Activation of the proto-oncogene p60c-src by point mutations in the SH2 domain.

Abstract

To investigate the importance of a conserved region spanning residues 137 to 241 in the noncatalytic domain of p60&csrc (SH2 region), we used oligonucleotide-directed mutagenesis to change residues that are highly conserved in this region. Chicken The proto-oncogene p60c-src, like its viral counterpart p60v-src, is a membrane-bound tyrosine-specific protein kinase of molecular weight 60,000 (for a review, see reference 20). However, p6c-src is not transforming even when overexpressed (19,41,51 proteins. A newly described oncogene, crk, has sequence similarities to tyrosine-specific protein kinases in the SH2 and SH3 regions but has no catalytic domain (38). Transformation by this oncogene does increase the level of phosphorylation on tyrosine within the cell, presumably indirectly. One isozyme of phospholipase C (phospholipase C--y) has two copies of SH2 and one of SH3 (54). In addition, GTPase-activating protein (GAP), which activates GTPase in normal but not oncogenic p2lras, has two copies of SH2 (57). GAP has been shown to have increased phosphorylation on tyrosine in cells transformed by v-src or other tyrosine kinases (11).Experiments with deletion mutants of p60vsrc indicate that the SH2 region is important for transformation (2,6,28,44,58,60). The mutants are transformation defective, incompletely transforming, or temperature sensitive. Various linker insertions and deletions in the SH2 region of fps produce host-dependent phenotypes or impaired kinase activity (9,29,49). Recently, mutations of SH2 in p60v-src have been found that also give a host-dependent transformationdefective phenotype (8,56). It has been suggested that the SH2 region interacts with a host cell protein (42).We were interested to see whether disrupting the SH2 region by changing the most conserved residues would affect the transforming ability of p60csrc. We therefore carried out oligonucleotide-directed mutagenesis of four well-conserved residues in SH2 and characterized the resulting mutants. Several of these are activated, as measured by morphological transformation, anchorage independence, and elevated kinase activities in vivo and in vitro. MATERIALS AND METHODSPlasmid constructions. The template used for mutagenesis was a modification of p5H in which the MluI site downstream of the c-src coding region was removed for convenient manipulation (gift of R. Jove; 22). This plasmid contains the complete coding sequence of chicken c-src and

New insights into protein‐tyrosine kinase receptor signaling complexes

“…In support of this, high-affinity binding phosphopeptides have been shown to be able to stimulate Src PTK activity in vitro (Lui et al, 1993). These data fit well with the earlier finding that point mutations within the SH2 domain (which from the crystal structure are predicted to disrupt the overall structure of the domain, or which involve key residues in binding phosphotyrosine) were both able to activate the protooncogene pp60c-src (O'Brien et al, 1990).…”

The catalytic activity of Src‐family tyrosine kinase is required for B cell antigen receptor signaling

“…These results are in good agreement with the previous report that deletion of the SH2 domain of Lyn causes an increase in tyrosine phosphorylation in NIH 3T3 cells despite its lower in vitro kinase activity [16]. In contrast to the lower kinase activity of the SH2 mutants of Fyn and Lyn, mutations in Src SH2 domain lead to the dramatic increase in kinase activity [17,18]. These observations suggest that the enzymatic regulation of Fyn and Lyn is different from that of Src, and that the SH2 domain of Fyn and Lyn may play a role in repressing the ability of Fyn and Lyn to phosphorylate substrates through a mechanism independent of regulating intrinsic kinase activity of Fyn and Lyn.…”

Deletions in the SH2 domain of p60v-src prevent association with the detergent-insoluble cellular matrix.

“…Therefore, there must be another factor determining the association of p6Osrc with the cytoskeleton in addition to the presence of this sequence. b Colony formation was assayed by seeding infected cells in soft agar as described previously (33). ' See reference 5 for a description of NY310, NY311, and NY320.…”

“…For the double-infection experiments, we therefore produced virus stocks of subgroup A or B by transfecting with the appropriate version of pSR-REP (27). Soft agar colony formation was performed as described previously (33).…”

“…Mutations of the SH2 regions of v-src and v-fps produce transformation-defective or temperature-sensitive tyrosine kinase proteins (2,5,25,26,36,44). Point mutations in the SH2 region of the proto-oncogene p60c-src have been shown to elevate kinase activity and transforming ability (17,33). Certain SH2 mutations give host-dependent phenotypes (7,8,42), indicating that the SH2 region may interact with host cell proteins.…”