AUTOPHOSPHORYLATION OF PURIFIED C-SRC AT ITS PRIMARY NEGATIVE REGULATION SITE

The phosphorylating and transforming activities of c-Src are negatively regulated by phosphorylation at Tyr-527 near its carboxyl terminus, Previous studies have indicated that c-Src preferentially autophosphorylates Tyr-416, a residue in the middle of the catalytic domain, in vitro, and that Tyr-527 is phosphorylated by the carboxyl-terminal Src kinase, Csk. However, indirect evidence suggests that c-Src may also autophosphorylate Tyr-527 as part of a negative feedback loop, While some in vivo evidence suggests that Tyr-527 can be autophosphorylated in an intermolecular interaction, it has not previously been possible to directly demonstrate significant autophosphorylation in vitro, Here we show that c-Src purified from recombinant bacteria can autophosphorylate Tyr-527 to high levels in vitro when incubated with sufficiently high concentrations of ATP (K-m(Mg2+/ATP) approximate to 20 mu M) that are well above those that have been used previously. In vitro Tyr-527 autophosphorylation can occur both as an intra- and intermolecular interaction; higher enzyme concentrations are required for intermolecular Tyr-527 phosphorylation than for Tyr-416 autophosphorylation, These results support the possibility that, Like G-proteins, c-Src can switch itself off in vivo by its own enzymatic activity.