Adenovirus-mediated Overexpression of C-terminal Src Kinase (Csk) in Type I Astrocytes Interferes with Cell Spreading and Attachment to Fibronectin

To examine the role of C-terminal Src kinase (Csk), a negative regulatory kinase of Src family tyrosine kinases, in the cell adhesion mechanism of the nervous system, wild-type Csk (Csk), and a kinase-deficient mutant of Csk (Csk-ΔK) were overexpressed in primary cultured type I astrocytes by infecting them with the recombinant adenovirus. Overexpression of Csk repressed the in vitro kinase activity of Src to as little as 10% that of control cells and interfered with cell spreading and cell attachment to fibronectin. Focal adhesion assembly and the organization of actin stress fibers were also disrupted in cells overexpressing Csk. On the other hand, overexpression of Csk-ΔK induced tyrosine phosphorylation of cellular proteins, including the paxillin and focal adhesion kinase (FAK) and enhanced to some extent the cytoskeletal organization and the rate of cell spreading on fibronectin, indicating that Src or its relatives was functionally activated in the cells. Paxillin was also tyrosine-phosphorylated in Csk-overexpressing cells, indicating that it can serve as a substrate of Csk. The phosphorylation state of paxillin in cells overexpressing Csk was indistinguishable from that in cells expressing Csk-ΔK in that both phosphorylated paxillins bound equally to SH2 domain of Csk and were co-immunoprecipitated with Csk. In contrast, tyrosine phosphorylation of FAK and its in vitroautophosphorylation activity were increased only in cells expressing Csk-ΔK. In Csk-expressing cells, the kinase activity of FAK was substantially decreased to 20–30% that of control cells, even though the expression level of FAK was rather increased. These findings suggest that Csk regulates Src family tyrosine kinases that play essential roles in the regulation of cell adhesion via a FAK-dependent mechanism and that the tyrosine phosphorylation of paxillin alone may not be sufficient for the regulation of the cell adhesion mechanism in astrocytes.

[1]  高山 喜晴.  Adenovirus-mediated overexpression of C-terminal Src kinase (Csk) in type I astrocytes interferes with cell spreading and attachment to fibronectin : correlation with tyrosine phosphorylations of paxillin and FAK , 1999 .

[2]  A. Kazlauskas,et al.  A Role for Src in Signal Relay by the Platelet-derived Growth Factor α Receptor* , 1998, The Journal of Biological Chemistry.

[3]  J. Parsons,et al.  Inhibition of cell spreading by expression of the C-terminal domain of focal adhesion kinase (FAK) is rescued by coexpression of Src or catalytically inactive FAK: a role for paxillin tyrosine phosphorylation , 1997, Molecular and cellular biology.

[4]  Y. Takayama,et al.  Role of Csk in neural differentiation of the embryonic carcinoma cell line P19 , 1997, FEBS letters.

[5]  Michael J. Eck,et al.  Three-dimensional structure of the tyrosine kinase c-Src , 1997, Nature.

[6]  T. Hunter,et al.  Evidence for in vivo phosphorylation of the Grb2 SH2-domain binding site on focal adhesion kinase by Src-family protein-tyrosine kinases , 1996, Molecular and cellular biology.

[7]  Y. Yazaki,et al.  Csk enhances insulin-stimulated dephosphorylation of focal adhesion proteins , 1996, Molecular and cellular biology.

[8]  J. Guan,et al.  Stimulation of cell migration by overexpression of focal adhesion kinase and its association with Src and Fyn. , 1996, Journal of cell science.

[9]  Y. Kanegae,et al.  Efficient generation of recombinant adenoviruses using adenovirus DNA-terminal protein complex and a cosmid bearing the full-length virus genome. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[10]  M. Okada,et al.  Depolarization-induced tyrosine phosphorylation of paxillin in PC12h cells. , 1996, European journal of biochemistry.

[11]  S. Aizawa,et al.  Reduced cell motility and enhanced focal adhesion contact formation in cells from FAK-deficient mice , 1995, Nature.

[12]  E. Kandel,et al.  Focal adhesion kinase in the brain: novel subcellular localization and specific regulation by Fyn tyrosine kinase in mutant mice. , 1995, Genes & development.

[13]  Sheila M. Thomas,et al.  Specific and redundant roles of Src and Fyn in organizing the cytoskeleton , 1995, Nature.

[14]  J. Parsons,et al.  pp125FAK-dependent tyrosine phosphorylation of paxillin creates a high-affinity binding site for Crk , 1995, Molecular and cellular biology.

[15]  J. Brugge,et al.  Integrins and signal transduction pathways: the road taken. , 1995, Science.

[16]  T. Hunter,et al.  Integrin-mediated signal transduction linked to Ras pathway by GRB2 binding to focal adhesion kinase , 1994, Nature.

[17]  S. Aizawa,et al.  Identification of major tyrosine-phosphorylated proteins in Csk-deficient cells. , 1994, Oncogene.

[18]  J. Pouysségur,et al.  Activation of Src family kinase activity by the G protein-coupled thrombin receptor in growth-responsive fibroblasts. , 1994, The Journal of biological chemistry.

[19]  Philippe Soriano,et al.  NCAM-dependent neurite outgrowth is inhibited in neurons from Fyn-minus mice , 1994, The Journal of cell biology.

[20]  H. Varmus,et al.  Association of the amino‐terminal half of c‐Src with focal adhesions alters their properties and is regulated by phosphorylation of tyrosine 527. , 1994, The EMBO journal.

[21]  Philippe Soriano,et al.  Impaired neurite outgrowth of src-minus cerebellar neurons on the cell adhesion molecule L1 , 1994, Neuron.

[22]  J. Parsons,et al.  Autophosphorylation of the focal adhesion kinase, pp125FAK, directs SH2-dependent binding of pp60src , 1994, Molecular and cellular biology.

[23]  T. Yagi,et al.  Initial events of myelination involve Fyn tyrosine kinase signalling , 1994, Nature.

[24]  M. Fournel,et al.  Negative regulation of T-cell receptor signalling by tyrosine protein kinase p50csk , 1993, Nature.

[25]  Jonathan A. Cooper,et al.  The when and how of Src regulation , 1993, Cell.

[26]  Philippe Soriano,et al.  Disruption of the csk gene, encoding a negative regulator of Src family tyrosine kinases, leads to neural tube defects and embryonic lethality in mice , 1993, Cell.

[27]  T. Yagi,et al.  Constitutive activation of Src family kinases in mouse embryos that lack Csk , 1993, Cell.

[28]  R. Perlmutter,et al.  Functional coupling of the src-family protein tyrosine kinases p59fyn and p53/56lyn with the interleukin 2 receptor: implications for redundancy and pleiotropism in cytokine signal transduction. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[29]  Martin Gs,et al.  Intracellular targeting of pp60src expression: localization of v-src to adhesion plaques is sufficient to transform chicken embryo fibroblasts. , 1992 .

[30]  H. Hanafusa,et al.  Activation of c-Src in cells bearing v-Crk and its suppression by Csk , 1992, Molecular and cellular biology.

[31]  D. Shalloway,et al.  Regulation of focal adhesion-associated protein tyrosine kinase by both cellular adhesion and oncogenic transformation , 1992, Nature.

[32]  T. Yamamoto,et al.  CSK: a protein-tyrosine kinase involved in regulation of src family kinases. , 1991, The Journal of biological chemistry.

[33]  Yamamura Ken-ichi,et al.  Efficient selection for high-expression transfectants with a novel eukaryotic vector , 1991 .

[34]  Jonathan A. Cooper,et al.  Cloning of a complementary DNA for a protein-tyrosine kinase that specifically phosphorylates a negative regulatory site of p60c-src , 1991, Nature.

[35]  J. Bishop,et al.  The product of the protooncogene c-src is modified during the cellular response to platelet-derived growth factor. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[36]  J. Parsons,et al.  Genetics of src: structure and functional organization of a protein tyrosine kinase. , 1989, Current topics in microbiology and immunology.