Inhibition of Src family kinases by a combinatorial action of 5 '-AMP and small heat shock proteins, identified from the adult heart

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.