p60v-src has been shown to associate with a detergent-insoluble cellular matrix containing cytoskeletal proteins, but p60csrc does not bind to this matrix. We analyzed the association of mutant src proteins with the matrix and found that mutants which lack an amino-terminal portion (residues 149 to 169) of the SH2 domain cannot bind to the matrix. Neither the SH3 region nor other portions of the SH2 region were required for association. We also tested protein kinase-defective mutants and chimeras of p6O-src and p60csrc. We found a strong correlation between the kinase activity of p6Osrc and its association with the detergent-insoluble matrix. Double infection of kinase-defective and kinase-active mutants did not result in matrix binding of the kinase-defective src proteins. We also found that Tyr-416, the major site of autophosphorylation in p60vsrc, was not required for matrix association.Rous sarcoma virus (RSV) encodes the oncogene v-src. The product of v-src, p60vsrc, is a tyrosine-specific protein kinase (19). p60vsrc is myristoylated on a glycine residue at the amino terminus and localizes in the membrane fraction (19). A number of cellular proteins are phosphorylated on tyrosine upon transformation with p60vsrc. Most of these are also in the membrane fraction (13), suggesting that specific localization of p60v-src is important for cell transformation.Burr et al. (3) have demonstrated association of p60v-src with a detergent-resistant subcellular structure that consists mostly of cytoskeleton. It has been previously reported that most transforming variants of p60vsrc associate with this structure but that nontransforming variants, including the cellular proto-oncogene p60csrc, do not (12,30). However, it is not known which sequences of p60v-src are necessary for its association with this structure.Two regions of sequence conservation have been identified in the amino termini of cytoplasmic tyrosine kinases, including p6Osrc (32, 36). These regions are SH2 (src homology 2, residues 137 through 241 in src; see Fig. lA) and SH3 (src homology 3, residues 84 through 114). SH2 sequences are also present in phospholipase C--y (39), GAP (ras GTPase-activating protein) (43), and the crk oncogene product (32). 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. Recently, SH2 sequences have been shown to bind to phosphotyrosine-containing proteins (31).SH3 sequences are present in most proteins containing SH2, as well as in a-spectrin (28), Acanthamoeba myosin-IB (34), and the actin binding protein of Saccharomyces cerevisiae, ABPlp (9). The latter three proteins are all associated with the membrane cytoskeleton, suggesting that the SH3 sequence mediat...

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

GTPase-activating protein (GAP) enhances the rate of GTP hydrolysis by cellular Ras proteins and is implicated in mitogenic signal transduction. GAP is phosphorylated on tyrosine in cells transformed by Rous sarcoma virus and serves as an in vitro substrate of the viral Src (v-Src) kinase. Our previous studies showed that GAP complexes stably with normal cellular Src (c-Src), although its association with v-Src is less stable. To further investigate the molecular basis for interactions between GAP and the Src kinases, we examined GAP association with and phosphorylation by a series of c-Src and v-Src mutants. Analysis of GAP association with c-Src/v-Src chimeric proteins demonstrates that GAP associates stably with Src proteins possessing low kinase activity and poorly with activated Src kinases, especially those that lack the carboxy-terminal segment of c-Src containing the regulatory amino acid Tyr-527. Phosphorylated Tyr-527 is a major determinant of c-Src association with GAP, as demonstrated by c-Src point mutants in which Tyr-527 is changed to Phe. While the isolated amino-terminal half of the c-Src protein is insufficient for stable GAP association, analysis of point substitutions of highly conserved amino acid residues in the c-Src SH2 region indicate that this region also influences Src-GAP complex formation. Therefore, our results suggest that both Tyr-527 phosphorylation and the SH2 region contribute to stable association of c-Src with GAP. Analysis of in vivo phosphorylation of GAP by v-Src mutants containing deletions encompassing the SH2, SH3, and unique regions suggests that the kinase domain of v-Src contains sufficient substrate specificity for GAP phosphorylation. Even though tyrosine phosphorylation of GAP correlates to a certain extent with the transforming ability of various c-Src and v-Src mutants, our data suggest that other GAP-associated proteins may also have roles in Src-mediated oncogenic transformation. These findings provide additional evidence for the specificity of Src interactions with GAP and support the hypothesis that these interactions contribute to the biological functions of the Src kinases.

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p60v-src has been shown to associate with a detergent-insoluble cellular matrix containing cytoskeletal proteins, but p60c-src does not bind to this matrix. We analyzed the association of mutant src proteins with the matrix and found that mutants which lack an amino-terminal portion (residues 149 to 169) of the SH2 domain cannot bind to the matrix. Neither the SH3 region nor other portions of the SH2 region were required for association. We also tested protein kinase-defective mutants and chimeras of p60v-src and p60c-src. We found a strong correlation between the kinase activity of p60src and its association with the detergent-insoluble matrix. Double infection of kinase-defective and kinase-active mutants did not result in matrix binding of the kinase-defective src proteins. We also found that Tyr-416, the major site of autophosphorylation in p60v-src, was not required for matrix association.