Src homology 2 domain substitution modulates the kinase and transforming activities of the Fes protein-tyrosine kinase.
The c-fes proto-oncogene encodes a Mr 93,000 protein-tyrosine kinase (Fes) that is strongly expressed in myeloid cells and has been implicated in myelomonocytic differentiation. Fes autophosphorylation and transforming activity are highly restrained after ectopic expression in fibroblasts, indicating tight negative regulation of Fes kinase activity in vivo. Here we investigated the regulatory role of the Fes Src homology 2 (SH2) domain by producing a series of chimeric constructs in which the Fes SH2 domain was replaced with those of the transforming oncogenes v-Fps and v-Src or by the NH2-terminal SH2 domain of the Ras GTPase-activating protein. Wild-type and chimeric Fes proteins readily underwent tyrosine autophosphorylation in vitro and produced identical cyanogen bromide phosphopeptide cleavage patterns, indicating that the SH2 substitutions did not influence overall kinase activity or autophosphorylation site selection. However, metabolic labeling of Rat-2 fibroblasts expressing each construct showed that only the Fes/Src SH2 chimera was active in vivo. Consistent with this result, the Fes/Src SH2 domain chimera exhibited potent transforming activity in fibroblasts and enhanced differentiation-inducing activity in K-562 myeloid leukemia cells. In addition, the Fes/Src SH2 chimera exhibited constitutive localization to focal 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 domain in the regulation of Fes kinase activity and biological function in vivo.
Rogers
J A
JA
Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pennsylvania 15261, USA.
Cheng
H Y
HY
Smithgall
T E
TE
D019943:Amino Acid Substitution / Q000235:genetics*; D000818:Animals; D002448:Cell Adhesion; D002454:Cell Differentiation; D002455:Cell Division; D002460:Cell Line; D002471:Cell Transformation, Neoplastic; D003598:Cytoskeletal Proteins / Q000378:metabolism; D004789:Enzyme Activation; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism / Q000473:pathology; D022001:Focal Adhesions / Q000737:chemistry / Q000378:metabolism; D015681:Fusion Proteins, gag-onc / Q000737:chemistry / Q000235:genetics; D006412:Hematopoietic Stem Cells / Q000166:cytology; D006801:Humans; D022423:Myeloid Cells / Q000166:cytology / Q000378:metabolism / Q000473:pathology; D015688:Oncogene Protein pp60(v-src) / Q000737:chemistry / Q000235:genetics; D051419:Paxillin; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D021381:Protein Transport; D011505:Protein-Tyrosine Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D051578:Proto-Oncogene Proteins c-fes; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020703:ras GTPase-Activating Proteins / Q000737:chemistry / Q000235:genetics; D018909:src Homology Domains / Q000235:genetics*
D003598:Cytoskeletal Proteins; D015681:Fusion Proteins, gag-onc; C000715470:MAS1 protein, human; C496061:PXN protein, human; D051419:Paxillin; D010750:Phosphoproteins; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins; C496063:Pxn protein, rat; D011993:Recombinant Fusion Proteins; D020703:ras GTPase-Activating Proteins; D011505:Protein-Tyrosine Kinases; C496645:FES protein, human; D015688:Oncogene Protein pp60(v-src); D051578:Proto-Oncogene Proteins c-fes; C068415:v-fps oncogene protein, Fujinami sarcoma virus