Autophosphorylation of Src and Yes blocks their inactivation by Csk phosphorylation

Abstract

Csk phosphorylates Src family protein tyrosine kinases on a tyrosine residue near their C-terminus and downregulates their activity. We previously observed that this regulation requires a stoichiometric ratio of Csk : Src in a time-independent manner. In this report we examined this unusual kinetic behavior and found it to be caused by Src autophosphorylation. First, pre-incubation of Src with ATP-Mg led to time-dependent autophosphorylation of Src, activation of its kinase activity and loss of its ability to be inactivated by Csk. However, the autophosphorylated 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 dephosphorylates the Src autophosphorylation site and allows for Src regulation by Csk. Finally, Yes, another member of the Src family, was also only partially inactivated when a sub-stoichiometric amount of Csk was used. Mutation of the tyrosine autophosphorylation site of Yes to a phenylalanine resulted in a mutant Yes enzyme that can be fully inactivated by a sub-stoichiometric amount of Csk in a time-dependent manner. These results demonstrate that Csk phosphorylation inactivates Src and Yes only when they are not previously autophosphorylated and Src autophosphorylation can block the inactivation by Csk phosphorylation. This conclusion suggests a dynamic model for the regulation of the Src family protein tyrosine kinases, which is discussed in the context of previously reported observations on the regulation of Src family protein tyrosine kinases.

Altered regulation of Src upon cell detachment protects human lung adenocarcinoma cells from anoikis

“…Phosphorylation of Tyr-419/416 can also activate Src activity in the context of simultaneous phosphorylation at Tyr-530/527 (Boerner et al, 1996;Sun et al, 1998), suggesting that active Src phosphorylated at Tyr-419/416 can not be fully inactivated by phosphorylation at Tyr-530/527 alone. This supports the findings that inactive Src may be the target of other kinases that can phosphorylate Tyr-419/416 and cause Src activation (Chiang and Sefton, 2000).…”

A Novel Non-catalytic Mechanism Employed by the C-terminal Src-homologous Kinase to Inhibit Src-family Kinase Activity

“…30). Furthermore, autophosphorylation permits SFKs to remain active even when their Tyr T is phosphorylated (31). Recently, Lerner and Smithgall (32) reported that human immunodeficiency virus Nef binding or mutations of the SH2 kinase linker caused Hck activation by autophosphorylation even when Tyr T remains phosphorylated and bound to the SH2 domain.…”

Variant estrogen receptor–c-Src molecular interdependence and c-Src structural requirements for endothelial NO synthase activation

“…3A) may favor homotypic (26,27) and heterotypic (24,25) protein interactions with c-Src and further enforce c-Src ''opening.'' Indeed, it has been demonstrated that doubly phosphorylated c-Src can be active, such that Y419 phosphorylation can override the p-Y530-directed inhibition (28). The balance of tyrosine kinase (Csk and Chk) and phosphatase (PTP␣) activity, in both basal and stimulated states, is partially responsible for the state of Src activation.…”