THE ROLE OF THE LINKER BETWEEN THE SH2 DOMAIN AND CATALYTIC DOMAIN INTHE REGULATION AND FUNCTION OF SRC

The crystal structures of the regulated Src and Hck tyrosine kinases s how intramolecular interactions between the phosphorylated tail and th e SH2 domain as well as between the SH3 domain, the SH2-catalytic doma in linker (SH2-CD linker) and the catalytic domain, The relative contr ibution of these interactions to regulation of activity is poorly unde rstood. Mutational analysis of Src and Lck revealed that interaction o f the SH2-CD linker with the SH3 domain is crucial for regulation, Mor eover, three sites of interaction of the linker with the catalytic dom ain, one at the beginning (Trp260) and two at the back of the small lo be, opposite the catalytic cleft (beta 2/beta 3 loop; alpha C/beta 4 l oop), impinge on Src activity, Other activating mutations map to the f ront of the catalytic domain in the loop preceding the alpha C-helix ( beta 3/alpha C loop), SH2-CD linker mutants are deregulated in mammali an cells but transform fibroblasts weakly, suggesting that the linker may bind cellular components. Interpretation of our results on the bas is of the crystal structure of Src favours a model in which the correc tly positioned SH2-CD linker exerts an inhibitory function on catalysi s of Src family members by facilitating displacement of the alpha C-he lix, This study may provide a template for the generation of deregulat ed versions of other protein kinases.