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Originally published In Press as doi:10.1074/jbc.M002022200 on August 8, 2000

J. Biol. Chem., Vol. 275, Issue 43, 33353-33364, October 27, 2000
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Modulation of the Catalytic Activity of the Src Family Tyrosine Kinase Hck by Autophosphorylation at a Novel Site in the Unique Domain*

Timothy M. JohnsonDagger §, Nicholas A. WilliamsonDagger , Glen Scholz, Anthony Jaworowski||**, Richard E. H. WettenhallDagger , Ashley R. Dunn, and Heung-Chin ChengDagger Dagger Dagger

From the Dagger  Department of Biochemistry and Molecular Biology, the || Department of Medicine, University of Melbourne, Parkville, Victoria 3052 and the  Ludwig Institute of Cancer Research, Melbourne Tumour Biology Branch, P. O. Royal Melbourne Hospital, Victoria 3050, Australia

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 activity. Hck was found to autophosphorylate readily to a stoichiometry of 1.3 mol of phosphate per mol of enzyme, indicating that the kinase autophosphorylated 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 following two sites: (i) Tyr-388, which is located to the consensus autophosphorylation site commonly found in the activation loop of many protein kinases, and (ii) Tyr-29, which is located in the unique domain of Hck. Hck purified from mouse bone marrow-derived macrophages could also autophosphorylate in vitro at both Tyr-388 and Tyr-29, indicating that naturally occurring Hck can also autophosphorylate at Tyr-29. Furthermore, Hck transiently expressed in human embryonic kidney 293T cells was found to be phosphorylated at Tyr-29 and Tyr-388, proving that Hck can also undergo autophosphorylation at both sites in vivo. The recombinant enzyme carrying the mutation of Tyr-388 to Phe was also able to autophosphorylate at Tyr-29, albeit at a significantly slower rate. A 2-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 that autophosphorylation of Tyr-29 contributes significantly to the activation 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 family tyrosine kinases.

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Supported by a post-graduate studentship from the Anti-cancer Council of Victoria.

** Present address: the Macfarlane Burnet Centre for Medical Research, Fairfield, Victoria, Australia.

Dagger Dagger Supported by grants from the National Health and Medical Research Council of Australia and the Anti-cancer Council of Victoria, Australia. To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria 3052, Australia. Tel.: 61-3-9344-5947; Fax: 61-3-9347-7730; E-mail: h.cheng@biochemistry.unimelb.edu.au.

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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