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Originally published In Press as doi:10.1074/jbc.C000606200 on September 26, 2000

J. Biol. Chem., Vol. 275, Issue 49, 38127-38130, December 8, 2000
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ACCELERATED PUBLICATION
Chemical Rescue of a Mutant Protein-tyrosine Kinase*

Daniel M. Williams, Dongxia Wang, and Philip A. ColeDagger §

From the Dagger  Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

Protein-tyrosine kinases contain a catalytic loop Arg residue located either two or four positions downstream of a highly conserved Asp residue. In this study, the role of this Arg (Arg-318) in the protein-tyrosine kinase C-terminal Src kinase (Csk) was investigated. The observed kcat for phosphorylation of the random copolymer poly(Glu,Tyr) substrate by Csk R318A is ~3000-fold smaller compared with that of wild type Csk, whereas the Km values for ATP and poly(Glu,Tyr) are only mildly affected. The kcat value for poly(Glu,Tyr) phosphorylation by the Csk double mutant A316R,R318A is 100-fold greater than the kcat value for the single R318A mutant, suggesting that an Arg positioned at the alternative location fulfills a similar function as in wild type. Csk R318A kinase activity can also be partially recovered by several exogenous small molecules including guanidinium and imidazole. These molecules contain key features whose roles in catalysis can be rationalized from a known x-ray structure of the insulin receptor tyrosine kinase. Imidazole is the best of these activators, enhancing phosphorylation rates by Csk R318A up to 100-fold for poly(Glu,Tyr) and significantly stimulating Csk R318A phosphorylation of the physiologic substrate Src. This chemical rescue of mutant protein kinase activity might find applications in cell signal transduction experiments.

* This work was supported in part by Johns Hopkins University in-house National Institutes of Health Training and Anti-Cancer Drug Development Grant T32CA09243, National Institutes of Health Grant CA74305-01 (to P. A. C.), and by the Burroughs-Wellcome foundation (to P. A. C.).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.

§ To whom correspondence should be addressed: Dept. of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Rm. 316, Hunterian Bldg., 725 N. Wolfe St., Baltimore, MD 21205. Tel.: 410-614-0540; Fax: 410-614-7717; E-mail: pcole@jhmi.edu.

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