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Belli S, Esposito D, Servetto A, Pesapane A, Formisano L, Bianco R. c-Src and EGFR Inhibition in Molecular Cancer Therapy: What Else Can We Improve? Cancers (Basel) 2020; 12:E1489. [PMID: 32517369 PMCID: PMC7352780 DOI: 10.3390/cancers12061489] [Citation(s) in RCA: 48] [Impact Index Per Article: 9.6] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/13/2020] [Revised: 06/02/2020] [Accepted: 06/04/2020] [Indexed: 02/06/2023] Open
Abstract
The proto-oncogene c-Src is a non-receptor tyrosine kinase playing a key role in many cellular pathways, including cell survival, migration and proliferation. c-Src de-regulation has been observed in several cancer types, making it an appealing target for drug discovery efforts. Recent evidence emphasizes its crucial role not only in promoting oncogenic traits, but also in the acquisition and maintenance of cancer resistance to various chemotherapeutic or molecular target drugs. c-Src modulates epidermal growth factor receptor (EGFR) activation and amplifies its downstream oncogenic signals. In this review, we report several studies supporting c-Src kinase role in the intricate mechanisms of resistance to EGFR tyrosine kinase inhibitors (TKIs). We further highlighted pre- and clinical progresses of combined treatment strategies made in recent years. Several pre-clinical data have encouraged the use of c-Src inhibitors in combination with EGFR inhibitors. However, clinical trials provided controversial outcomes in some cancer types. Despite c-Src inhibitors showed good tolerability in cancer patients, no incontrovertible and consistent clinical responses were recorded, supporting the idea that a better selection of patients is needed to improve clinical outcome. Currently, the identification of biological markers predictive of therapy response and the accurate molecular screening of cancer patients aimed to gain most clinical benefits become decisive and mandatory.
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Affiliation(s)
| | | | | | | | - Luigi Formisano
- Department of Clinical Medicine and Surgery, University of Naples “Federico II”, 80131 Naples, Italy; (S.B.); (D.E.); (A.S.); (A.P.)
| | - Roberto Bianco
- Department of Clinical Medicine and Surgery, University of Naples “Federico II”, 80131 Naples, Italy; (S.B.); (D.E.); (A.S.); (A.P.)
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2
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Jin LL, Wybenga-Groot LE, Tong J, Taylor P, Minden MD, Trudel S, McGlade CJ, Moran MF. Tyrosine phosphorylation of the Lyn Src homology 2 (SH2) domain modulates its binding affinity and specificity. Mol Cell Proteomics 2015; 14:695-706. [PMID: 25587033 DOI: 10.1074/mcp.m114.044404] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Src homology 2 (SH2) domains are modular protein structures that bind phosphotyrosine (pY)-containing polypeptides and regulate cellular functions through protein-protein interactions. Proteomics analysis showed that the SH2 domains of Src family kinases are themselves tyrosine phosphorylated in blood system cancers, including acute myeloid leukemia, chronic lymphocytic leukemia, and multiple myeloma. Using the Src family kinase Lyn SH2 domain as a model, we found that phosphorylation at the conserved SH2 domain residue Y(194) impacts the affinity and specificity of SH2 domain binding to pY-containing peptides and proteins. Analysis of the Lyn SH2 domain crystal structure supports a model wherein phosphorylation of Y(194) on the EF loop modulates the binding pocket that engages amino acid side chains at the pY+2/+3 position. These data indicate another level of regulation wherein SH2-mediated protein-protein interactions are modulated by SH2 kinases and phosphatases.
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Affiliation(s)
- Lily L Jin
- From the ‡Molecular Structure and Function, The Hospital For Sick Children, 686 Bay Street, Toronto, M5G 0A4, Canada; ‡‡Departments of Molecular Genetics, Medical Science Building, Room 4386, 1 King's College Circle, Toronto, ON M5S 1A8, Canada
| | - Leanne E Wybenga-Groot
- §Cell Biology, The Hospital For Sick Children, 686 Bay Street, Toronto, M5G 0A4, Canada; ¶The Arthur and Sonia Labatt Brain Tumour Research Centre, The Hospital For Sick Children, 686 Bay Street, Toronto, M5G 0A4, Canada
| | - Jiefei Tong
- From the ‡Molecular Structure and Function, The Hospital For Sick Children, 686 Bay Street, Toronto, M5G 0A4, Canada
| | - Paul Taylor
- From the ‡Molecular Structure and Function, The Hospital For Sick Children, 686 Bay Street, Toronto, M5G 0A4, Canada
| | - Mark D Minden
- ‖Medical Biophysics, University of Toronto, Medical Science Building, Room 4386, 1 King's College Circle, Toronto, ON M5S 1A8, Canada; **The Princess Margaret Cancer Center, 610 University Avenue, M5G 2M9, Toronto, Canada
| | - Suzanne Trudel
- ‖Medical Biophysics, University of Toronto, Medical Science Building, Room 4386, 1 King's College Circle, Toronto, ON M5S 1A8, Canada; **The Princess Margaret Cancer Center, 610 University Avenue, M5G 2M9, Toronto, Canada
| | - C Jane McGlade
- §Cell Biology, The Hospital For Sick Children, 686 Bay Street, Toronto, M5G 0A4, Canada; ¶The Arthur and Sonia Labatt Brain Tumour Research Centre, The Hospital For Sick Children, 686 Bay Street, Toronto, M5G 0A4, Canada; ‖Medical Biophysics, University of Toronto, Medical Science Building, Room 4386, 1 King's College Circle, Toronto, ON M5S 1A8, Canada
| | - Michael F Moran
- From the ‡Molecular Structure and Function, The Hospital For Sick Children, 686 Bay Street, Toronto, M5G 0A4, Canada; ‡‡Departments of Molecular Genetics, Medical Science Building, Room 4386, 1 King's College Circle, Toronto, ON M5S 1A8, Canada; **The Princess Margaret Cancer Center, 610 University Avenue, M5G 2M9, Toronto, Canada
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3
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Unlocking Doors without Keys: Activation of Src by Truncated C-terminal Intracellular Receptor Tyrosine Kinases Lacking Tyrosine Kinase Activity. Cells 2014; 3:92-111. [PMID: 24709904 PMCID: PMC3980740 DOI: 10.3390/cells3010092] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/22/2013] [Revised: 02/07/2014] [Accepted: 02/07/2014] [Indexed: 01/07/2023] Open
Abstract
One of the best examples of the renaissance of Src as an open door to cancer has been the demonstration that just five min of Src activation is sufficient for transformation and also for induction and maintenance of cancer stem cells [1]. Many tyrosine kinase receptors, through the binding of their ligands, become the keys that unlock the structure of Src and activate its oncogenic transduction pathways. Furthermore, intracellular isoforms of these receptors, devoid of any tyrosine kinase activity, still retain the ability to unlock Src. This has been shown with a truncated isoform of KIT (tr-KIT) and a truncated isoform of VEGFR-1 (i21-VEGFR-1), which are intracellular and require no ligand binding, but are nonetheless able to activate Src and induce cell migration and invasion of cancer cells. Expression of the i21-VEGFR-1 is upregulated by the Notch signaling pathway and repressed by miR-200c and retinoic acid in breast cancer cells. Both Notch inhibitors and retinoic acid have been proposed as potential therapies for invasive breast cancer.
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Pagano MA, Tibaldi E, Palù G, Brunati AM. Viral proteins and Src family kinases: Mechanisms of pathogenicity from a “liaison dangereuse”. World J Virol 2013; 2:71-78. [PMID: 24175231 PMCID: PMC3785045 DOI: 10.5501/wjv.v2.i2.71] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/05/2012] [Revised: 01/07/2013] [Accepted: 01/24/2013] [Indexed: 02/05/2023] Open
Abstract
To complete their life cycle and spread, viruses interfere with and gain control of diverse cellular processes, this most often occurring through interaction between viral proteins (VPs) and resident protein partners. Among the latter, Src family kinases (SFKs), a class of non-receptor tyrosine kinases that contributes to the conversion of extracellular signals into intracellular signaling cascades and is involved in virtually all cellular processes, have recently emerged as critical mediators between the cell’s infrastructure and the viral demands. In this scenario, structural or ex novo synthesized VPs are able to bind to the different domains of these enzymes through specific short linear motifs present along their sequences. Proline-rich motifs displaying the conserved minimal consensus PxxP and recognizing the SFK Src homology (SH)3 domain constitute a cardinal signature for the formation of multiprotein complexes and this interaction may promote phosphorylation of VPs by SFKs, thus creating phosphotyrosine motifs that become a docking site for the SH2 domains of SFKs or other SH2 domain-bearing signaling molecules. Importantly, the formation of these assemblies also results in a change in the activity and/or location of SFKs, and these events are critical in perturbing key signaling pathways so that viruses can utilize the cell’s machinery to their own benefit. In the light of these observations, although VPs as such, especially those with enzyme activity, are still regarded as valuable targets for therapeutic strategies, multiprotein complexes composed of viral and host cell proteins are increasingly becoming objects of investigation with a view to deeply characterize the structural aspects that favor their formation and to develop new compounds able to contrast viral diseases in an alternative manner.
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Mainou BA, Dermody TS. In search of cathepsins: how reovirus enters host cells. DNA Cell Biol 2012; 31:1646-9. [PMID: 23134451 DOI: 10.1089/dna.2012.1868] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/27/2022] Open
Affiliation(s)
- Bernardo A Mainou
- Department of Pediatrics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA
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6
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Involvement of Src in the Adaptation of Cancer Cells under Microenvironmental Stresses. JOURNAL OF SIGNAL TRANSDUCTION 2012; 2012:483796. [PMID: 22988500 PMCID: PMC3439988 DOI: 10.1155/2012/483796] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 02/24/2012] [Revised: 05/16/2012] [Accepted: 06/04/2012] [Indexed: 01/03/2023]
Abstract
Protein-tyrosine phosphorylation, which is catalyzed by protein-tyrosine kinase (PTK), plays a pivotal role in a variety of cellular functions related to health and disease. The discovery of the viral oncogene Src (v-Src) and its cellular nontransforming counterpart (c-Src), as the first example of PTK, has opened a window to study the relationship between protein-tyrosine phosphorylation and the biology and medicine of cancer. In this paper, we focus on the roles played by Src and other PTKs in cancer cell-specific behavior, that is, evasion of apoptosis or cell death under stressful extracellular and/or intracellular microenvironments (i.e., hypoxia, anoikis, hypoglycemia, and serum deprivation).
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7
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The role of MAPK in drug-induced kidney injury. JOURNAL OF SIGNAL TRANSDUCTION 2012; 2012:463617. [PMID: 22523682 PMCID: PMC3317229 DOI: 10.1155/2012/463617] [Citation(s) in RCA: 44] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 08/15/2011] [Revised: 11/02/2011] [Accepted: 11/04/2011] [Indexed: 12/23/2022]
Abstract
This paper focuses on the role that mitogen-activated protein kinases (MAPKs) play in drug-induced kidney injury. The MAPKs, of which there are four major classes (ERK, p38, JNK, and ERK5/BMK), are signalling cascades which have been found to be broadly conserved across a wide variety of organisms. MAPKs allow effective transmission of information from the cell surface to the cytosolic or nuclear compartments. Cross talk between the MAPKs themselves and with other signalling pathways allows the cell to modulate responses to a wide variety of external stimuli. The MAPKs have been shown to play key roles in both mediating and ameliorating cellular responses to stress including xenobiotic-induced toxicity. Therefore, this paper will discuss the specific role of the MAPKs in the kidney in response to injury by a variety of xenobiotics and the potential for therapeutic intervention at the level of MAPK signalling across different types of kidney disease.
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8
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Regulation of SRC family kinases in human cancers. JOURNAL OF SIGNAL TRANSDUCTION 2011; 2011:865819. [PMID: 21776389 PMCID: PMC3135246 DOI: 10.1155/2011/865819] [Citation(s) in RCA: 156] [Impact Index Per Article: 11.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 12/15/2010] [Accepted: 02/08/2011] [Indexed: 11/22/2022]
Abstract
The nonreceptor protein tyrosine kinase Src plays a crucial role in the signal transduction pathways involved in cell division, motility, adhesion, and survival in both normal and cancer cells. Although the Src family kinases (SFKs) are activated in various types of cancers, the exact mechanisms through which they contribute to the progression of individual tumors remain to be defined. The activation of Src in human cancers may occur through a variety of mechanisms that include domain interaction and structural remodeling in response to various activators or upstream kinases and phosphatastes. Because of Src's prominent roles in invasion and tumor progression, epithelial-to-mesenchymal transition, angiogenesis, and the development of metastasis, Src is a promising target for cancer therapy. Several small molecule inhibitors of Src are currently being investigated in clinical trials. In this article, we will summarize the mechanisms regulating Src kinase activity in normal and cancer cells and discuss the status of Src inhibitor development against various types of cancers.
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9
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Quintela-Fandino M, González-Martín A, Colomer R. Targeting cytoskeleton reorganisation as antimetastatic treatment. Clin Transl Oncol 2011; 12:662-9. [PMID: 20947480 DOI: 10.1007/s12094-010-0575-8] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
Metastatic relapse is responsible for 90% of cancer-related deaths. The process of distant spreading is a cascade of events that is regulated in a highly complex manner; one cellular phenomenon underlying all the events is cytoskeletal reorganisation. Despite the fact that the ability to leave the primary site and establish a viable mass in a distant site is a hallmark of cancer, targeting cytoskeletal reorganisation is an emerging field. In this review we describe the key signalling pathways controlling cytoskeletal reorganisation and the current targeted therapies against the "druggable" nodes. Finally, we discuss potential implications of trial design that can play a role in detecting the specific activity of this drug class.
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Affiliation(s)
- Miguel Quintela-Fandino
- Breast Cancer Unit, Clinical Research Programme CNIO-Spanish National Cancer Research Center C/ Melchor Fernández Almagro, 3 ES-28029 Madrid, Spain.
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10
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Abstract
Reovirus cell entry is initiated by viral attachment to cell surface glycans and junctional adhesion molecule A. Following receptor engagement, reovirus is internalized into cells by receptor-mediated endocytosis using a process dependent on β1 integrin. Endocytosed virions undergo stepwise disassembly catalyzed by cathepsin proteases, followed by endosomal membrane penetration and delivery of transcriptionally active core particles into the cytoplasm. Cellular factors that mediate reovirus endocytosis are poorly defined. We found that both genistein, a broad-spectrum tyrosine kinase inhibitor, and PP2, a specific Src-family kinase inhibitor, diminish reovirus infectivity by blocking a cell entry step. Although neither inhibitor impedes internalization of reovirus virions, both inhibitors target virions to lysosomes. Reovirus colocalizes with Src during cell entry, and reovirus infection induces phosphorylation of Src at the activation residue, tyrosine 416. Diminished Src expression by RNA interference reduces reovirus infectivity, suggesting that Src is required for efficient reovirus entry. Collectively, these data provide evidence that Src kinase is an important mediator of signaling events that regulate the appropriate sorting of reovirus particles in the endocytic pathway for disassembly and cell entry.
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11
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Cayer MP, Proulx M, Ma XZ, Sakac D, Giguère JF, Drouin M, Néron S, Branch DR, Jung D. c-Src tyrosine kinase co-associates with and phosphorylates signal transducer and activator of transcription 5b which mediates the proliferation of normal human B lymphocytes. Clin Exp Immunol 2009; 156:419-27. [PMID: 19438593 DOI: 10.1111/j.1365-2249.2009.03917.x] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022] Open
Abstract
c-Src is the normal human cellular protein homologue of the viral oncogene v-src. c-Src activity was reported recently to increase in CD40-activated human B lymphocytes, suggesting its involvement in proliferation. To elucidate the exact role of c-Src in this process, we investigated the effects of c-Src over-expression on normal B lymphocyte growth. B lymphocytes purified from human peripheral blood were infected with Ad5/F35 vector encoding either a constitutively active c-Src (c-Src/dominant-positive) or a dominant-negative c-Src (c-Src/DN). Little variation of B lymphocytes expansion could be observed between control enhanced yellow fluorescent protein and c-Src/dominant-positive-infected cells. In contrast, over-expression of c-Src/DN results in a 40% inhibition of B lymphocyte expansion. These results suggest that DN c-Src may compete with endogenous c-Src, resulting in partial inhibition of a transcriptional pathway involved in B lymphocyte proliferation. We demonstrate further that c-Src can phosphorylate signal transducer and activator of transcription 5b (STAT5b) on tyrosine 699 and that c-Src and STAT5b co-associate during B lymphocyte proliferation. These results confirm an important role for c-Src in the expansion of normal human B lymphocytes in vitro, in which c-Src may regulate STAT5b in the intracellular signalling pathway important for the proliferation of normal human B lymphocytes.
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12
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McLachlan RW, Kraemer A, Helwani FM, Kovacs EM, Yap AS. E-cadherin adhesion activates c-Src signaling at cell-cell contacts. Mol Biol Cell 2007; 18:3214-23. [PMID: 17553930 PMCID: PMC1949350 DOI: 10.1091/mbc.e06-12-1154] [Citation(s) in RCA: 116] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023] Open
Abstract
Cadherin-based cell-cell contacts are prominent sites for phosphotyrosine signaling, being enriched in tyrosine-phosphorylated proteins and tyrosine kinases and phosphatases. The functional interplay between cadherin adhesion and tyrosine kinase signaling, however, is complex and incompletely understood. In this report we tested the hypothesis that cadherin adhesion activates c-Src signaling and sought to assess its impact on cadherin function. We identified c-Src as part of a cadherin-activated cell signaling pathway that is stimulated by ligation of the adhesion receptor. However, c-Src has a biphasic impact on cadherin function, exerting a positive supportive role at lower signal strengths, but inhibiting function at high signal strengths. Inhibiting c-Src under circumstances when it is activated by cadherin adhesion decreased several measures of cadherin function. This suggests that the cadherin-activated c-Src signaling pathway serves positively to support cadherin function. Finally, our data implicate PI3-kinase signaling as a target for cadherin-activated c-Src signaling that contributes to its positive impact on cadherin function. We conclude that E-cadherin signaling is an important activator of c-Src at cell-cell contacts, providing a key input into a signaling pathway where quantitative changes in signal strength may result in qualitative differences in functional outcome.
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Affiliation(s)
- Robert W. McLachlan
- *Division of Molecular Cell Biology, Institute for Molecular Bioscience, and
| | - Astrid Kraemer
- *Division of Molecular Cell Biology, Institute for Molecular Bioscience, and
| | - Falak M. Helwani
- *Division of Molecular Cell Biology, Institute for Molecular Bioscience, and
| | - Eva M. Kovacs
- School for Biomedical Science, The University of Queensland, St. Lucia, Brisbane, Queensland, Australia 4072
| | - Alpha S. Yap
- *Division of Molecular Cell Biology, Institute for Molecular Bioscience, and
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Abstract
Although oncogenes and their transformation mechanisms have been known for 30 years, we are just now using our understanding of protein function to abrogate the activity of these genes to block cancer growth. The advent of specific small-molecule inhibitors has been a tremendous step in the fight against cancer and their main targets are the cellular counterparts of viral oncogenes. The best-known example of a molecular therapeutic is Gleevec (imatinib). In the early 1990s, IFN-alpha treatment produced a sustained cytologic response in approximately 33% of chronic myelogenous leukemia patients. Today, with Gleevec targeting the kinase activity of the proto-oncogene abl, the hematologic response rate in chronic myelogenous leukemia patients is 95% with 89% progression-free survival at 18 months. There are still drawbacks to the new therapies, such as drug resistance after a period of treatment, but the drawbacks are being studied experimentally. New drugs and combination therapies are being designed that will bypass the resistance mechanisms.
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Affiliation(s)
- Kathleen M Diehl
- Department of Urology, University of Michigan, 1500 East Medical Center Drive, Ann Arbor, MI 48109-0940, USA
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14
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Néron S, Suck G, Ma XZ, Sakac D, Roy A, Katsman Y, Dussault N, Racine C, Branch DR. B cell proliferation following CD40 stimulation results in the expression and activation of Src protein tyrosine kinase. Int Immunol 2006; 18:375-87. [PMID: 16415104 DOI: 10.1093/intimm/dxh377] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022] Open
Abstract
Resting normal human B cells express negligible c-src mRNA or Src protein tyrosine kinase; however, upon induction of proliferation, these cells express high levels of both mRNA and protein and show a concomitant increase in tyrosine kinase activity of immunoprecipitated Src. Src expression was most pronounced upon stimulation with CD154, and to a lesser extent CD70, Staphylococcus aureus, Cowan strain I and phorbol ester, and correlated with the activation of the cells. Transfection of cDNA for human wild-type or kinase-dead Src into Raji B cells resulted in an increase and decrease, respectively, of the cell numbers in culture, showing a direct correlation of proliferation to the expression of Src that was corroborated using anti-sense oligodeoxynucleotides and chemical inhibitors. Furthermore, the human B cell lines, Namalwa, Daudi and Raji express low levels of Src but express very high levels of Src after stimulation with CD154 that showed a correlation with increased activation. This is the first report of Src detectable in normal B cells. The finding that Src expression is inducible and correlates with stimulation by CD154 and the proliferation of the B cells suggests that Src may play a specific role in normal and transformed B cell activation/proliferation pathways mediated primarily through CD40 stimulation.
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Affiliation(s)
- Sonia Néron
- Héma-Québec, Recherche et Développement, Sainte-Foy, Québec, Canada.
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Miyake I, Hakomori Y, Misu Y, Nakadate H, Matsuura N, Sakamoto M, Sakai R. Domain-specific function of ShcC docking protein in neuroblastoma cells. Oncogene 2005; 24:3206-15. [PMID: 15735675 DOI: 10.1038/sj.onc.1208523] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/02/2023]
Abstract
ShcC is a family member of the Shc docking proteins that possess two different phosphotyrosine-binding motifs and conduct signals as Grb2-binding substrates of various receptor tyrosine kinases. We have recently shown that some neuroblastoma cell lines, such as NB-39-nu cells, express a protein complex of hyperphosphorylated ShcC and anaplastic lymphoma kinase (ALK), which is self-activated by gene amplification. Here, we demonstrate that the expression of a mutant ShcC lacking Grb2-binding sites, 3YF-ShcC, significantly impaired the survival, differentiation and motility of NB-39-nu cells by blocking the ERK and Akt pathways. On the other hand, cells overexpressing ShcC or 3YF-ShcC, but not a mutant ShcC that lacks SH2, showed decreased anchorage independency and in vivo tumorigenicity, suggesting a novel ShcC-specific suppressive effect through its SH2 domain on cell transformation. Notably, overexpression of ShcC suppressed the sustained phosphorylation of Src family kinase after cell detachment, which might be independent of phosphorylation of Grb2-binding site. It was indicated that the Src/Fyn-Cas pathway is modulated as a target of these suppressive effects by ShcC. Reciprocal change of ShcC expression and phosphorylation observed in malignant neuroblastoma cell lines might be explained by these phosphotyrosine-dependent and -independent functions of ShcC.
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Affiliation(s)
- Izumi Miyake
- Growth Factor Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan
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16
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Abstract
More than a quarter of a century has elapsed since the identification of the c-src proto-oncogene. During that period, we have learned that cancer arises as the result of mutations in proto-oncogenes and tumor suppressor genes, and we are now seeing the first fruits of these discoveries, in the form of targeted therapies directed against activated tyrosine kinases such as Bcr-Abl, c-Kit and the EGF receptor. But the discovery of the c-src proto-oncogene was in turn based on decades of study on an avian RNA tumor virus, Rous sarcoma virus (RSV). Here I review the work that led up to the identification of the RSV transforming gene and its protein product, and how this information in turn led to the discovery of cellular Src.
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Affiliation(s)
- G Steven Martin
- University of California at Berkeley, Department of Molecular and Cell Biology, 16 Barker Hall # 3204, Berkeley, CA 94720-3204, USA.
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Ahn BH, Kim SY, Kim EH, Choi KS, Kwon TK, Lee YH, Chang JS, Kim MS, Jo YH, Min DS. Transmodulation between phospholipase D and c-Src enhances cell proliferation. Mol Cell Biol 2003; 23:3103-15. [PMID: 12697812 PMCID: PMC153190 DOI: 10.1128/mcb.23.9.3103-3115.2003] [Citation(s) in RCA: 81] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023] Open
Abstract
Phospholipase D (PLD) has been implicated in the signal transduction pathways initiated by several mitogenic protein tyrosine kinases. We demonstrate for the first time that most notably PLD2 and to a lesser extent the PLD1 isoform are tyrosine phosphorylated by c-Src tyrosine kinase via direct association. Moreover, epidermal growth factor induced tyrosine phosphorylation of PLD2 and its interaction with c-Src in A431 cells. Interaction between these proteins is via the pleckstrin homology domain of PLD2 and the catalytic domain of c-Src. Coexpression of PLD1 or PLD2 with c-Src synergistically enhances cellular proliferation compared with expression of either molecule. While PLD activity as a lipid-hydrolyzing enzyme is not affected by c-Src, wild-type PLDs but not catalytically inactive PLD mutants significantly increase c-Src kinase activity, up-regulating c-Src-mediated paxillin phosphorylation and extracellular signal-regulated kinase activity. These results demonstrate the critical role of PLD catalytic activity in the stimulation of Src signaling. In conclusion, we provide the first evidence that c-Src acts as a kinase of PLD and PLD acts as an activator of c-Src. This transmodulation between c-Src and PLD may contribute to the promotion of cellular proliferation via amplification of mitogenic signaling pathways.
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Affiliation(s)
- Bong-Hyun Ahn
- Department of Physiology, College of Medicine, The Catholic University of Korea, Seoul, Korea
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Boyer B, Bourgeois Y, Poupon MF. Src kinase contributes to the metastatic spread of carcinoma cells. Oncogene 2002; 21:2347-56. [PMID: 11948418 DOI: 10.1038/sj.onc.1205298] [Citation(s) in RCA: 91] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/19/2001] [Revised: 12/20/2001] [Accepted: 01/08/2002] [Indexed: 12/31/2022]
Abstract
The involvement of Src kinase during carcinoma metastasis has been explored by using the NBT-II rat carcinoma cell line, which can be induced to scatter in vitro through Src activity. Here we show that Src activity was not required for growth of tumors derived from NBT-II cells injected into nude mice. In contrast, the presence of micrometastases was strictly dependent on Src, since the percentage of mice bearing metastases was dramatically reduced by the expression of a dominant-negative mutant of Src (SrcK-) or of Csk, the natural inhibitor of Src. Furthermore, metastatic cells originating from NBT-II cells displayed a Src activity higher than the parental cells, confirming that Src gives a selective advantage during the metastatic process. Finally, anatomopathological analysis of the primary tumors arising from NBT-II cells expressing Csk or SrcK- constructs revealed a highly differentiated epithelial phenotype contrasting with the poor differentiation of tumors derived from parental cells. The differentiated phenotype correlated with the presence of desmosomes at the cell periphery and the absence of vimentin intermediate filaments. Altogether, these data demonstrate that Src activity correlates with the loss of epithelial differentiation concomitantly with the increase of the metastatic potential of carcinoma cells.
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Affiliation(s)
- Brigitte Boyer
- UMR 146 CNRS, Institut Curie, Section de Recherche, Bâtiment 110 Centre Universitaire Paris-Sud 91405 Orsay Cedex, France.
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Crostella L, Lidder S, Williams R, Skouteris GG. Hepatocyte Growth Factor/scatter factor-induces phosphorylation of cortactin in A431 cells in a Src kinase-independent manner. Oncogene 2001; 20:3735-45. [PMID: 11439336 DOI: 10.1038/sj.onc.1204474] [Citation(s) in RCA: 33] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/19/2000] [Revised: 03/12/2001] [Accepted: 03/21/2001] [Indexed: 11/08/2022]
Abstract
The Hepatocyte Growth Factor receptor transduces proliferating and scattering signals in epithelial and endothelial cells. We have explored potential interactions of the HGF/SF receptor beta-subunit (p145(beta MET)) with F-actin binding partners aiming to identify novel downstream effectors implicated in HGF/SF pluripotent signalling. Cortactin, a p80/85 F-actin binding protein, was found phosphorylated on tyrosine in response to HGF-SF in A431 human epidermoid carcinoma cells, expressing the HGF/SF receptor (c-MET). The HGF/SF receptor was enriched in the detergent-insoluble fraction and was found to co-precipitate with cortactin and to associate in vitro with cortactin. The Grb2 small adapter protein known to associate via its Src homology 2 domain (SH2) with the MET C-terminus, was also associated with cortactin. Transient transfection of A431 cells with dominant-negative Grb2 constructs has revealed that the Grb2-C-SH3 domain possesses a central role in cortactin phosphorylation in response to HGF/SF. Finally, tyrosine phosphorylation of cortactin was found uncoupled of endogenous c-Src kinase activity, thus further supporting the hypothesis that cortactin is a direct target of the MET kinase. We propose that cortactin may constitute a docking site for MET-derived signals within the cytoskeleton.
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Affiliation(s)
- L Crostella
- Laboratory of Cell Biology, Institute of Hepatology, Department of Medicine, Royal Free and University College London Medical School, London WC1E 6HX, UK
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20
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Sakai T, Jove R, Fässler R, Mosher DF. Role of the cytoplasmic tyrosines of beta 1A integrins in transformation by v-src. Proc Natl Acad Sci U S A 2001; 98:3808-3813. [PMID: 11259684 PMCID: PMC31134 DOI: 10.1073/pnas.240456398] [Citation(s) in RCA: 62] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/22/2000] [Accepted: 09/25/2000] [Indexed: 12/27/2022] Open
Abstract
GD25 cells lacking beta 1 integrins or expressing beta 1A with mutations of conserved cytoplasmic tyrosines (Y783, Y795) to phenylalanine have poor directed migration to platelet-derived growth factor or lysophosphatidic acid when compared with GD25 cells expressing wild-type beta 1A. We studied the effects of v-src on these cells. Transformation with v-src caused tyrosine and serine phosphorylation of wild-type beta1 A but not of Y783/795F doubly mutated beta 1A. v-src-transformed cells had rounded and/or fusiform morphology and poor assembly of fibronectin matrix. Adhesion to fibronectin or laminin and coupling of focal contacts to actin-containing cytoskeleton were preserved in transformed Y783/795F cells but lost on transformation when beta 1A was wild type. Transformed Y783/795F cells also retained ability, albeit limited, to migrate across filters, whereas transformed cells with wild-type beta 1A were unable to transverse filters. Studies of single tyrosine mutants showed that the more important tyrosine for retaining ability to adhere, assemble focal contacts, and migrate is Y783. These results suggest that overactive phosphorylation of cytoplasmic residues of beta 1A, particularly Y783, accounts in part for the phenotype of v-src-transformed cells.
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Affiliation(s)
- T Sakai
- Department of Medicine and University of Wisconsin Comprehensive Cancer Center, University of Wisconsin, 1300 University Avenue, Madison, WI 53706, USA
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21
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Harashima N, Tanaka K, Sasatomi T, Shimizu K, Miyagi Y, Yamada A, Tamura M, Yamana H, Itoh K, Shichijo S. Recognition of the Lck tyrosine kinase as a tumor antigen by cytotoxic T lymphocytes of cancer patients with distant metastases. Eur J Immunol 2001; 31:323-32. [PMID: 11180095 DOI: 10.1002/1521-4141(200102)31:2<323::aid-immu323>3.0.co;2-0] [Citation(s) in RCA: 55] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
The Lck protein (p56(lck)), a src family tyrosine kinase that is essential for T cell development and function, is aberrantly expressed in metastatic colon cancers. p56(lck) seems to facilitate the malignant transformation of epithelial cells through initiation of anchorage-independent proliferation. We demonstrate that the lck gene encodes antigenic epitopes recognized by the HLA class I-restricted and tumor-specific CTL of metastatic cancer patients. Lck peptides augmented CTL activity in peripheral blood mononuclear cells (PBMC) of colon and other epithelial cancer patients with distant metastases, but not those without distant metastases. CTL precursors recognizing the Lck peptide were identified in freshly prepared PBMC of patients with distant metastases, and their frequency was significantly augmented by stimulation with the peptide. Thus, Lck peptides could be useful in developing a specific immunotherapy for cancer patients with distant metastases.
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Affiliation(s)
- N Harashima
- Department of Immunology, Kurume University School of Medicine, Kurume, Japan
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22
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Xing L, Venegas AM, Chen A, Garrett-Beal L, Boyce BF, Varmus HE, Schwartzberg PL. Genetic evidence for a role for Src family kinases in TNF family receptor signaling and cell survival. Genes Dev 2001; 15:241-53. [PMID: 11157779 PMCID: PMC312612 DOI: 10.1101/gad.840301] [Citation(s) in RCA: 85] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
Mutant src(-/-) mice have osteopetrosis resulting from defective osteoclasts, the cells that resorb bone. However, signaling pathways involving Src family members in osteoclasts remain unclear. We demonstrate that expression of a truncated Src molecule, Src251, lacking the kinase domain, induces osteopetrosis in wild-type and src(+/-) mice and worsens osteopetrosis in src(-/-) mice by a novel mechanism, increased osteoclast apoptosis. Induction of apoptosis by Src251 requires a functional SH2, but not an SH3, domain and is associated with reduced AKT kinase activity. Expression of Src251 dramatically reduces osteoclast survival in response to RANKL/TRANCE/OPGL, providing evidence that Src family kinases are required in vivo for survival signaling pathways downstream from TNF family receptors.
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Affiliation(s)
- L Xing
- Department of Pathology, University of Rochester, Rochester, New York 14627, USA
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23
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Violette SM, Guan W, Bartlett C, Smith JA, Bardelay C, Antoine E, Rickles RJ, Mandine E, van Schravendijk MR, Adams SE, Lynch BA, Shakespeare WC, Yang M, Jacobsen VA, Takeuchi CS, Macek KJ, Bohacek RS, Dalgarno DC, Weigele M, Lesuisse D, Sawyer TK, Baron R. Bone-targeted Src SH2 inhibitors block Src cellular activity and osteoclast-mediated resorption. Bone 2001; 28:54-64. [PMID: 11165943 DOI: 10.1016/s8756-3282(00)00427-0] [Citation(s) in RCA: 35] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
Abstract
Src, a nonreceptor tyrosine kinase, is an important regulator of osteoclast-mediated resorption. We have investigated whether compounds that bind to the Src SH2 domain inhibit Src activity in cells and decrease osteoclast-mediated resorption. Compounds were examined for binding to the Src SH2 domain in vitro using a fluorescence polarization binding assay. Experiments were carried out with compounds demonstrating in vitro binding activity (nmol/L range) to determine if they inhibit Src SH2 binding and Src function in cells, demonstrate blockade of Src signaling, and lack cellular toxicity. Cell-based assays included: (1) a mammalian two-hybrid assay; (2) morphological reversion and growth inhibition of cSrcY527F-transformed cells; and (3) inhibition of cortactin phosphorylation in csk-/- cells. The Src SH2 binding compounds inhibit Src activity in all three of these mechanism-based assays. The compounds described were synthesized to contain nonhydrolyzable phosphotyrosine mimics that bind to bone. These compounds were further tested and found to inhibit rabbit osteoclast-mediated resorption of dentine. These results indicate that compounds that bind to the Src SH2 domain can inhibit Src activity in cells and inhibit osteoclast-mediated resorption.
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Affiliation(s)
- S M Violette
- ARIAD Pharmaceuticals Inc., Cambridge, MA 02139, USA
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24
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Burnham MR, Bruce-Staskal PJ, Harte MT, Weidow CL, Ma A, Weed SA, Bouton AH. Regulation of c-SRC activity and function by the adapter protein CAS. Mol Cell Biol 2000; 20:5865-78. [PMID: 10913170 PMCID: PMC86064 DOI: 10.1128/mcb.20.16.5865-5878.2000] [Citation(s) in RCA: 96] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/03/2023] Open
Abstract
SRC family kinases play essential roles in a variety of cellular functions, including proliferation, survival, differentiation, and apoptosis. The activities of these kinases are regulated by intramolecular interactions and by heterologous binding partners that modulate the transition between active and inactive structural conformations. p130(CAS) (CAS) binds directly to both the SH2 and SH3 domains of c-SRC and therefore has the potential to structurally alter and activate this kinase. In this report, we demonstrate that overexpression of full-length CAS in COS-1 cells induces c-SRC-dependent tyrosine phosphorylation of multiple endogenous cellular proteins. A carboxy-terminal fragment of CAS (CAS-CT), which contains the c-SRC binding site, was sufficient to induce c-SRC-dependent protein tyrosine kinase activity, as measured by tyrosine phosphorylation of cortactin, paxillin, and, to a lesser extent, focal adhesion kinase. A single amino acid substitution located in the binding site for the SRC SH3 domain of CAS-CT disrupted CAS-CT's interaction with c-SRC and inhibited its ability to induce tyrosine phosphorylation of cortactin and paxillin. Murine C3H10T1/2 fibroblasts that expressed elevated levels of tyrosine phosphorylated CAS and c-SRC-CAS complexes exhibited an enhanced ability to form colonies in soft agar and to proliferate in the absence of serum or growth factors. CAS-CT fully substituted for CAS in mediating growth in soft agar but was less effective in promoting serum-independent growth. These data suggest that CAS plays an important role in regulating specific signaling pathways governing cell growth and/or survival, in part through its ability to interact with and modulate the activity of c-SRC.
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Affiliation(s)
- M R Burnham
- Department of Microbiology and Cancer Center, Health Sciences Center, University of Virginia, Charlottesville, Virginia 22908, Trinity College, Dublin 2, Ireland.
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25
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Violette SM, Shakespeare WC, Bartlett C, Guan W, Smith JA, Rickles RJ, Bohacek RS, Holt DA, Baron R, Sawyer TK. A Src SH2 selective binding compound inhibits osteoclast-mediated resorption. CHEMISTRY & BIOLOGY 2000; 7:225-35. [PMID: 10712930 DOI: 10.1016/s1074-5521(00)00090-9] [Citation(s) in RCA: 51] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/24/2022]
Abstract
BACKGROUND The observations that Src(-/-) mice develop osteopetrosis and Src family tyrosine kinase inhibitors decrease osteoclast-mediated resorption of bone have implicated Src in the regulation of osteoclast-resorptive activity. We have designed and synthesized a compound, AP22161, that binds selectively to the Src SH2 domain and demonstrated that it inhibits Src-dependent cellular activity and inhibits osteoclast-mediated resorption. RESULTS AP22161 was designed to bind selectively to the Src SH2 domain by targeting a cysteine residue within the highly conserved phosphotyrosine-binding pocket. AP22161 was tested in vitro for binding to SH2 domains and was found to bind selectively and with high affinity to the Src SH2 domain. AP22161 was further tested in mechanism-based cellular assays and found to block Src SH2 binding to peptide ligands, inhibit Src-dependent cellular activity and diminish osteoclast resorptive activity. CONCLUSIONS These results indicate that a compound that selectively inhibits Src SH2 binding can be used to inhibit osteoclast resorption. Furthermore, AP22161 has the potential to be further developed for treating osteoporosis.
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Affiliation(s)
- S M Violette
- ARIAD Pharmaceuticals Inc., Biogen, Cambridge, Cambridge, MA 02139, USA.
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26
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Burnham MR, Harte MT, Bouton AH. The role of SRC-CAS interactions in cellular transformation: ectopic expression of the carboxy terminus of CAS inhibits SRC-CAS interaction but has no effect on cellular transformation. Mol Carcinog 1999; 26:20-31. [PMID: 10487518 DOI: 10.1002/(sici)1098-2744(199909)26:1<20::aid-mc3>3.0.co;2-m] [Citation(s) in RCA: 18] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/16/2023]
Abstract
Several lines of evidence indicate that the adapter molecule p130CAS (crk-associated substrate (CAS)) is required for src-mediated cellular transformation. CAS has been shown to be heavily tyrosine-phosphorylated in src-transformed cells, and genetic variants of src that are deficient in CAS binding are also unable to mediate cellular transformation. In this report, we investigated whether CAS phosphorylation and/or its association with src are required elements of the transformation process. Expression of the carboxy-terminal src binding domain of CAS in Rat 1 fibroblasts expressing a temperature-sensitive allele of v-src inhibited the formation of src-CAS complexes and also inhibited tyrosine phosphorylation of CAS. However, expression of this protein had no effect on morphological transformation, src-mediated actin rearrangements, or anchorage-independent growth of these cells when grown at the src-permissive temperature. Thus, the ability of activated src to mediate cellular transformation is either largely independent of endogenous CAS phosphorylation and/or its association with CAS or, alternatively, the carboxy-terminus of CAS may substitute for endogenous CAS in the process of src-mediated transformation.
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Affiliation(s)
- M R Burnham
- Department of Microbiology and Cancer Center, University of Virginia Health Science Center, Charlottesville 22908, USA
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27
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Loo LWM, Kanemitsu MY, Lau AF. In vivo association of pp60v-src and the gap-junction protein connexin 43 in v-src-transformed fibroblasts. Mol Carcinog 1999. [DOI: 10.1002/(sici)1098-2744(199907)25:3<187::aid-mc5>3.0.co;2-o] [Citation(s) in RCA: 40] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/10/2022]
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28
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Abstract
Xenopus laevis larvae with an elevated expression of c-src were generated by mating a transgenic X. laevis male frog carrying proviral Rous sarcoma virus (RSV) long terminal repeat (LTR) and most of the pol gene sequences in its sperm DNA and a normal X. laevis female frog. Offspring (15-20%) with a higher dosage of c-Src, detected in disorganized myotomal musculature and in cerebral and spinal neuronal cells by immunohistochemical analysis, developed abnormally, with edemas (in most cases), head deformities, and eye and axial system defects. In the remaining embryos, a small increase in c-src expression seemed to be compatible with normal embryogenesis. The dosage of c-Src correlated with the dosage of RSV LTR integrated in frog DNA as revealed by Southern and polymerase chain reaction (PCR) analyses. Authenticity of the integrated RSV LTR including enhancer sequence was proved by sequencing. Probing of total RNA from aberrant larvae demonstrated several times higher dosage of c-src mRNA in their tissues than in control tadpoles. We hypothesize that the integrated RSV regulatory sequences can stimulate the expression of c-src proto-oncogene of X. laevis above a threshold that interferes with the early developmental program of frog embryos.
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Affiliation(s)
- M Takác
- Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague
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30
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Provenzano C, Gallo R, Carbone R, Di Fiore PP, Falcone G, Castellani L, Alemà S. Eps8, a tyrosine kinase substrate, is recruited to the cell cortex and dynamic F-actin upon cytoskeleton remodeling. Exp Cell Res 1998; 242:186-200. [PMID: 9665816 DOI: 10.1006/excr.1998.4095] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Eps8 is a recently identified substrate of receptor and nonreceptor tyrosine kinases implicated in the control of cell proliferation. To investigate potential functions of Eps8, its intracellular localization has been examined in several cell types. In cycling fibroblasts immunolabeling with antibodies to Eps8 reveals a punctate pattern within the perinuclear region and staining of motile peripheral cell extensions and cell-cell contact regions. Stimulation of quiescent Swiss 3T3 fibroblasts with serum induces a striking reorganization of the actin cytoskeleton which is accompanied by the enrichment of Eps8 and cortactin in membrane ruffles and lamellipodia. A similar accumulation of Eps8 to membrane ruffles is observed in cells treated with phorbol esters, which also induce marked changes of the F-actin cytoskeleton. The localization of Eps8 at the cell cortex is largely independent from the binding of Eps8 to an EGFR/ErbB-2 chimeric receptor. Moreover, fractionation studies reveal that a portion of the Eps8 molecules present in the cell periphery, unlike cortactin and the receptor, is resistant to mild extraction with detergent. Upon cellular transformation by the tyrosine kinase v-Src, a pool of Eps8 is recruited to newly formed specialized regions of the cytoskeleton, such as actin bodies in terminally differentiated myotubes and podosomes in fibroblasts, where cortactin and a variety of cytoskeletal proteins are also found. Extraction with Triton X-100 preserves the association of Eps8 to podosomes and leaves the majority of the v-Src tyrosine-phosphorylated Eps8 in the detergent-resistant fraction. The observed recruitment of Eps8 to highly dynamic cytoskeletal structures of normal and transformed cells suggests that Eps8 may play a role in the reorganization of the cytoskeleton, perhaps acting as a docking site for other signaling molecules.
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Affiliation(s)
- C Provenzano
- Istituto di Biologia Cellulare, CNR, Università di Roma Tor Vergata, Rome, Italy
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31
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Gonfloni S, Williams JC, Hattula K, Weijland A, Wierenga RK, Superti-Furga G. The role of the linker between the SH2 domain and catalytic domain in the regulation and function of Src. EMBO J 1997; 16:7261-71. [PMID: 9405355 PMCID: PMC1170326 DOI: 10.1093/emboj/16.24.7261] [Citation(s) in RCA: 125] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023] Open
Abstract
The crystal structures of the regulated Src and Hck tyrosine kinases show intramolecular interactions between the phosphorylated tail and the SH2 domain as well as between the SH3 domain, the SH2-catalytic domain linker (SH2-CD linker) and the catalytic domain. The relative contribution of these interactions to regulation of activity is poorly understood. Mutational analysis of Src and Lck revealed that interaction of the SH2-CD linker with the SH3 domain is crucial for regulation. Moreover, three sites of interaction of the linker with the catalytic domain, one at the beginning (Trp260) and two at the back of the small lobe, opposite the catalytic cleft (beta2/beta3 loop; alphaC/beta4 loop), impinge on Src activity. Other activating mutations map to the front of the catalytic domain in the loop preceding the alphaC-helix (beta3/alphaC loop). SH2-CD linker mutants are deregulated in mammalian cells but transform fibroblasts weakly, suggesting that the linker may bind cellular components. Interpretation of our results on the basis of the crystal structure of Src favours a model in which the correctly positioned SH2-CD linker exerts an inhibitory function on catalysis of Src family members by facilitating displacement of the alphaC-helix. This study may provide a template for the generation of deregulated versions of other protein kinases.
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Affiliation(s)
- S Gonfloni
- European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117 Heidelberg, Germany
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32
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Insogna KL, Sahni M, Grey AB, Tanaka S, Horne WC, Neff L, Mitnick M, Levy JB, Baron R. Colony-stimulating factor-1 induces cytoskeletal reorganization and c-src-dependent tyrosine phosphorylation of selected cellular proteins in rodent osteoclasts. J Clin Invest 1997; 100:2476-85. [PMID: 9366562 PMCID: PMC508448 DOI: 10.1172/jci119790] [Citation(s) in RCA: 86] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023] Open
Abstract
Colony-stimulating factor-1 (CSF-1) stimulates motility and cytoplasmic spreading in mature osteoclasts. Therefore, we examined the cellular events and intracellular signaling pathways that accompany CSF-1-induced spreading in normal osteoclasts. To explore the role c-src plays in these processes, we also studied osteoclasts prepared from animals with targeted disruption of the src gene. In normal osteoclasts, CSF-1 treatment induces rapid cytoplasmic spreading, with redistribution of F-actin from a well-delineated central attachment ring to the periphery of the cell. CSF-1 increases membrane phosphotyrosine staining in osteoclasts and induces the phosphorylation of several cellular proteins in cultured, osteoclast-like cells, including c-fms, c-src, and an 85-kD Grb2-binding protein. Src kinase activity is increased threefold after CSF-1 treatment. In src- cells, no attachment ring is present, and CSF-1 fails to induce spreading or a change in the pattern of F-actin distribution. Although c-fms becomes phosphorylated after CSF-1 treatment, the 85-kD protein is significantly less phosphorylated in src- osteoclast-like cells. These results indicate that c-src is critical for the normal cytoskeletal architecture of the osteoclast, and, in its absence, the spreading response induced by CSF-1 is abrogated, and downstream signaling from c-fms is altered.
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Affiliation(s)
- K L Insogna
- Department of Medicine, Yale School of Medicine, New Haven, Connecticut 06520-8020, USA.
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33
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Boyer B, Roche S, Denoyelle M, Thiery JP. Src and Ras are involved in separate pathways in epithelial cell scattering. EMBO J 1997; 16:5904-13. [PMID: 9312048 PMCID: PMC1170221 DOI: 10.1093/emboj/16.19.5904] [Citation(s) in RCA: 116] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023] Open
Abstract
We have demonstrated previously that Src controls the epidermal growth factor (EGF)-induced dispersion of NBT-II carcinoma epithelial cells. Here we show that while only Src and Yes were expressed and activated by EGF, microinjected kinase-inactive mutants of Src (SrcK-) and Fyn (FynK-) were able to exert a dominant-negative effect on the scattering response. Both SH2 and SH3 domains of FynK- were required for inhibition of cell scattering. Expression of dominant-negative N17Ras also abrogated EGF-induced dispersion, showing that Ras is another regulator of cell dispersion. Expression of SrcK- did not alter EGF-evoked Shc tyrosine phosphorylation, Shc-Grb2 complex formation and MAPK activation, three elements of the Ras pathway. Furthermore, the expression of Jun-Fos and Slug rescued the block induced by N17Ras but not by SrcK-, showing that Src kinases and Ras operate in separate pathways. In addition, actinomycin D inhibition of RNA synthesis repressed the ability of the activated mutant L61Ras but not that of F527Src to induce epithelial cell scattering. Since tyrosine phosphorylation of cytoskeleton-associated proteins pp125FAK and cortactin were abolished in EGF-stimulated SrcK- cells, we concluded that, in contrast to Ras, Src kinases may control epithelial cell dispersion in the absence of gene expression and by directly regulating the organization of the cortical cytoskeleton.
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Affiliation(s)
- B Boyer
- Laboratoire de Compartimentation et Dynamique cellulaires, UMR CNRS 144, Institut Curie Section de Recherche, 26 rue d'Ulm F-75248 Paris Cedex 05, France.
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34
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Tian M, Martin GS. The role of the Src homology domains in morphological transformation by v-src. Mol Biol Cell 1997; 8:1183-93. [PMID: 9243500 PMCID: PMC276145 DOI: 10.1091/mbc.8.7.1183] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/04/2023] Open
Abstract
The Src homology (SH2 and SH3) domains of v-Src are required for transformation of Rat-2 cells and for wild-type (morphr) transformation of chicken embryo fibroblasts (CEFs). We report herein that the N-terminal domains of v-Src, when expressed in trans, cannot complement the transformation defect of a deletion mutant lacking the "unique," SH3, and SH2 regions. However, the same regions of Src can promote transformation when translocated to the C terminus of v-Src, although the transformation of CEFs is somewhat slower. We conclude that the SH3 and SH2 domains must be present in cis to the catalytic domain to promote transformation but that transformation is not dependent on the precise intramolecular location of these domains. In CEFSs and in Rat-2 cells, the expression of wild-type v-Src results in tyrosine phosphorylation of proteins that bind to the v-Src SH3 and SH2 domains in vitro; mutations in the SH2 or SH3 and SH2 domains prevent the phosphorylation of these proteins. These findings are most consistent with models in which the SH3 and SH2 domains of v-Src directly or indirectly target the catalytic domain to substrates involved in transformation. However, the N-terminal domains of v-Src can promote tyrosine phosphorylation of certain proteins, in particular p130Cas, even when expressed in the absence of the catalytic domain, indicating that the N-terminal domains of v-Src have effects that are independent of the catalytic domain.
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Affiliation(s)
- M Tian
- Department of Molecular and Cell Biology, University of California at Berkeley 94720-3204, USA
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35
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Abstract
The viral and cellular forms of the Src protein tyrosine kinases take a prototypic role in oncology and signal transduction research, by virtue of being holders of an impressive number of 'firsts'. Our understanding of the biochemistry and physiology of Src has therefore always been used as a reference for our general advancement in the field of protein phosphorylation and growth control. The recent solution of the crystal structure of two members of the Src family represents a milestone in these disciplines and, as usual, provides a general lookout post for developments to come.
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36
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Verderame MF. pp60v-src transformation of rat cells but not chicken cells strongly correlates with low-affinity phosphopeptide binding by the SH2 domain. Mol Biol Cell 1997; 8:843-54. [PMID: 9168470 PMCID: PMC276133 DOI: 10.1091/mbc.8.5.843] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/04/2023] Open
Abstract
Substrates critical for transformation by pp60v-src remain unknown, as does the precise role of the src homology 2 (SH2) domain in this process. To continue exploring the role of the SH2 domain in pp60v-src-mediated transformation, site-directed mutagenesis was used to create mutant v-src alleles predicted to encode proteins with overall structural integrity intact but with reduced ability to bind phosphotyrosine-containing peptides. Arginine-175, which makes critical contacts in the phosphotyrosine-binding pocket, was mutated to lysine or alanine. Unexpectedly, both mutations created v-src alleles that transform chicken cells with wild-type (wt) efficiency and are reduced for transformation of rat cells; these alleles are host dependent for transformation. Additionally, these alleles resulted in a round morphological transformation of chicken cells, unlike 12 of the 13 known host-dependent src SH2 mutations that result in a fusiform morphology. Analysis of phosphopeptide binding by the mutant SH2 domains reveal that the in vitro ability to bind phosphopeptides known to have a high affinity for wt src SH2 correlates with wt (round) morphological transformation in chicken cells and in vitro ability to bind phosphopeptides known to have a low affinity for wt src SH2 correlates with rat cell transformation. These results suggest that the search for critical substrates in rat cells should be among proteins that interact with pp60v-src with low affinity.
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Affiliation(s)
- M F Verderame
- Department of Medicine, College of Medicine, Pennsylvania State University, Hershey 17033, USA
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37
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Kulik G, Klippel A, Weber MJ. Antiapoptotic signalling by the insulin-like growth factor I receptor, phosphatidylinositol 3-kinase, and Akt. Mol Cell Biol 1997; 17:1595-606. [PMID: 9032287 PMCID: PMC231885 DOI: 10.1128/mcb.17.3.1595] [Citation(s) in RCA: 794] [Impact Index Per Article: 28.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/03/2023] Open
Abstract
We have found that insulin-like growth factor I (IGF-I) can protect fibroblasts from apoptosis induced by UV-B light. Antiapoptotic signalling by the IGF-I receptor depended on receptor kinase activity, as cells overexpressing kinase-defective receptor mutants could not be protected by IGF-I. Overexpression of a kinase-defective receptor which contained a mutation in the ATP binding loop functioned as a dominant negative and sensitized cells to apoptosis. The antiapoptotic capacity of the IGF-I receptor was not shared by other growth factors tested, including epidermal growth factor (EGF) and thrombin, although the cells expressed functional receptors for all the agonists. However, EGF was antiapoptotic for cells overexpressing the EGF receptor, and expression of activated pp60v-src also was protective. There was no correlation between protection from apoptosis and activation of mitogen-activated protein kinase, p38/HOG1, or p70S6 kinase. On the other hand, protection by any of the tyrosine kinases against UV-induced apoptosis was blocked by wortmannin, implying a role for phosphatidylinositol 3-kinase (PI3 kinase). To test this, we transiently expressed constitutively active or kinase-dead PI3 kinase and found that overexpression of activated phosphatidylinositol 3-kinase (PI3 kinase) was sufficient to provide protection against apoptosis. Because Akt/PKB is believed to be a downstream effector for PI3 kinase, we also examined the role of this serine/threonine protein kinase in antiapoptotic signalling. We found that membrane-targeted Akt was sufficient to protect against apoptosis but that kinase-dead Akt was not. We conclude that the endogenous IGF-I receptor has a specific antiapoptotic signalling capacity, that overexpression of other tyrosine kinases can allow them also to be antiapoptotic, and that activation of PI3 kinase and Akt is sufficient for antiapoptotic signalling.
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Affiliation(s)
- G Kulik
- Department of Microbiology and Cancer Center, University of Virginia Health Sciences Center, Charlottesville 22908, USA
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38
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Woodring PJ, Garrison JC. Transformation of Rat-1 fibroblasts with the v-src oncogene induces inositol 1,4,5-trisphosphate 3-kinase expression. Biochem J 1996; 319 ( Pt 1):73-80. [PMID: 8870651 PMCID: PMC1217737 DOI: 10.1042/bj3190073] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/02/2023]
Abstract
Transformation of Rat-1 fibroblasts with the v-src oncogene leads to a 6- to 8-fold enhancement of the activity of the Ins(1,4,5)P3 3-kinase in cytosolic extracts [Johnson, Wasilenko, Mattingly, Weber and Garrison (1989) Science 246, 121-124]. This study confirms these results using another v-src-transformed Rat-1 cell line (B31 cells) and investigates the molecular mechanism by which pp60v-src activates Ins(1,4,5)P3 3-kinase. The mRNA and protein levels for two rat isoforms of Ins(1,4,5)P3 3-kinase were determined in the v-src-transformed cell line. Both the mRNA and protein levels for isoform A were elevated in v-src-transformed Rat-1 cells while those for isoform B were not significantly affected. Moreover, stable expression of either form of Ins(1,4,5)P3 3-kinase in the B31 v-src-transformed Rat-1 cell line did not result in tyrosine phosphorylation of Ins(1,4,5)P3 3-kinase A or B. These results suggest that at least one mechanism by which the v-src oncogene increases the activity of the Ins(1,4,5)P3 3-kinase in the Rat-1 transformed fibroblast is by increasing the level of expression of Ins(1,4,5)P3 3-kinase A.
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Affiliation(s)
- P J Woodring
- Department of Pharmacology, University of Virginia School of Medicine, Charlottesville 22908, USA
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39
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Tobe K, Sabe H, Yamamoto T, Yamauchi T, Asai S, Kaburagi Y, Tamemoto H, Ueki K, Kimura H, Akanuma Y, Yazaki Y, Hanafusa H, Kadowaki T. Csk enhances insulin-stimulated dephosphorylation of focal adhesion proteins. Mol Cell Biol 1996; 16:4765-72. [PMID: 8756634 PMCID: PMC231477 DOI: 10.1128/mcb.16.9.4765] [Citation(s) in RCA: 47] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/02/2023] Open
Abstract
Insulin has pleiotropic effects on the regulation of cell physiology through binding to its receptor. The wide variety of tyrosine phosphorylation motifs of insulin receptor substrate 1 (IRS-1), a substrate for the activated insulin receptor tyrosine kinase, may account for the multiple functions of insulin. Recent studies have shown that activation of the insulin receptor leads to the regulation of focal adhesion proteins, such as a dephosphorylation of focal adhesion kinase (pp125FAK). We show here that C-terminal Src kinase (Csk), which phosphorylates C-terminal tyrosine residues of Src family protein tyrosine kinases and suppresses their kinase activities, is involved in this insulin-stimulated dephosphorylation of focal adhesion proteins. We demonstrated that the overexpression of Csk enhanced and prolonged the insulin-induced dephosphorylation of pp125FAK. Another focal adhesion protein, paxillin, was also dephosphorylated upon insulin stimulation, and a kinase-negative mutant of Csk was able to inhibit the insulin-induced dephosphorylation of pp125FAK and paxillin. Although we have shown that the Csk Src homology 2 domain can bind to several tyrosine-phosphorylated proteins, including pp125FAK and paxillin, a majority of protein which bound to Csk was IRS-1 when cells were stimulated by insulin. Our data also indicated that tyrosine phosphorylation levels of IRS-1 appear to be paralleled by the dephosphorylation of the focal adhesion proteins. We therefore propose that the kinase activity of Csk, through the insulin-induced complex formation of Csk with IRS-1, is involved in insulin's regulation of the phosphorylation levels of the focal adhesion proteins, possibly through inactivation of the kinase activity of c-Src family kinases.
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Affiliation(s)
- K Tobe
- Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Japan
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40
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O'Connell JC, McCallum JF, McPhee I, Wakefield J, Houslay ES, Wishart W, Bolger G, Frame M, Houslay MD. The SH3 domain of Src tyrosyl protein kinase interacts with the N-terminal splice region of the PDE4A cAMP-specific phosphodiesterase RPDE-6 (RNPDE4A5). Biochem J 1996; 318 ( Pt 1):255-61. [PMID: 8761480 PMCID: PMC1217616 DOI: 10.1042/bj3180255] [Citation(s) in RCA: 88] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/02/2023]
Abstract
The PDE4A (type IV) cAMP-specific, rolipram-inhibited phosphodiesterase RPDE-6 (RNPDE4A5), when transiently expressed in COS7 cells, could be complexed with the v-Src-SH3 domain expressed as a glutathione S-transferase (GST) fusion protein. RPDE-6 did not interact with GST itself. This complex was not disrupted by treatment with high NaCl concentration together with Triton X-100. Interaction was apparently determined by the N-terminal splice region of RPDE-6, as the PDE4A splice variant RPDE-39, which differs from RPDE-6 at the extreme N-terminus, failed to associate with v-Src-SH3; met26RD1 (where RD1 is rat 'dunc-like' PDE), which has the N-terminal splice region deleted, failed to associate with v-Src-SH3, and the association of RPDE-6 and v-Src-SH3 was blocked by a fusion protein formed from the N-terminal splice region. RDPE-6 showed binding to GST fusion proteins of both the intact Src kinase and an SH2-SH3 construct but did not bind to the Src-SH2 domain or to the adaptor protein Grb-2. RPDE-6 could be co-immunoprecipitated from cytosol extracts of transfected cells by using anti-Src antiserum. RPDE-6 exhibited selectivity in binding to the SH3 domains of c-Abl, Crk, Csk, Lck, Lyn, Fyn and v-Src, with binding to the SH3 regions of the Src-related tyrosyl kinases Lyn and Fyn being the most effective. The binding of RPDE-6 to the SH3 domains of Crk, Csk and Lck led to a marked reduction in PDE activity, but no change was apparent in complexes with other species. Endogenous RPDE-6 from brain, but not endogenous RPDE-39 from testis, bound to the Src-SH3 domain. We suggest that the PDE4A splice variant RPDE-6 has a propensity for interaction with selective SH3 domains, in particular those from Src and the Src-related tyrosyl kinases Lyn and Fyn. This interaction seems to be governed by alternative splicing of the PDE4A gene, because RPDE-39, a splice variant that lacks the proline-rich N-terminal splice region of RPDE-6, does not interact with these SH3 domains. It is proposed that the binding site on RPDE-6 for SH3 domains lies within the unique first 102 residues of its N-terminal splice domain, where two motifs representing Class I SH3 binding sites with selectivity for Src kinase SH3 domains can be identified and one motif for a putative Class II SH3 binding site.
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Affiliation(s)
- J C O'Connell
- Division of Biochemistry and Molecular Biology, I.B.L.S., University of Glasgow, Scotland, U.K
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41
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Liu WW, Mattingly RR, Garrison JC. Transformation of Rat-1 fibroblasts with the v-src oncogene increases the tyrosine phosphorylation state and activity of the alpha subunit of Gq/G11. Proc Natl Acad Sci U S A 1996; 93:8258-63. [PMID: 8710857 PMCID: PMC38657 DOI: 10.1073/pnas.93.16.8258] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/01/2023] Open
Abstract
Two major intermediaries in signal transduction pathways are pp60v-sre family tyrosine kinases and heterotrimeric guanine nucleotide-binding proteins. In Rat-1 fibroblasts transformed by the v-src oncogene, endothelin-1 (ET-1)-induced inositol 1,4,5-trisphosphate accumulation is increased 6-fold, without any increases in the numbers of ET-1 receptors or in the response to another agonist, thrombin. This ET-1 hyperresponse can be inhibited by an antibody directed against the carboxyl terminus of the Gq/G11 alpha subunit, suggesting that the Gq/G11 protein couples ET-1 receptors to phospholipase C (PLC). While v-src transformation did not increase the expression of the Gq/G11 alpha subunit, immunoblotting with anti-phosphotyrosine antibodies and phosphoamino acid analysis demonstrated that the Gq/G11 alpha subunit becomes phosphorylated on tyrosine residues in v-src-transformed cells. Moreover, when the Gq/G11 protein was extracted from control and transformed cell lines and reconstituted with exogenous PLC, AIF*4-stimulated Gq/G11 activity was markedly increased in extracts from v-src-transformed cells. Our results demonstrate that the process of v-src transformation can increase the tyrosine phosphorylation state of the Gq/G11 alpha-subunit in intact cells and that the process causes an increase in the Gq/G11 alpha-subunit's ability to stimulate PLC following activation with AIF-4.
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Affiliation(s)
- W W Liu
- Department of Pharmacology, School of Medicine, University of Virginia, Charlottesville 22908, USA
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42
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Lau AF, Kurata WE, Kanemitsu MY, Loo LW, Warn-Cramer BJ, Eckhart W, Lampe PD. Regulation of connexin43 function by activated tyrosine protein kinases. J Bioenerg Biomembr 1996; 28:359-68. [PMID: 8844333 DOI: 10.1007/bf02110112] [Citation(s) in RCA: 96] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/02/2023]
Abstract
Gap junctions are specialized membrane structures that are involved in the normal functioning of numerous mammalian tissues and implicated in several human disease processes. This mini-review focuses on the regulation of gap junctions through phosphorylation of connexin43 induced by the v-Src or epidermal growth factor receptor tyrosine kinases. These tyrosine kinases markedly disrupt gap junctional communication in mammalian cells. here, we describe work correlating the alteration of connexin43 function with the ability of the v-Src tyrosine kinase to phosphorylate connexin43 directly on two distinct tyrosine sites in mammalian cells (Y247 and Y265). We also present evidence that proline-rich regions and phosphotyrosine sites of connexin43 may mediate interactions with the SH3 and SH2 domains of v-Src. In contrast to v-Src, the activated epidermal growth factor receptor acts indirectly through activated MAP kinase which may stimulate phosphorylation of connexin43 exclusively on serine. This phosphorylation event is complex because MAP kinase phosphorylates three serine sites in connexin43 (S255, S279, and S282). These findings suggest novel interactions between connexin43, the v-Src tyrosine kinase, and activated MAP kinase that set the stage for future investigations into the regulation of gap junctions by protein phosphorylation.
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Affiliation(s)
- A F Lau
- Cancer Research Center, University of Hawaii at Manoa, Honolulu, Hawaii
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43
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Wang HC. Induction of down-regulation of the kinase activities of Mek, p42Erk, p90RSK, and p63SAMK in chicken embryo fibroblast at the late stage of src-induced cellular transformation. J Cell Physiol 1996; 168:87-96. [PMID: 8647927 DOI: 10.1002/(sici)1097-4652(199607)168:1<87::aid-jcp11>3.0.co;2-m] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/01/2023]
Abstract
Two distinct stages in regulation of protein kinases are detectable upon cellular transformation of CEF induced by pp60v-src. Upon cellular transformation induced by ts v-src mutants, the kinase activities of Mek and p42Erk are rapidly induced at the early stage and significantly decreased at the late stage of cellular transformation. In contrast, a novel p63SAMK is partially activated at the early stage and is fully activated at the late stage of cellular transformation. However, p90RSK is activated through the entire course of cellular transformation. In this study, I detect a transient down-regulation of p90RSK activity that is inducible in cultures at the late stage of the src-induced cellular transformation by an increase of extracellular pH value from 7 to 8 and unidentified components in DMEM, but not in cultures which are at the early stage. Concomitant with down-regulation of p90RSK activity, the kinase activities of Mek, p42Erk, and p63SAMK are also down-regulated. Blockage of down-regulation of p90RSK activity by pretreatment of cells with different phosphatase inhibitors correlates with blockage of the down-regulation of either p42Erk or p63SAMK activity. Multiple pathways appear to involve in regulation of p90RSK activity. The discrepancy in regulation of protein kinase activity between the early and late stages of cellular transformation induced by pp60src may indicate a change in signaling cascades during the progress of cellular transformation. The induction of the down-regulation event in this study may provide a new approach to investigate the regulation not only of protein kinases but also phosphatases in transformed cells.
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Affiliation(s)
- H C Wang
- Department of Cellular and Developmental Biology, Harvard University, Cambridge, Massachusetts 02138, USA
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44
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Yonehara M, Minami Y, Kawata Y, Nagai J, Yahara I. Heat-induced chaperone activity of HSP90. J Biol Chem 1996; 271:2641-5. [PMID: 8576234 DOI: 10.1074/jbc.271.5.2641] [Citation(s) in RCA: 138] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/31/2023] Open
Abstract
The 90-kDa stress protein, HSP90, is a major cytosolic protein ubiquitously distributed in all species. Using two substrate proteins, dihydrofolate reductase (DHFR) and firefly luciferase, we demonstrate here that HSP90 newly acquires a chaperone activity when incubated at temperatures higher than 46 degrees C, which is coupled with self-oligomerization of HSP90. While chemically denatured DHFR refolds spontaneously upon dilution from denaturant, oligomerized HSP90 bound DHFR during the process of refolding and prevented it from renaturation. DHFR was released from the complex with HSP90 by incubating with GroEL/ES complexes in an ATP-dependent manner and refolded into the native form. alpha-Casein inhibited the binding of DHFR to HSP90 and also chased DHFR from the complex with HSP90. These results suggest that HSP90 binds substrates to maintain them in a folding-competent structure. Furthermore, we found that HSP90 prevents luciferase from irreversible thermal denaturation and enables it to refold when postincubated with reticulocyte lysates. This heat-induced chaperone activity of HSP90 associated with its oligomerization may have a pivotal role in protection of cells from thermal damages.
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Affiliation(s)
- M Yonehara
- Department of Cell Biology, Tokyo Metropolitan Institute of Medical Science, Japan
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45
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Odai H, Sasaki K, Hanazono Y, Ueno H, Tanaka T, Miyagawa K, Mitani K, Yazaki Y, Hirai H. c-Cbl is inducibly tyrosine-phosphorylated by epidermal growth factor stimulation in fibroblasts, and constitutively tyrosine-phosphorylated and associated with v-Src in v-src-transformed fibroblasts. Jpn J Cancer Res 1995; 86:1119-26. [PMID: 8635998 PMCID: PMC5920666 DOI: 10.1111/j.1349-7006.1995.tb03303.x] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/01/2023] Open
Abstract
The c-cbl gene was cloned as the cellular homolog of the v-cbl oncogene that is the transforming component of a murine tumorigenic retrovirus, CAS NS-1, though the biological roles of c-Cbl remain to be elucidated. We have previously reported that c-Cbl is implicated in the signal transduction triggered by granulocyte-macrophage colony-stimulating factor or erythropoietin in hematopoietic cells. Here, we observed tyrosine phosphorylation of C-cbl in cells expressing epidermal growth factor receptor depending on EGF stimulation and in v-src transformed cells. Furthermore, c-Cbl was revealed to associate with v-Src in vivo. By means of binding experiments using glutathione S-transferase fusion proteins, we have found that the SH2 and SH3 domains of many proteins bind to c-Cbl. These findings strongly suggest that c-Cbl is implicated in a wide variety of signal transduction pathways, including those of EGF receptor and Src protein, as well as in the signaling pathways of hematopoietic cells.
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Affiliation(s)
- H Odai
- Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo
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46
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Crowley E, Horwitz AF. Tyrosine phosphorylation and cytoskeletal tension regulate the release of fibroblast adhesions. J Cell Biol 1995; 131:525-37. [PMID: 7593176 PMCID: PMC2199981 DOI: 10.1083/jcb.131.2.525] [Citation(s) in RCA: 107] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/26/2023] Open
Abstract
We have investigated the mechanisms by which fibroblasts release their adhesions to the extracellular matrix substrata using a permeabilized cell system in which the adhesions remain relatively stable. A large number of different molecules were assayed for their effect on focal adhesion stability using immunofluorescence with antibodies against different focal adhesion constituents. ATP uniquely stimulates a rapid breakdown of focal adhesions, and at high ATP concentrations (> 5 mM), many cells are released from the dish. The remaining cells appear contracted with talin, alpha-actinin, and vinculin localized diffusely throughout the cell. Integrin containing tracks of variable intensity outline the regions where cells had resided before they detached from the substratum. At lower ATP concentrations (0.5-5 mM) the cells remain spread; however the focal adhesion components, including integrin, show an array of phenotypes ranging from diffusely localized throughout the cell to a localization in small, thin focal adhesions. Okadaic acid, a serine, threonine phosphatase inhibitor, enhances the contracted phenotype, even at low concentrations (0.5 mM) of ATP. The localization of focal adhesion components is different in okadaic acid-treated cells. In highly contracted cells, integrin is present in tracks where the cells resided before the contraction; however focal adhesions are no longer apparent. Talin, vinculin, and alpha-actinin localize in trabecular networks toward the periphery of the cell. Interestingly, phosphotyrosine staining as well as nascent, intracellular integrin precedes the recruitment of focal adhesion constituents into the trabecular network. The ATP-stimulated focal adhesion breakdown appears to operate through two mechanisms. First, ATP stimulates the tyrosine phosphorylation of several cytoskeletally associated proteins. These tyrosine phosphorylations correlated well with focal adhesion breakdown. Furthermore, addition of a recombinant, constitutively active tyrosine phosphatase inhibits both the tyrosine phosphorylations and the breakdown of the focal adhesions. None of the major tyrosine phosphoproteins are FAK, integrin, tensin, paxillin, or other phosphoproteins implicated in focal adhesion assembly. The second mechanism is cell contraction. High ATP concentrations, or lower ATP concentrations in the presence of okadaic acid induce cell contraction. Inhibiting the contraction by addition of a heptapeptide IRICRKG, which blocks the actin-myosin interaction, also inhibits focal adhesion breakdown. Neither the peptide nor the phosphatase inhibits focal adhesion breakdown under all conditions suggesting that both tension and tyrosine phosphorylations mediate the release of adhesions.
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Affiliation(s)
- E Crowley
- Department of Cell and Structural Biology, University of Illinois, Urbana 61801, USA
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47
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Castellani L, Reedy MC, Gauzzi MC, Provenzano C, Alemà S, Falcone G. Maintenance of the differentiated state in skeletal muscle: activation of v-Src disrupts sarcomeres in quail myotubes. J Biophys Biochem Cytol 1995; 130:871-85. [PMID: 7642704 PMCID: PMC2199955 DOI: 10.1083/jcb.130.4.871] [Citation(s) in RCA: 23] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/26/2023] Open
Abstract
We have used quail skeletal myotubes expressing a temperature-sensitive allele of the v-src oncogene to address the issue of the homeostasis of sarcomeric myofibrils in differentiated muscle cells. Reactivation of the v-Src tyrosine kinase by shifting the cultures to the permissive temperature leads within minutes to the formation of F-actin-containing bodies (ABs), that originate in the ventral region of the myotubes and increase in number concomitantly with the dismantling of the I-Z-I complex of the sarcomeres. This process is detailed by confocal and electron microscopy. Indirect immunofluorescence reveals that ABs contain muscle-specific protein isoforms associated with the I-Z-I complexes and vinculin, a component of the cytoskeletal network. Anti-phosphotyrosine antibodies label proteins in ABs and Z-discs. Evidence is presented indicating that this phenomenon specifically depends on the persistent activation of v-Src, rather than on a general increase in phosphotyrosine content such as that induced by vanadate. AB formation is prevented by activation of protein kinase C by phorbol ester or by treatment with the kinase inhibitor 2-aminopurine, without any detectable effect on tyrosine phosphorylation. Taken together these findings indicate that phosphorylation of specific target proteins by v-Src, although necessary, is not sufficient per se to induce AB formation. In addition, the signal transduction cascade that culminates in MAP kinase activation and its nuclear translocation is activated both by v-Src and phorbol ester, and is relatively unaffected by 2-aminopurine. These findings imply that both phorbol esters and 2-aminopurine operate, at least in part, at the level of alternative pathways that may diverge upstream of the MAP kinase and are presumably mediating the early effects of v-Src on the differentiated phenotype.
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Affiliation(s)
- L Castellani
- Dipartimento di Medicina Sperimentale e Scienze Biochimiche, Università di Roma Tor Vergata, Italy
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48
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Verderame MF, Guan JL, Woods Ignatoski KM. Transformation and pp60v-src autophosphorylation correlate with SHC-GRB2 complex formation in rat and chicken cells expressing host-range and kinase-active, transformation-defective alleles of v-src. Mol Biol Cell 1995; 6:953-66. [PMID: 7579711 PMCID: PMC301255 DOI: 10.1091/mbc.6.8.953] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/26/2023] Open
Abstract
The biochemical properties of several pp60v-src substrates believed to participate in src-mediated transformation were examined in cells expressing a kinase-active, transformation-defective v-src allele (v-src-F172 delta/Y416F) and its parental allele, v-src-F172 delta, a host-range--dependent allele that transforms chicken cells to a fusiform morphology, but does not transform rat cells. Because pp60v-src-F172 delta is dependent on autophosphorylation for transforming ability, these alleles provide a unique opportunity to examine the role of pp60v-src autophosphorylation in regulating substrate interactions. Increased pp125FAK tyrosine phosphorylation and high levels of pp60v-src-associated phosphotidylinositol-3' kinase activity were detected specifically in chicken cells exhibiting round, refractile transformation but not in cells transformed to a fusiform morphology. Increased pp125FAK kinase activity, but not increased pp125FAK tyrosine-phosphorylation correlated with pp60v-src autophosphorylation and increased anchorage-independent growth. Thus, pp125FAK and PI3'K may participate in morphological transformation by v-src. Furthermore, association of phosphorylated SHC with the adapter GRB2 correlated with increased anchorage-independent growth (and autophosphorylation) in both rat and chicken cells independent of the morphological phenotype induced. Therefore, host-range dependence for transformation may be regulated through association of SHC with GRB2, thus implicating SHC as a crucial substrate for src-dependent transformation.
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Affiliation(s)
- M F Verderame
- Department of Microbiology and Immunology, Pennsylvania State University College of Medicine, Hershey 17033, USA
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49
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Chang JH, Gill S, Settleman J, Parsons SJ. c-Src regulates the simultaneous rearrangement of actin cytoskeleton, p190RhoGAP, and p120RasGAP following epidermal growth factor stimulation. J Cell Biol 1995; 130:355-68. [PMID: 7542246 PMCID: PMC2199934 DOI: 10.1083/jcb.130.2.355] [Citation(s) in RCA: 211] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2023] Open
Abstract
Analysis of C3H10T1/2 murine fibroblasts overexpressing wild type and dominant negative variants of c-Src has demonstrated a requirement for c-Src in EGF-induced mitogenesis. Correlating with the ability of c-Src variants to potentiate or inhibit EGF-dependent DNA synthesis is the phosphotyrosine content of multiple cellular proteins, including p190-RhoGAP, a protein thought to regulate growth factor-induced actin cytoskeleton remodeling by modulating the activity of the small GTP binding protein, Rho. Because the in vivo phosphotyrosine content of p190 varies with the level of active c-Src and not with EGF treatment, p190 is considered to be a preferred substrate of c-Src. To determine whether tyrosyl phosphorylation of p190 (by c-Src) could influence EGF-dependent actin remodeling, we used conventional and confocal immunofluorescence microscopy to examine the intracellular distribution of p190, actin, and p120RasGAP in EGF-stimulated or unstimulated 10T1/2 Neo control cells and cells that stably overexpress wild-type (K+) or kinase-defective (K-) c-Src. We found that in all cell lines, EGF induced a rapid and transient condensation of p190 and RasGAP into cytoplasmic, arclike structures. However, in K+ cells the rate of appearance and number of cells exhibiting arcs increased when compared with control cells. Conversely, K- cells exhibited delayed arc formation and a reduction in number of cells forming arcs. EGF-induced actin stress fiber disassembly and reassembly occurred with the same kinetics and frequency as did p190 and RasGAP rearrangements in all three cell lines. These results, together with the documented Rho-GAP activity intrinsic to p190 and the ability of Rho to modulate actin stress fiber formation, suggest that c-Src regulates EGF-dependent actin cytoskeleton reorganization through phosphorylation of p190.
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Affiliation(s)
- J H Chang
- Department of Microbiology, University of Virginia Health Sciences Center, Charlottesville 22908, USA
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50
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Kharbanda S, Saleem A, Yuan Z, Emoto Y, Prasad KV, Kufe D. Stimulation of human monocytes with macrophage colony-stimulating factor induces a Grb2-mediated association of the focal adhesion kinase pp125FAK and dynamin. Proc Natl Acad Sci U S A 1995; 92:6132-6. [PMID: 7597091 PMCID: PMC41656 DOI: 10.1073/pnas.92.13.6132] [Citation(s) in RCA: 60] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/26/2023] Open
Abstract
Macrophage colony-stimulating factor (M-CSF) is required for the growth and differentiation of mononuclear phagocytes. In the present studies using human monocytes, we show that M-CSF induces interaction of the Grb2 adaptor protein with the focal adhesion kinase pp125FAK. The results demonstrate that tyrosine-phosphorylated pp125FAK directly interacts with the SH2 domain of Grb2. The findings indicate that a pYENV site at Tyr-925 in pp125FAK is responsible for this interaction. We also demonstrate that the Grb2-FAK complex associates with the GTPase dynamin. Dynamin interacts with the SH3 domains of Grb2 and exhibits M-CSF-dependent tyrosine phosphorylation in association with pp125FAK. These findings suggest that M-CSF-induced signaling involves independent Grb2-mediated pathways, one leading to Ras activation and another involving pp125FAK and a GTPase implicated in receptor internalization.
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Affiliation(s)
- S Kharbanda
- Division of Cancer Pharmacology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA
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