Modulation of the catalytic activity of the Src family tyrosine kinase Hckby autophosphorylation at a novel site in the unique domain

Autophosphorylation is a key event in the activation of protein kinases, In this study, we demonstrate that autophosphorylation of the recombinant Src family kinase Hck leads to a 20-fold increase in its specific enzymatic ac tivity. Hck was found to autophosphorylate readily to a stoichiometry of 1. 3 mol of phosphate per mol of enzyme, indicating that the kinase autophosph orylated at more than one site. Solid phase sequencing and two-dimensional mapping of the phosphopeptide fragments derived from the autophosphorylated enzyme revealed that the kinase can undergo autophosphorylation at the fol lowing two sites: (i) Tyr-388, which is located to the consensus autophosph orylation site commonly found in the activation loop of many protein kinase s, and (ii) Tyr-29, which is located in the unique domain of Hck, lick puri fied from mouse bone marrow-derived macrophages could also autophosphorylat e in vitro at both Tyr-388 and Tyr-29, indicating that naturally occurring Hck can also autophosphorylate at Tyr-29, Furthermore, Hck transiently expr essed in human embryonic kidney 293T cells was found to be phosphorylated a t Tyr-29 and Tyr-388, proving that lick can also undergo autophosphorylatio n at both sites in vivo. The recombinant enzyme carrying the mutation of TS T-388 to Phe was also able to autophosphorylate at Tyr-29, albeit at a sign ificantly slower rate. A a-fold increase in the specific enzymatic activity was seen with this mutant despite the stoichiometry of autophosphorylation only approaching 0.2 mol of phosphate per mol of enzyme. This indicates th at autophosphorylation of Tyr-29 contributes significantly to the activatio n of Hck. Regulation of the catalytic activity by phosphorylation of Tyr-29 in the unique domain may represent a new mechanism of regulation of Src fa mily tyrosine kinases.