pp60v-src transformation of rat cells but not chicken cells strongly correlates with low-affinity phosphopeptide binding by the SH2 domain.
Substrates critical for transformation by pp60v-src remain unknown, as does the precise role of the src homology 2 (SH2) domain in this process. To continue exploring the role of the SH2 domain in pp60v-src-mediated transformation, site-directed mutagenesis was used to create mutant v-src alleles predicted to encode proteins with overall structural integrity intact but with reduced ability to bind phosphotyrosine-containing peptides. Arginine-175, which makes critical contacts in the phosphotyrosine-binding pocket, was mutated to lysine or alanine. Unexpectedly, both mutations created v-src alleles that transform chicken cells with wild-type (wt) efficiency and are reduced for transformation of rat cells; these alleles are host dependent for transformation. Additionally, these alleles resulted in a round morphological transformation of chicken cells, unlike 12 of the 13 known host-dependent src SH2 mutations that result in a fusiform morphology. Analysis of phosphopeptide binding by the mutant SH2 domains reveal that the in vitro ability to bind phosphopeptides known to have a high affinity for wt src SH2 correlates with wt (round) morphological transformation in chicken cells and in vitro ability to bind phosphopeptides known to have a low affinity for wt src SH2 correlates with rat cell transformation. These results suggest that the search for critical substrates in rat cells should be among proteins that interact with pp60v-src with low affinity.
Verderame
M F
MF
Department of Medicine, College of Medicine, Pennsylvania State University, Hershey 17033, USA.
D000818:Animals; D002460:Cell Line; D002471:Cell Transformation, Neoplastic; D002642:Chick Embryo; D015870:Gene Expression; D009154:Mutation; D015688:Oncogene Protein pp60(v-src) / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D010748:Phosphopeptides / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D013045:Species Specificity; D018909:src Homology Domains
D010748:Phosphopeptides; D015688:Oncogene Protein pp60(v-src)