Mayer, Bruce J. and Baltimore, David (1994) Mutagenic analysis of the roles of SH2 and SH3 domains in regulation of the Abl tyrosine kinase. Molecular and Cellular Biology, 14 (5). pp. 2883-2894. ISSN 0270-7306. http://resolver.caltech.edu/CaltechAUTHORS:MAYmcb94
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We have used in vitro mutagenesis to examine in detail the roles of two modular protein domains, SH2 and SH3, in the regulation of the Abl tyrosine kinase. As previously shown, the SH3 domain suppresses an intrinsic transforming activity of the normally nontransforming c-Abl product in vivo. We show here that this inhibitory activity is extremely position sensitive, because mutants in which the position of the SH3 domain within the protein is subtly altered are fully transforming. In contrast to the case in vivo, the SH3 domain has no effect on the in vitro kinase activity of the purified protein. These results are consistent with a model in which the SH3 domain binds a cellular inhibitory factor, which in turn must physically interact with other parts of the kinase. Unlike the SH3 domain, the SH2 domain is required for transforming activity of activated Abl alleles. We demonstrate that SH2 domains from other proteins (Ras-GTPase-activating protein, Src, p85 phosphatidylinositol 3-kinase subunit, and Crk) can complement the absence of the Abl SH2 domain and that mutants with heterologous SH2 domains induce altered patterns of tyrosine-phosphorylated proteins in vivo. The positive function of the SH2 domain is relatively position independent, and the effect of multiple SH2 domains appears to be additive. These results suggest a novel mechanism for regulation of tyrosine kinases in which the SH2 domain binds to, and thereby enhances the phosphorylation of, a subset of proteins phosphorylated by the catalytic domain. Our data also suggest that the roles of the SH2 and SH3 domains in the regulation of Abl are different in several respects from the roles proposed for these domains in the closely related Src family of tyrosine kinases.
|Additional Information:||Copyright © 1994 by the American Society for Microbiology. Received 22 November 1993/Returned for modification 3 January 1994/Accepted 25 January 1994. We thank R. Ren for critically reading the manuscript and M. Scott, W. Sha, T. Fujita, and other members of the laboratory for many helpful suggestions. We are also grateful to T. Miller for advice about quantitative kinase assays, P. Jackson for the K290M mutant, and S. Nagata for the human elongation factor la expression vector. This work was supported by PHS grants CA 08875 to B.J.M. and CA 51462 to D.B.|
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