CSK SUPPRESSION OF SRC INVOLVES MOVEMENT OF CSK TO SITES OF SRC ACTIVITY

Csk phosphorylates Src family members at a key regulatory tyrosine in the C-terminal tail and suppresses their activities. It is not known w hether Csk activity is regulated. To examine the features of Csk requi red for Src suppression, we expressed Csk mutants in a cell line with a disrupted fsk gene, Expression of wild-type Csk suppressed Src, but Csk with mutations in the SH2, SH3, and catalytic domains did not supp ress Src. An SH3 deletion mutant of Csk was fully active against in vi tro substrates, but two SH2 domain mutants were essentially inactive. Whereas Src repressed by Csk was predominantly perinuclear, the activa ted Src in cells lacking Csk was localized to structures resembling po dosomes, Activated mutant Src was also in podosomes, even in the prese nce of Csk. When Src was not active, Csk was diffusely located in the cytosol, but when Src was active, Csk colocalized with activated Src t o podosomes. Csk also localizes to podosomes of cells transformed by a n activated Src that lacks the major tyrosine autophosphorylation site , suggesting that the relocalization of Csk is not a consequence of th e binding of the Csk SH2 domain to phosphorylated Src. A catalytically inactive Csk mutant also localized with Src to podosomes, but SH3 and SH2 domain mutants did not, suggesting that the SH3 and SH2 domains a re both necessary to target Csk to places where Src is active. The fai lure of the catalytically active SH3 mutant of Csk to regulate Src may be due to its inability to colocalize with active Src.