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

To examine the role of C-terminal Src kinase (Csk), a negative regulatory k inase 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-Delta K) were overexpressed in primary cultured type I astrocytes by i nfecting them with the recombinant adenovirus, Overexpression of Csk repres sed 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 fibronecti n. Focal adhesion assembly and the organization of actin stress fibers were also disrupted in cells overexpressing Csk. On the other hand, overexpress ion of Csk-Delta K induced tyrosine phosphorylation of cellular proteins, i ncluding the paxillin and focal adhesion kinase (FAK) and enhanced to some extent the cytoskeletal organization and the rate of cell spreading on fibr onectin, 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 phosphoryla tion state of paxillin in cells overexpressing Csk was indistinguishable fr om that in cells expressing Csk-Delta K in that both phosphorylated paxilli ns bound equally to SH2 domain of Csk and were co-immunoprecipitated with C sk, In contrast, tyrosine phosphorylation of FAK and its in vitro autophosp horylation activity were increased only in cells expressing Csk-Delta K, In Csk-expressing cells, the kinase activity of FAK was substantially decreas ed to 20-30% that of control cells, even though the expression level of FAK : was rather increased. These findings suggest that Csk regulates Src famil y tyrosine kinases that play essential roles in the regulation of cell adhe sion via a FAK-dependent mechanism and that the tyrosine phosphorylation of paxillin alone may not be sufficient for the regulation of the cell adhesi on mechanism in astrocytes.