Src homology 2 domain substitution modulates the kinase and transforming activities of the fes protein-tyrosine kinase

The c-fes proto-oncogene encodes a M-r 93,000 protein-tyrosine kinase (Fes) that is strongly expressed in myeloid cells and has been implicated in mye lomonocytic differentiation. Fes autophosphorylation and transforming activ ity are highly restrained after ectopic expression in fibroblasts, indicati ng tight negative regulation of Fes kinase activity in vivo. Here we invest igated the regulatory role of the Fes Src homology 2 (SH2) domain by produc ing a series of chimeric constructs in which the Fes SH2 domain was replace d with those of the transforming oncogenes v-Fps and v-Src or by the NH2-te rminal SH2 domain of the Ras GTPase-activating protein. Wild-type and chime ric Fes proteins readily underwent tyrosine autophosphorylation in vitro an d produced identical cyanogen bromide phosphopeptide cleavage patterns, ind icating that the SH2 substitutions did not influence overall kinase activit y or autophosphorylation site selection. However, metabolic labeling of Rat -2 fibroblasts expressing each construct showed that only the Fes/Src SH2 c himera was active in vivo. Consistent with this result, the Fes/Src SH2 dom ain chimera exhibited potent transforming activity in fibroblasts and enhan ced differentiation-inducing activity in K-562 myeloid leukemia cells. In a ddition, the Fes/Src SH2 chimera exhibited constitutive localization to foc al adhesions in Rat-2 fibroblasts and induced the attachment and spreading of TF-1 myeloid cells. These data demonstrate a central role for the SH2 do main in the regulation of Fes kinase activity and biological function in vi vo.