CSK-MEDIATED PHOSPHORYLATION OF SUBSTRATES IS REGULATED BY SUBSTRATE TYROSINE PHOSPHORYLATION

Csk is a cellular protein tyrosine kinase (PTK) that has been shown to specifically regulate the activity of Src kinase family members by ph osphorylation of a carboxy-terminal tyrosine residue. The molecular me chanisms controlling Csk regulation and its substrate specificity have not been elucidated. Here we report a novel type of overlay kinase as say that allows to probe for Csk-mediated phosphorylation of cellular substrates separated by polyacrylamide gel electrophoresis and transfe rred to nitrocellulose filters. Most of the cell lines analyzed with t his method revealed only a few potential Csk substrates. However, an i ncreased number of Csk substrates was detected in NIH3T3 cells express ing a constitutively activated form of the Src kinase Lck or in PC12 a nd NIH3T3 cells that had been treated with pervanadate. These cells al l display an increased level of cellular protein tyrosine phosphorylat ion which led to the conclusion that Csk preferentially phosphorylates tyrosine-phosphorylated proteins. To verify this hypothesis we analyz ed Csk-mediated phosphorylation of recombinant Lck, a known Csk substr ate. Results demonstrated that autophosphorylation of Lck (at Tyr394) facilitates Csk-mediated phosphorylation of Lck at its regulatory site (Tyr505). Subsequent peptide binding studies revealed that Csk can bi nd to a peptide corresponding to the Lck-autophosphorylation site only when it is phosphorylated. These findings suggest that autophosphoryl ation of Lck at Tyr394 triggers an interaction with Csk and thereby fa cilitates subsequent phosphorylation and inactivation of Lck. The phos phorylation of other cellular Csk substrates may be regulated by a sim ilar mechanism. (C) 1998 Elsevier Science S.A. All rights reserved.