Abstract
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-DeltaK) 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-DeltaK 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-DeltaK 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 vitro autophosphorylation activity were increased only in cells expressing Csk-DeltaK. 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.
Highlights
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-⌬K) were overexpressed in primary cultured type I astrocytes by infecting them with the recombinant adenovirus
In Csk-expressing cells, the kinase activity of focal adhesion kinase (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
We have shown that adenovirus-mediated overexpression of Csk in type I astrocytes inhibited cell spreading and attachment to the extracellular matrix (ECM), and these effects were accompanied by defects in the cell adhesions and cytoskeletal organization
Summary
The cell adhesion mechanism allows cells to adhere to one another and transduces signals involved in the regulation of cytoskeletal organization and gene expression In such a signaling cascade, the nonreceptor tyrosine kinase FAK is regarded as a critical signaling mole-. A recent study has indicated that tyrosine phosphorylation of paxillin by Src may be a critical step in focal adhesion assembly [26] These observations suggest that Src is an important mediator of signals originating from cell adhesion through FAK, the molecular mechanism leading to the regulation of cytoskeletal organization and gene expression has yet to be elucidated. Biochemical links between the phenotype and the tyrosine phosphorylation of cell adhesion proteins were investigated
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