Csk suppression of Src involves movement of Csk to sites of Src activity.

Abstract

Csk phosphorylates Src family members at a key regulatory tyrosine in the C-terminal tail and suppresses their activities. It is not known whether Csk activity is regulated. To examine the features of Csk required for Src suppression, we expressed Csk mutants in a cell line with a disrupted csk gene. Expression of wild-type Csk suppressed Src, but Csk with mutations in the SH2, SH3, and catalytic domains did not suppress Src. An SH3 deletion mutant of Csk was fully active against in vitro substrates, but two SH2 domain mutants were essentially inactive. Whereas Src repressed by Csk was predominantly perinuclear, the activated Src in cells lacking Csk was localized to structures resembling podosomes. Activated mutant Src was also in podosomes, even in the presence of Csk. When Src was not active, Csk was diffusely located in the cytosol, but when Src was active, Csk colocalized with activated Src to podosomes. Csk also localizes to podosomes of cells transformed by an activated Src that lacks the major tyrosine autophosphorylation site, suggesting that the relocalization of Csk is not a consequence of the 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 are both necessary to target Csk to places where Src is active. The failure of the catalytically active SH3 mutant of Csk to regulate Src may be due to its inability to colocalize with active Src.

Diminished TCR signaling in cutaneous T cell lymphoma is associated with decreased activities of Zap70, Syk and membrane-associated Csk

“…Furthermore, the reduced responsiveness was not due to diminished levels of CD3⑀, as indicated before (Figure 1), or low expression of sev- we went on to determine if the stunted responsiveness is a general characteristic of this population of memory T cells. As shown in Figure 2e, ␣CD3 stimulation triggered a similar increase in the intensity of tyrosyl-phosphorylated proteins in previous report 34 ␣CD3⑀ caused redistribution of Csk as shown by a loss in kinase activity in the cytosolic fraction and CD4 + and CD4 + /CD45RO + selected subtype of normal T cells purified from the same donor. This is in spite of the fact that a gain in the membranous fraction (Figure 2b,c, normal T cells and the CTCL of patient BU).…”

c-Src enhances the spreading of src-/- fibroblasts on fibronectin by a kinase-independent mechanism.

“…The portion of c-Src that remains phosphorylated at Y527 may be a subpopulation that is not activated during cellular adhesion; alternatively, the rapid rephosphorylation of this site, presumably by CSK, may occur following activation /Okada et al 1991). Recent reports that CSK and c-Src simultaneously associate with focal adhesions are consistent with the latter possibility (Howell and Cooper 1994;Sabe et al 1994). …”

Podosomes at a glance

“…Indeed, one of the best ways to visualize podosomes is by staining phosphorylated tyrosine (Linder and Apfelbacher, 2003), which is highly enriched in some types of adhesive structure. Not surprisingly, cellular tyrosine kinases such as Src and Csk play major roles in podosome regulation (Howell and Cooper, 1994), and tools for podosome manipulation include Src kinase inhibitors, which disrupt podosomes (Linder et al, 2000b), and vanadate, a phosphotyrosine phosphatase inhibitor, which is able to induce podosome formation (Marchisio et al, 1988).…”