AUTOPHOSPHORYLATION OF SRC AND YES BLOCKS THEIR INACTIVATION BY CSK PHOSPHORYLATION

Csk phosphorylates Src family protein tyrosine kinases on a tyrosine r esidue near their C-terminus and down-regulates their activity. We pre viously observed that this regulation requires a stoichiometric ratio of Csk:Src in a time-independent manner. In this report we examined th is unusual kinetic behavior and found it to be caused by Src autophosp horylation, First, pre-incubation of Src with ATP-Mg led to time-depen dent autophosphorylation of Src, activation of its kinase activity and loss of its ability to be inactivated by Csk, However, the autophosph orylated Src can still be phosphorylated by Csk. The SH2 binding site for phospho-Tyr of this hyperactive and doubly phosphorylated form of Src is not accessible. Second, dephosphorylation of autophosphorylated Src by protein tyrosine phosphatase 1B allowed Src to be inactivated by Csk, Third, protein tyrosine phosphatase 1B preferentially dephosph orylates the Src autophosphorylation site and allows for Src regulatio n by Csk, Finally, Yes, another member of the Src family, was also onl y partially inactivated when a sub-stoichiometric amount of Csk was us ed. Mutation of the tyrosine autophosphorylation site of Yes to a phen ylalanine resulted in a mutant Yes enzyme that can be fully inactivate d by a sub-stoichiometric amount of Csk in a time-dependent manner. Th ese results demonstrate that Csk phosphorylation inactivates Src and Y es only when they are not previously autophosphorylated and Src autoph osphorylation can block the inactivation by Csk phosphorylation, This conclusion suggests a dynamic model for the regulation of the Src fami ly protein tyrosine kinases, which is discussed in the context of prev iously reported observations on the regulation of Src family protein t yrosine kinases.