STRUCTURAL REQUIREMENTS FOR THE EFFICIENT REGULATION OF THE SRC PROTEIN-TYROSINE KINASE BY CSK

Protein tyrosine kinases of the Src family are negatively regulated by phosphorylation in the C-terminal tail of the molecule, A different p rotein tyrosine kinase, Csk, is largely responsible for this regulatio n, The phosphorylated tail of c-Src engages with the SH2 domain in a c onformation that is associated with low kinase activity and which invo lves stabilization by the SH3 domain, Inducible expression of c-Src in fission yeast is lethal unless Csk is coexpressed, Using this assay w e present evidence that Src regulation by C-terminal phosphorylation d oes not require the myristylation signal or the unique domain at the N -terminus of the Src protein, Mutagenesis of the SH3 and SH2 domains o f Csk show that neither are necessary in yeast or in vitro for efficie nt regulation of Src, Mutation of Tyr416 of Src, a site of autophospho rylation common to most protein tyrosine kinases, abolished the abilit y of Src to arrest growth of S. pombe and dramatically reduces the abi lity to phosphorylate endogenous proteins, Tyr416 had the same effect on a shorter form of Src consisting of the kinase domain only, indicat ing that the mutation affects a property intrinsic to the catalytic do main, The residual activity of full-length Src mutated at Tyr416 is ef ficiently repressed by Csk action, suggesting that regulation by C-ter minal phosphorylation does not act by preventing phosphorylation at Ty r416.