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 (KM(Mg2+/ATP) approximately equal to 20 microM) 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.
Osusky
M
M
Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853, USA.
Taylor
S J
SJ
Shalloway
D
D
D004926:Escherichia coli / Q000235:genetics; D009154:Mutation; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000096:biosynthesis; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism
D011994:Recombinant Proteins; D019000:Phosphotyrosine; D014443:Tyrosine; D016392:Proto-Oncogene Proteins pp60(c-src)