests that a FAK/Grb7 complex is not involved in the cell cycle regulation either, which is supported by direct analysis of cells expressing a dominant negative Grb7 construct. Finally, we provide evidence that the Src-dependent association of FAK with Grb2 and p130(Cas) are both required for the regulation of cell cycle progression by FAK. Together, these studies identify important FAK downstream signaling pathways in cell cycle regulation.
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Affiliation(s)
- H R Reiske
- Cancer Biology Laboratories, Department of Molecular Medicine, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA
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41
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Escalante M, Courtney J, Chin WG, Teng KK, Kim JI, Fajardo JE, Mayer BJ, Hempstead BL, Birge RB. Phosphorylation of c-Crk II on the negative regulatory Tyr222 mediates nerve growth factor-induced cell spreading and morphogenesis. J Biol Chem 2000; 275:24787-97. [PMID: 10825157 DOI: 10.1074/jbc.m000711200] [Citation(s) in RCA: 44] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
The Crk family of adaptor proteins participate in diverse signaling pathways that regulate growth factor-induced proliferation, anchorage-dependent DNA synthesis, and cytoskeletal reorganization, important for cell adhesion and motility. Using kidney epithelial 293T cells for transient co-transfection studies and the nerve growth factor (NGF)-responsive PC12 cell line as a model system for neuronal morphogenesis, we demonstrate that the non-receptor tyrosine kinase c-Abl is an intermediary for NGF-inducible c-Crk II phosphorylation on the negative regulatory Tyr(222). Transient expression of a c-Crk II Tyr(222) point mutant (c-Crk Y222F) in 293T cells induces hyperphosphorylation of paxillin on Tyr(31) and enhances complex formation between c-Crk Y222F and paxillin as well as c-Crk Y222F and c-Abl, suggesting that c-Crk II Tyr(222) phosphorylation induces both the dissociation of the Crk SH2 domain from paxillin and the Crk SH3 domain from c-Abl. Interestingly, examination of the early kinetics of NGF stimulation in PC12 cells showed that c-Crk II Tyr(222) phosphorylation preceded paxillin Tyr(31) phosphorylation, followed by a transient initial dissociation of the c-Crk II paxillin complex. PC12 cells overexpressing c-Crk Y222F manifested a defect in cellular adhesion and neuritogenesis that led to detachment of cells from the extracellular matrix, thus demonstrating the biological significance of c-Crk II tyrosine phosphorylation in NGF-dependent morphogenesis. Whereas previous studies have shown that Crk SH2 binding to paxillin is critical for cell adhesion and migration, our data show that the phosphorylation cycle of c-Crk II determines its dynamic interaction with paxillin, thereby regulating turnover of multiprotein complexes, a critical aspect of cytoskeletal plasticity and actin dynamics.
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Affiliation(s)
- M Escalante
- Laboratory of Molecular Oncology, The Rockefeller University, New York, New York 10021, USA
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Buist A, Blanchetot C, Tertoolen LG, den Hertog J. Identification of p130cas as an in vivo substrate of receptor protein-tyrosine phosphatase alpha. J Biol Chem 2000; 275:20754-61. [PMID: 10787408 DOI: 10.1074/jbc.m001626200] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
We have employed a substrate trapping strategy to identify physiological substrates of the receptor protein-tyrosine phosphatase alpha (RPTPalpha). Here we report that a substrate-trapping mutant of the RPTPalpha membrane proximal catalytic domain (D1), RPTPalpha-D1-C433S, specifically bound to tyrosine-phosphorylated proteins from pervanadate-treated cells. The membrane distal catalytic domain of RPTPalpha (D2) and mutants thereof did not bind to tyrosine-phosphorylated proteins. The pattern of tyrosine-phosphorylated proteins that bound to RPTPalpha-D1-C433S varied between cell lines, but a protein of approximately 130 kDa was pulled down from every cell line. This protein was identified as p130(cas). Tyrosine-phosphorylated p130(cas) from fibronectin-stimulated NIH3T3 cells bound to RPTPalpha-D1-C433S as well, suggesting that p130(cas) is a physiological substrate of RPTPalpha. RPTPalpha dephosphorylated p130(cas) in vitro, and RPTPalpha co-localized with a subpopulation of p130(cas) to the plasma membrane. Co-transfection experiments with activated SrcY529F, p130(cas), and RPTPalpha or inactive, mutant RPTPalpha indicated that RPTPalpha dephosphorylated p130(cas) in vivo. Tyrosine-phosphorylated epidermal growth factor receptor was not dephosphorylated by RPTPalpha under these conditions, suggesting that p130(cas) is a specific substrate of RPTPalpha in living cells. In conclusion, our results provide evidence that p130(cas) is a physiological substrate of RPTPalpha in vivo.
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Affiliation(s)
- A Buist
- Hubrecht Laboratory, Netherlands Institute for Developmental Biology, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands
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43
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Nishio H, Matsui K, Tsuji H, Tamura A, Suzuki K. Immunohistochemical study of tyrosine phosphorylation signaling in Hassall's corpuscles of the human thymus. Acta Histochem 1999; 101:421-9. [PMID: 10611930 DOI: 10.1016/s0065-1281(99)80042-7] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/28/2022]
Abstract
Tyrosine phosphorylation signaling has been reported to play a key role in thymocyte development. However, the physiological role of signaling in thymus stroma is poorly understood, and there is lack of information on the in situ localization of elements of the signaling pathway in thymus stroma. In the present study, we have found by immunohistochemical analysis that tyrosine-phosphorylated proteins are present in high amounts in Hassall's corpuscles of the thymus medulla. Hassall's corpuscles represent end stages of maturation of thymic medullary epithelium. We have also investigated the localization of the src family that is involved in tyrosine phosphorylation signaling in Hassall's corpuscles. A member of the src family protein tyrosine kinases, p59fyn, was shown to be abundantly expressed in the outer layer of Hassall's corpuscles. Another member of the family, p60c-src, was highly expressed in the entire Hassall's corpuscles. Furthermore, p50csk and p130cas, both of which are involved in the pathway, were shown to be preferably expressed in the outer layer of Hassall's corpuscles. These findings suggest that tyrosine phosphorylation signaling may play a role in thymic medullary epithelial maturation and that the src family is involved in the process.
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Affiliation(s)
- H Nishio
- Department of Legal Medicine, Osaka Medical College, Takatsuki, Japan
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44
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Owen JD, Ruest PJ, Fry DW, Hanks SK. Induced focal adhesion kinase (FAK) expression in FAK-null cells enhances cell spreading and migration requiring both auto- and activation loop phosphorylation sites and inhibits adhesion-dependent tyrosine phosphorylation of Pyk2. Mol Cell Biol 1999; 19:4806-18. [PMID: 10373530 PMCID: PMC84279 DOI: 10.1128/mcb.19.7.4806] [Citation(s) in RCA: 319] [Impact Index Per Article: 12.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/09/1998] [Accepted: 04/22/1999] [Indexed: 12/29/2022] Open
Abstract
Focal adhesion kinase (FAK) is a nonreceptor protein tyrosine kinase involved in integrin-mediated control of cell behavior. Following cell adhesion to components of the extracellular matrix, FAK becomes phosphorylated at multiple sites, including tyrosines 397, 576, and 577. Tyr-397 is an autophosphorylation site that promotes interaction with c-Src or Fyn. Tyr-576 and Tyr-577 lie in the putative activation loop of the kinase domain, and FAK catalytic activity may be elevated through phosphorylation of these residues by associated Src family kinase. Recent studies have implicated FAK as a positive regulator of cell spreading and migration. To further study the mechanism of adhesion-induced FAK activation and the possible role and signaling requirements for FAK in cell spreading and migration, we utilized the tetracycline repression system to achieve inducible expression of either wild-type FAK or phosphorylation site mutants in fibroblasts derived from FAK-null mouse embryos. Using these Tet-FAK cells, we demonstrated that both the FAK autophosphorylation and activation loop sites are critical for maximum adhesion-induced FAK activation and FAK-enhanced cell spreading and migration responses. Negative effects on cell spreading and migration, as well as decreased phosphorylation of the substrate p130(Cas), were observed upon induced expression of the FAK autophosphorylation site mutant. These negative effects appear to result from an inhibition of integrin-mediated signaling by the FAK-related kinase Pyk2/CAKbeta/RAFTK/CadTK.
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Affiliation(s)
- J D Owen
- Department of Cell Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA
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45
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Schlaepfer DD, Hauck CR, Sieg DJ. Signaling through focal adhesion kinase. PROGRESS IN BIOPHYSICS AND MOLECULAR BIOLOGY 1999; 71:435-78. [PMID: 10354709 DOI: 10.1016/s0079-6107(98)00052-2] [Citation(s) in RCA: 940] [Impact Index Per Article: 36.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
Integrin receptor binding to extracellular matrix proteins generates intracellular signals via enhanced tyrosine phosphorylation events that are important for cell growth, survival, and migration. This review will focus on the functions of the focal adhesion kinase (FAK) protein-tyrosine kinase (PTK) and its role in linking integrin receptors to intracellular signaling pathways. FAK associates with several different signaling proteins such as Src-family PTKs, p130Cas, Shc, Grb2, PI 3-kinase, and paxillin. This enables FAK to function within a network of integrin-stimulated signaling pathways leading to the activation of targets such as the ERK and JNK/mitogen-activated protein kinase pathways. Focus will be placed on the structural domains and sites of FAK tyrosine phosphorylation important for FAK-mediated signaling events and how these sites are conserved in the FAK-related PTK, Pyk2. We will review what is known about FAK activation by integrin receptor-mediated events and also non-integrin stimuli. In addition, we discuss the emergence of a consensus FAK substrate phosphorylation sequence. Emphasis will also be placed on the role of FAK in generating cell survival signals and the cleavage of FAK during caspase-mediated apoptosis. An in-depth discussion will be presented of integrin-stimulated signaling events occurring in the FAK knockout fibroblasts (FAK-) and how these cells exhibit deficits in cell migration. FAK re-expression in the FAK- cells confirms the role of this PTK in the regulation of cell morphology and in promoting cell migration events. In addition, these results reinforce the potential role for FAK in promoting an invasive phenotype in human tumors.
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Affiliation(s)
- D D Schlaepfer
- Scripps Research Institute, Department of Immunology, La Jolla, CA 92037, USA.
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Weng LP, Wang X, Yu Q. Transmembrane tyrosine phosphatase LAR induces apoptosis by dephosphorylating and destabilizing p130Cas. Genes Cells 1999; 4:185-96. [PMID: 10320483 DOI: 10.1046/j.1365-2443.1999.00251.x] [Citation(s) in RCA: 44] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
BACKGROUND LAR is a transmembrane receptor-like protein tyrosine phosphatase (PTP). Genetic studies of Drosophila LAR suggest that LAR may function to regulate cell adhesions or adhesion-mediated signal transduction. The over-expression of LAR in mammalian tissue culture cells does not affect cell adhesion but induces caspase-dependent apoptosis. This study investigates molecular mechanisms of LAR-induced apoptosis by searching for in vivo substrates of LAR which are responsible for LAR-induced apoptosis. RESULTS The over-expression of LAR in tissue culture cells specifically decreased the steady state protein level of p130Cas, a multifunctional signal assembly protein in signal transduction, by reducing the tyrosine phosphorylation and protein stability of p130Cas. The reduction of p130Cas protein level could be inhibited by tyrosine phosphatase inhibitors. Phosphatase domain-deleted mutant LARs had no effect on p130Cas. LAR also preferentially dephosphorylated p130Cas in vitro. Subcellularly, LAR and p130Cas were co-localized along stress fibres and at focal adhesions. LAR over-expression eliminated p130Cas from focal adhesions without affecting focal adhesion assembly. Restoring the level of p130Cas alleviated LAR-induced apoptosis. CONCLUSIONS p130Cas is an in vivo substrate of LAR. LAR specifically dephosphorylates and destabilizes p130Cas and may play a role in regulating cell adhesion-mediated cell survival. The function of p130Cas in focal adhesions may not be to regulate focal adhesion assembly and cell adhesion but rather to transduce the cell adhesion-generated signals which are essential for cell survival.
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Affiliation(s)
- L P Weng
- Pulmonary Center, Department of Medicine, and Department of Biochemistry, Boston University Medical Center, Boston, Massachusetts 02118, USA
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Garcia-Guzman M, Dolfi F, Russello M, Vuori K. Cell adhesion regulates the interaction between the docking protein p130(Cas) and the 14-3-3 proteins. J Biol Chem 1999; 274:5762-8. [PMID: 10026197 DOI: 10.1074/jbc.274.9.5762] [Citation(s) in RCA: 62] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022] Open
Abstract
Integrin ligand binding induces a signaling complex formation via the direct association of the docking protein p130(Cas) (Cas) with diverse molecules. We report here that the 14-3-3zeta protein interacts with Cas in the yeast two-hybrid assay. We also found that the two proteins associate in mammalian cells and that this interaction takes place in a phosphoserine-dependent manner, because treatment of Cas with a serine phosphatase greatly reduced its ability to bind 14-3-3zeta. Furthermore, the Cas-14-3-3zeta interaction was found to be regulated by integrin-mediated cell adhesion. Thus, when cells are detached from the extracellular matrix, the binding of Cas to 14-3-3zeta is greatly diminished, whereas replating the cells onto fibronectin rapidly induces the association. Consistent with these results, we found that the subcellular localization of Cas and 14-3-3 is also regulated by integrin ligand binding and that the two proteins display a significant co-localization during cell attachment to the extracellular matrix. In conclusion, our results demonstrate that 14-3-3 proteins participate in integrin-activated signaling pathways through their interaction with Cas, which, in turn, may contribute to important biological responses regulated by cell adhesion to the extracellular matrix.
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Affiliation(s)
- M Garcia-Guzman
- La Jolla Cancer Research Center, The Burnham Institute, La Jolla, California 92037, USA
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Sieg DJ, Ilić D, Jones KC, Damsky CH, Hunter T, Schlaepfer DD. Pyk2 and Src-family protein-tyrosine kinases compensate for the loss of FAK in fibronectin-stimulated signaling events but Pyk2 does not fully function to enhance FAK- cell migration. EMBO J 1998; 17:5933-47. [PMID: 9774338 PMCID: PMC1170921 DOI: 10.1093/emboj/17.20.5933] [Citation(s) in RCA: 278] [Impact Index Per Article: 10.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022] Open
Abstract
The focal adhesion kinase (FAK) protein-tyrosine kinase (PTK) links transmembrane integrin receptors to intracellular signaling pathways. We show that expression of the FAK-related PTK, Pyk2, is elevated in fibroblasts isolated from murine fak-/- embryos (FAK-) compared with cells from fak+/+ embryos (FAK+). Pyk2 was localized to perinuclear regions in both FAK+ and FAK- cells. Pyk2 tyrosine phosphorylation was enhanced by fibronectin (FN) stimulation of FAK- but not FAK+ cells. Increased Pyk2 tyrosine phosphorylation paralleled the time-course of Grb2 binding to Shc and activation of ERK2 in FAK- cells. Pyk2 in vitro autophosphorylation activity was not enhanced by FN plating of FAK- cells. However, Pyk2 associated with active Src-family PTKs after FN but not poly-L-lysine replating of the FAK- cells. Overexpression of both wild-type (WT) and kinase-inactive (Ala457), but not the autophosphorylation site mutant (Phe402) Pyk2, enhanced endogenous FN-stimulated c-Src in vitro kinase activity in FAK- cells, but only WT Pyk2 overexpression enhanced FN-stimulated activation of co-transfected ERK2. Interestingly, Pyk2 overexpression only weakly augmented FAK- cell migration to FN whereas transient FAK expression promoted FAK- cell migration to FN efficiently compared with FAK+ cells. Significantly, repression of endogenous Src-family PTK activity by p50(csk) overexpression inhibited FN-stimulated cell spreading, Pyk2 tyrosine phosphorylation, Grb2 binding to Shc, and ERK2 activation in the FAK- but not in FAK+ cells. These studies show that Pyk2 and Src-family PTKs combine to promote FN-stimulated signaling events to ERK2 in the absence of FAK, but that these signaling events are not sufficient to overcome the FAK- cell migration defects.
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Affiliation(s)
- D J Sieg
- The Scripps Research Institute, Department of Immunology, IMM-26, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA
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Sorokin A, Reed E. Insulin stimulates the tyrosine dephosphorylation of docking protein p130cas (Crk-associated substrate), promoting the switch of the adaptor protein crk from p130cas to newly phosphorylated insulin receptor substrate-1. Biochem J 1998; 334 ( Pt 3):595-600. [PMID: 9729467 PMCID: PMC1219728 DOI: 10.1042/bj3340595] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
The docking protein p130(cas) (Crk-associated substrate) forms a stable complex with the adaptor protein CrkII in a tyrosine-phosphorylation-dependent manner. Insulin-induced tyrosine phosphorylation of insulin receptor substrates results in the redistribution of CrkII between p130(cas) and insulin receptor substrate-1. A decrease in the association between CrkII and p130(cas) in response to insulin stimulation was detected in CHO cells stably expressing insulin receptor or insulin receptor substrate-1, and in L6 rat myoblasts. Along with the decrease in the association of CrkII with p130(cas), the amount of tyrosine-phosphorylated insulin receptor substrate-1 co-precipitated with CrkII increased in all cell types studied. The insulin-induced decrease in the CrkII-p130(cas) association was further confirmed by Far Western Blot analysis with the Src homology 2 (SH2) domain of CrkII. Insulin regulates the association of CrkII with p130(cas) by tyrosine dephosphorylation of p130(cas) and co-ordinated tyrosine phosphorylation of insulin receptor substrate-1. Tyrosine-phosphorylated insulin receptor substrate-1 serves as a docking protein for multiple adaptor proteins and competes with p130(cas) for CrkII.
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Affiliation(s)
- A Sorokin
- Department of Medicine and Cardiovascular Research Center, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
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Nakamura I, Jimi E, Duong LT, Sasaki T, Takahashi N, Rodan GA, Suda T. Tyrosine phosphorylation of p130Cas is involved in actin organization in osteoclasts. J Biol Chem 1998; 273:11144-9. [PMID: 9556601 DOI: 10.1074/jbc.273.18.11144] [Citation(s) in RCA: 105] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Integrin-mediated interaction with the extracellular matrix plays a critical role in the function of osteoclasts, the bone-resorbing cells. This study examines the role of p130Cas (Crk-associated substrate (Cas)) in actin organization in osteoclasts. Multinucleated osteoclast-like cells (OCLs) were obtained in a co-culture of murine bone marrow cells and primary osteoblasts. After plating on culture dishes, OCLs formed a ringlike structure consisting of F-actin dots at cell periphery (actin ring). The percentage of OCLs with actin rings and its diameter increased with time and cell spreading. Tyrosine phosphorylation of a protein (p130) increased with actin ring formation. Treatment with cytochalasin D disrupted actin rings and reduced tyrosine phosphorylation of p130. Using specific antibodies, p130 was identified as Cas. By immunocytochemistry, Cas was localized to the peripheral regions of OCLs and its distribution overlapped that of F-actin. In OCLs derived from Src(-/-) mice, in which osteoclast activity is severely compromised, tyrosine phosphorylation of Cas was markedly reduced. Moreover, Cas was diffusely distributed in the cytoplasm and actin ring formation is not observed. These findings suggest that Src-dependent tyrosine phosphorylation of Cas is involved in the adhesion-induced actin organization associated with osteoclast activation.
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Affiliation(s)
- I Nakamura
- Department of Biochemistry, School of Dentistry, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142, Japan
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