CSK PHOSPHORYLATION AND INACTIVATION IN-VITRO BY THE CAMP-DEPENDENT PROTEIN-KINASE

Csk is a protein tyrosine kinase that phosphorylates other protein tyr osine kinases of the Src family and down-regulates their activities. I t is not known how Csk is regulated. We investigated the possibility t hat Csk is regulated through phosphorylation by examining if Csk can s erve as an in vitro substrate for a panel of protein kinases, We found that Csk was phosphorylated by the cAMP-dependent protein kinase (PKA ), but not by protein kinase C, Src, or the fibroblast growth factor r eceptor kinase. Csk phosphorylation in vitro by PKA is on a serine res idue(s) and can reach a stoichiometry of approximately 0.6 mol phospha te per mole of enzyme, Furthermore, incubation with PKA in the presenc e of ATP ansi magnesium ion results in a time-dependent decrease: in C sk kinase activity, A sixfold decrease in Csk activity (expressed as V -max/K-m ratio) was achieved due to a threefold increase in its K-m an d a twofold decrease in its V-max value within 1 h of incubation with the catalytic subunit of PRA and ATP-Mg. Both phosphorylation and inac tivation by PKA were blocked by a PKA-specific inhibitor. Csk mutants with a deleted SH2 or SH3 domain retained their ability to be phosphor ylated and inactivated by PRA, indicating that the phosphorylation sit e is located within the catalytic domain. These studies suggest that t he cAMP-dependent protein kinase can regulate Csk activity. (C) 1997 A cademic Press.