title: Effects of SH2 and SH3 deletions on the functional activities of wild-type and transforming variants of c-Src.
abstract: The amino-termina, noncatalytic half of Src contains two domains, designated the Src homology 2 (SH2) and Src homology 3 (SH3) domains, that are highly conserved among members of the Src family of tyrosine kinases. The SH2 domain (which can be further divided into the B and C homology boxes) and the SH3 domain (also referred to as the A box) are also found in several proteins otherwise unrelated to protein tyrosine kinases. It is believed that these domains are important for directing specific protein-protein interactions necessary for the proper functioning of Src. To determine the importance of the SH2 and SH3 domains in regulating the functions of c-Src, we evaluated mutants of c-Src lacking the A box (residues 88 to 137), the B box (residues 148 to 187) or the C box (residues 220 to 231). Each of these deletions caused a 14- to 30-fold increase in the in vitro level of kinase activity of c-Src. Chicken embryo fibroblasts expressing the deletion mutants displayed a transformed cell morphology, formed colonies in soft agar, and contained elevated levels of cellular phosphotyrosine-containing proteins. Src substrates p36, p85, p120, p125, the GTPase-activating protein (GAP), and several GAP-associated proteins were phosphorylated on tyrosine in cells expressing the A, B, or C box deletion mutant. p110 was highly phosphorylated in cells expressing the C box mutant, was weakly phosphorylated in cells expressing the B box mutant, and was not phosphorylated in cells expressing the A box mutant. Expression of the mutant proteins caused a reorganization of the actin cytoskeleton similar to that seen in v-Src-transformed cells. In addition, deletion of the A, B, or C box did not diminish the transforming or enzymatic activity of an activated variant of c-Src, E378G. These data indicate that deletion of the A, B, or C homology box causes an activation of the catalytic and transforming potential of c-Src and that while these mutations caused subtle differences in substrate phosphorylation, the homology boxes are not required for many of the phenotypic changes associated with transformation by Src.
authors.affiliation: Howard Hughes Medical Institute, University of Pennsylvania School of Medicine, Philadelphia 19104.
authors.forename: C
authors.initials: C
authors.lastname: Seidel-Dugan
authors.forename: B E
authors.initials: BE
authors.lastname: Meyer
authors.forename: S M
authors.initials: SM
authors.lastname: Thomas
authors.forename: J S
authors.initials: JS
authors.lastname: Brugge
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D002471:Cell Transformation, Neoplastic / Q000235:genetics*; D002642:Chick Embryo; D003599:Cytoskeleton / Q000378:metabolism; D004252:DNA Mutational Analysis; D005347:Fibroblasts; D016391:Genes, src / Q000235:genetics*; D009024:Morphogenesis; D015688:Oncogene Protein pp60(v-src) / Q000235:genetics*; D010449:Peptide Mapping; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases; D013329:Structure-Activity Relationship
chemical_list: D000199:Actins; D011505:Protein-Tyrosine Kinases; D015688:Oncogene Protein pp60(v-src)