Vs. Kasi et D. Kuppuswamy, Inhibition of Src family kinases by a combinatorial action of 5 '-AMP and small heat shock proteins, identified from the adult heart, MOL CELL B, 19(10), 1999, pp. 6858-6871
Src family kinases are implicated in cellular proliferation and transformat
ion. Terminally differentiated myocytes have lost the ability to proliferat
e, indicating the existence of a down-regulatory mechanism(s) for these mit
ogenic kinases, Here we show that feline cardiomyocyte lysate contains ther
mostable components that inhibit c-Src kinase in vitro. This inhibitory act
ivity, present predominantly in heart tissue, involves two components actin
g combinatorially. After purification by sequential chromatography, one com
ponent was identified by mass and nuclear magnetic resonance spectroscopies
as 5'-AMP, while the other was identified by peptide sequencing as a small
heat shock protein (sHSP), 5'-AMP and to a lesser extent 5'-ADP inhibit c-
Src when combined with either HSP-27 or HSP-32, Other HSPs, including alpha
B-crystalIin, HSP-70, and HSP-90, did not exhibit this effect. The inhibit
ion, observed preferentially on Src family kinases and independent of the S
rc tyrosine phosphorylation state, occurs via a direct interaction of the c
-Src catalytic domain with the inhibitory components. Our study indicates t
hat sHSPs increase the affinity of 5'-AMP for the c-Src ATP binding site, t
hereby facilitating the inhibition. In vivo, elevation of ATP levels in the
cardiomyocytes results in the tyrosine phosphorylation of cellular protein
s including c-Src at the activatory site, and this effect is blocked when t
he 5'-AMP concentration is raised, Thus, this study reveals a novel role fo
r sHSPs and 5'-AMP in the regulation of Src family kinases, presumably for
the maintenance of the terminally differentiated state.