Effects of SH2 and SH3 deletions on the functional activities of wild-type and transforming variants of c-Src.

Abstract

The amino-terminal, 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, p1209, 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. pllO 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.

Mutagenic analysis of Vav reveals that an intact SH3 domain is required for transformation

“…Thus, mutations in the SH3 domain of the adaptor v-Crk (Mayer and Hanafusa, 1990) reduced its transforming potential whereas deletion/mutations of this domain from the proto-oncogenes Abl (Jackson and Baltimore, 1989;Jackson et al, 1993) and Src (Erpel et al, 1995;Hirai and Varmus, 1990a,b;Kato et al, 1986;Weng et al, 1995) increased their transforming potential. The kinase activity of the SH3 mutant Src proteins increased relative to the intact protein and in addition, the cytoskeleton organization in cells with these proteins changed and resembled the morphology of v-src transformed cells (Erpel et al, 1995;Hirai and Varmus, 1990a,b;Kato et al, 1986;Seidel-Dugan et al, 1992;Weng et al, 1995). Detailed studies indicated that subtle amino-acid substitutions in the SH3 domain of Src changed their binding properties and therefore resulted in alteration of its biological functions (Erpel et al, 1995;Hirai and Varmus, 1990a,b;Kato et al, 1986;Seidel-Dugan et al, 1992;Weng et al, 1995).…”

“…The kinase activity of the SH3 mutant Src proteins increased relative to the intact protein and in addition, the cytoskeleton organization in cells with these proteins changed and resembled the morphology of v-src transformed cells (Erpel et al, 1995;Hirai and Varmus, 1990a,b;Kato et al, 1986;Seidel-Dugan et al, 1992;Weng et al, 1995). Detailed studies indicated that subtle amino-acid substitutions in the SH3 domain of Src changed their binding properties and therefore resulted in alteration of its biological functions (Erpel et al, 1995;Hirai and Varmus, 1990a,b;Kato et al, 1986;Seidel-Dugan et al, 1992;Weng et al, 1995).…”

Regulation of mRNA splicing and transport by the tyrosine kinase activity of src

“…In agreement with this model, the global level of phosphotyrosine induced by Y527F and Y527F/DSH2 are similar. In contrast, the E378G mutation leads to a signi®cant increase in tyrosine kinase activity in chicken embryo ®broblasts (CEF) by an uncharacterized mechanism (Seidel-Dugan et al, 1992). In NIH3T3 cells, we observed that this kinase activity can be further increased by the deletion of the SH2 domain suggesting that the Y527-SH2 regulatory interaction is still operative in the E378G context (hence the`closed' conformation of the E378G protein in Figure 7).…”

“…By contrast, the transport of immature transcripts, which leads to their accumulation in the cytoplasm, requires the presence of an activating mutation and is sensitive to the SH2 domain, indicating more speci®c requirement on the activity of the src gene. Similarly, Y527F/DSH2 has been reported to be poorly transforming in CEF cells (Kanner et al, 1991) in contrast with E378G/DSH2 (Seidel-Dugan et al, 1992). Therefore, the accumulation of partially spliced transcripts correlates with the transforming activity of src.…”

“…The contribution of these ligands to the e ector functions of src has not yet been fully delineated and, in particular, the role of the SH2 and SH3 domains in the establishment of the transformed phenotype is poorly understood. Indeed, they appear to be required or not, depending upon the nature of the activating mutation (Kanner et al, 1991;Seidel-Dugan et al, 1992) and/or the cellular context (Hirai and Varmus, 1990). The involvement of these domains in the regulation of the tyrosine kinase activity of src (Superti-Furga and Gon¯oni, 1997), may underly these divergent results.…”