pp60v-src transformation of rat cells but not chicken cells strongly correlates with low-affinity phosphopeptide binding by the SH2 domain.

Abstract

Substrates critical for transformation by pp6Ov-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 pp60vsrc-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 the 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 pp6ov-src with low affinity.

The role of the Src homology domains in morphological transformation by v-src.

“…The rationale for the use of this protein was as follows. We have shown previously that the R175H mutation reduces but does not eliminate the binding of the Src SH2 domain to phosphotyrosyl proteins, without affecting the ability of the mutant Src protein to transform CEFs to a wild-type (morphr) phenotype (Tian and Martin, 1996); similar observations have been made on the R175K and R175A mutants of v-Src (Tian and Martin, 1996;Verderame, 1997). Measurements of the affinity of the mutant Src proteins to phosphotyrosyl peptides indicate that the binding of the Src SH2 domain to lowaffinity phosphopeptides is not required for wild-type SH2 and SH3 in Transformation by v-src transformation of CEFs (Verderame, 1997).…”

“…We have shown previously that the R175H mutation reduces but does not eliminate the binding of the Src SH2 domain to phosphotyrosyl proteins, without affecting the ability of the mutant Src protein to transform CEFs to a wild-type (morphr) phenotype (Tian and Martin, 1996); similar observations have been made on the R175K and R175A mutants of v-Src (Tian and Martin, 1996;Verderame, 1997). Measurements of the affinity of the mutant Src proteins to phosphotyrosyl peptides indicate that the binding of the Src SH2 domain to lowaffinity phosphopeptides is not required for wild-type SH2 and SH3 in Transformation by v-src transformation of CEFs (Verderame, 1997). We reasoned therefore that a fusion protein containing the R175H SH2 domain might recognize a more restricted set of proteins than the wild-type SH2 domain and that this could simplify the characterization of proteins recognized by the Src N terminus, whose phosphorylation might correlate with transformation.…”

Roles of the SH2 and SH3 domains in the regulation of neuronal Src kinase functions

“…1D and Table 1). Previous studies [3,18–21] have shown that D99 (corresponding to D101 in n‐Src) in the SH3 domain of c‐Src forms a salt bridge with an arginine located three residues upstream of the conserved PXXP motif of the SH3 ligand. The D99N mutation prevents formation of this salt bridge and disrupts the SH3 binding specificity.…”

“…Mutation of R175 to lysine prevents this connection, and decreases SH2 interactions with its ligand. D99N and R175K mutations therefore inhibit interactions with ligands of the SH3 and SH2 domains, respectively, both intra‐ and inter‐molecularly, and thereby disrupt the overall functions of Src kinase [3,18–21].…”

Genome-Wide Prediction of SH2 Domain Targets Using Structural Information and the FoldX Algorithm

“…Although the differences in the binding constants for different phosphopeptides are often modest [9] , they are known to play an important role in regulating signal transduction in vivo [3] . For example, exchanging an SH2 domain for another with a different specificity can impair activation of the Ras pathway in Caenorhabditis elegans [10] , alter the transformation ability of the Abelson murine leukemia [11] and the Rous sarcoma viruses [12] and trigger mesoderm formation in Xenopus laevis [13] . Moreover, point mutations that induce changes in specificity are associated with diseases such as the X-linked alpha-gammaglobulinemia [14] , the X-linked lymphoproliferative syndrome [15] and the Noonan syndrome [16] .…”