Transformation of cells by the src oncogene results in elevated tyrosine phosphorylation of two related proteins, p80 and p85 (p80/85). Immunostaining with specific monoclonal antibodies revealed a striking change of subcellular localization of p80/85 in src-transformed cells. p80/85 colocalizes with F-actin in peripheral extensions of normal cells and rosettes (podosomes) of src-transformed cells. Sequence analysis of cDNA clones encoding p80/85 revealed an amino-terminal domain composed of six copies of a direct tandem repeat, each repeat containing 37 amino acids, a carboxyl-terminal SH3 domain, and an interdomain region composed of a highly charged acidic region and a region rich in proline, serine, and threonine. The multidomain structure of p80/85 and its colocalization with F-actin in normal and src-transformed cells suggest that these proteins may associate with components of the cytoskeleton and contribute to organization of cell structure.Transformation of cells by tyrosine kinase oncogenes leads to alterations of cell shape, cellular metabolism, growth control, and gene expression (16,31,54). A substantial body of evidence indicates that many if not all of these changes are a direct result of the tyrosine kinase activity of the oncogene product (reviewed in references 31, 54, and 66). Rous sarcoma virus (RSV) encodes an enzymatically activated, 60-kDa tyrosine protein kinase, pp60v"src (6,15,44). However, the product of the normal cellular homolog of src, pp60c-sc, is enzymatically down regulated and does not induce significant alterations in cellular growth or changes in cell morphology when overexpressed in rodent or avian cells (30,53,67). Oncogenic activation of c-src and concomitant activation of tyrosine kinase activity can be achieved by mutation of the regulatory site of tyrosine phosphorylation, 40,58,63). Thus, expression of pp6Ov-src or activated forms of the c-src protein pp60527F results in efficient cellular transformation and the increased tyrosine phosphorylation of approximately 15 to 30 cellular proteins (27,33,45,61).Genetic studies have shown that structural perturbation of several different domains of pp6Osrc leads to alterations in the pattern of tyrosine phosphorylation of specific cellular proteins and accompanying changes in morphological phenotypes (reviewed in references 31 and 54). For example, mutation of the site of myristylation (e.g., Gly-2 to Ala) of pp60v-src or pp60527F blocks cellular transformation (9, 32, 61) and the tyrosine phosphorylation of a 120-kDa cellular protein (35,45,61). pp60src contains two regions that share amino acid sequence similarity with other nonreceptor tyrosine protein kinases (55) and regulatory proteins such as phospholipase C--y, Crk, and GTPase-activating protein (GAP) (70,73,76). Structural alterations within these regions alter or abolish the transforming activity of src (18,28,51,59,77,79) Whereas recent experiments have shown that tyrosine phosphorylation of some cellular proteins appears to direct stable protein-protein interactio...
Cellular transformation by oncogenic retroviruses encoding protein tyrosine kinases coincides with the tyrosine-specific phosphorylation of multiple protein substrates. Previous studies have shown that tyrosine phosphorylation of a protein of 120 kDa, p120, correlated with src transformation in chicken embryo fibroblasts. Additionally, we previously identified two phosphotyrosine-containing cellular proteins, p130 and p110, that formed stable complexes with activated variants of pp605)C, the src-encoded tyrosine kinase. To study transformation-relevant tyrosine kinase substrates, we have generated monoclonal antibodies to individual tyrosine phosphoproteins, including p130, p120, p110, and five additional phosphoproteins (p210, p125, p118, p85, and p185/p64). These antibodies detected several of the same tyrosine phosphoproteins in chicken embryo fibroblasts transformed by avian retroviruses Y73 and CT10, encoding the yes and crk oncogenes, respectively. Protein substrates in mouse, rat, hamster, and human cells overexpressing activated variants of chicken pp6rc were also detected by several of the monoclonal antibodies.The role of tyrosine phosphorylation of cellular proteins by oncogene-encoded tyrosine kinases during transformation is largely unclear. Multiple protein substrates are phosphorylated on tyrosine in response to various cell stimuli, including growth factor activation (1-5), agonist stimulation of secretion (6), platelet activation (7-9), cell cycle changes (10, 11), and cell transformation induced by oncogene-encoded tyrosine kinases (12)(13)(14)(15)(16)(17)(18)(19)(20)(21). Although the majority of these cellular protein substrates have not been identified, recent studies have demonstrated the interactions of known tyrosine phosphoproteins thought to be involved in signal-transduction pathways (22). Direct identification of the individual proteins may provide information regarding their activities during cell growth processes and perhaps lead to an understanding of the relevance of tyrosine phosphorylation of proteins in vivo.The study of tyrosine-phosphorylated cellular proteins has recently relied on antibodies to phosphotyrosine (anti-P-Tyr) (13,15,16,23,24). Immunoblot analysis of cell proteins with anti-P-Tyr has revealed previously undetected P-Tyrcontaining proteins in both normal and transformed cells. Specifically, expression of the oncogene-encoded tyrosine kinase pp60src of Rous sarcoma virus (RSV) leads to the tyrosine phosphorylation of 15-20 different cellular substrates in several cell systems (13,16,17,19,20).We and others have demonstrated that one phosphoprotein of 120 kDa, p120, may be relevant to transformation of chicken embryo fibroblasts (CEFs) by activated pp60src (17,20). A mutation of the normal c-src gene that changes the Cterminal tyrosine residue (Tyr-527) to phenylalanine was sufficient to induce both cell transformation (25-29) and tyrosine phosphorylation of p120 (17,20). However, an additional mutation at the N-terminal glycine residue at position 2 (the ...
Transformation of chicken embryo cells with the tyrosine kinase oncogene src results in the tyrosine phosphorylation of numerous cellular proteins. We have recently generated monoclonal antibodies to individual tyrosine phosphorylated cellular src substrates, several of which are directed to the phosphotyrosine‐containing proteins p130 and p110. These proteins form stable complexes with activated variants of pp60src. Mutagenesis of the src homology domains (SH2 and SH3) of activated pp60src resulted in src variants with altered association with p130 and p110. Analysis of these variants showed that the SH3 domain was required for association of p110, while the SH2 domain contained residues necessary for the formation of the ternary complex involving p130, p110 and pp60src. Both the tyrosine phosphorylation status and pp60src association of p130 and p110 appeared to correlate, in part, with the extent of cell transformation. Biochemical analysis demonstrated that p130 and p110 were substrates of both serine/threonine and tyrosine kinases. In addition, p130 was redistributed from the nucleus to cellular membranes upon src transformation, whereas p110, which normally colocalized with cytoskeletal elements, was observed in adhesion plaques (podosomes) in src transformed cells. These data indicate that tyrosine phosphorylation of two different phosphoproteins may play a role during src transformation either by directing their interaction with pp60src, by redirecting subcellular distribution or both.
The phosphorylation of protein tyrosine kinases (PTKs) on tyrosine residues is a critical regulatory event that modulates catalytic activity and triggers the physical association of PTKs with Src homology 2 (SH2)-containing proteins. The integrin-linked focal adhesion kinase, pp125FAK, exhibits extracellular matrix-dependent phosphorylation on tyrosine and physically associates with two nonreceptor PTKs, pp60src and pp59fyn, via their SH2 domains. Herein, we identify Tyr-397 as the major site of tyrosine phosphorylation on pp125FAK both in vivo and in vitro. Tyrosine 397 is located at the juncture of the N-terminal and catalytic domains, a novel site for PTK autophosphorylation. Mutation of Tyr-397 to a nonphosphorylatable residue dramatically impairs the phosphorylation of pp125FAK on tyrosine in vivo and in vitro. The mutation of Tyr-397 to Phe also inhibits the formation of stable complexes with pp60src in cells expressing Src and FAK397F, suggesting that autophosphorylation of pp125FAK may regulate the association of pp125FAK with Src family kinases in vivo. The identification of Tyr-397 as a major site for FAK autophosphorylation provides one of the first examples of a cellular protein containing a high-affinity binding site for a Src family kinase SH2 domain. This finding has implications for models describing the mechanisms of action of pp125FAK, the regulation of the Src family of PTKs, and signal transduction through the integrins.
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