M. Osusky et al., AUTOPHOSPHORYLATION OF PURIFIED C-SRC AT ITS PRIMARY NEGATIVE REGULATION SITE, The Journal of biological chemistry, 270(43), 1995, pp. 25729-25732
The phosphorylating and transforming activities of c-Src are negativel
y regulated by phosphorylation at Tyr-527 near its carboxyl terminus,
Previous studies have indicated that c-Src preferentially autophosphor
ylates Tyr-416, a residue in the middle of the catalytic domain, in vi
tro, and that Tyr-527 is phosphorylated by the carboxyl-terminal Src k
inase, Csk. However, indirect evidence suggests that c-Src may also au
tophosphorylate Tyr-527 as part of a negative feedback loop, While som
e 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 autophosphoryl
ate Tyr-527 to high levels in vitro when incubated with sufficiently h
igh 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 int
eraction; higher enzyme concentrations are required for intermolecular
Tyr-527 phosphorylation than for Tyr-416 autophosphorylation, These r
esults support the possibility that, Like G-proteins, c-Src can switch
itself off in vivo by its own enzymatic activity.