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 ...

We used myristylated and nonmyristylated c-src-based variants and phosphotyrosine-specific antibodies to reevaluate the role of tyrosine phosphorylation in cellular transformation by pp60src. Prior methods used to detect tyrosine-phosphorylated proteins failed to discriminate predicted differences in tyrosine phosphorylation which are clearly observed with phosphotyrosine-specific antibodies and Western blotting (immunoblotting). Here we report the observation of a 120,000-Mr protein whose phosphorylation on tyrosine correlates with the induction of morphological transformation. p120 was not observed in cells overexpressing the regulated, nononcogenic pp60c-src, whereas phosphorylation of p120 was greatly enhanced in cells expressing activated, oncogenic pp60527F. Furthermore, phosphorylation of p120 was not induced by expression of the activated but nonmyristylated src variant pp602A/527F, which is transformation defective. p120 partitioned preferentially with cellular membranes, consistent with the observation that transforming src proteins are membrane associated. Although a number of additional putative substrates were identified and partially characterized with respect to intracellular localization, tyrosine phosphorylation of these proteins was not tightly linked to transformation.

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.

We have identified two phosphotyrosine-containing cellular proteins with relative molecular masses of 130,000 (ppl30) and 110,000 (ppllO) daltons in chicken embryo cells that coimmunoprecipitated with pp60v-src and activated forms of chicken pp6ocsrc (pp60527F). Most if not all of the tyrosine-phosphorylated forms of ppl30 and ppllO could be immunoprecipitated from lysates with any of several src protein-specific monoclonal antibodies directed against at least three spatially distinct epitopes. Consequently, of the more than 15 prominent phosphoproteins detected on immunoblots with phosphotyrosine-specific antibodies, ppl30 and ppl1O were selectively removed by src protein-specific immunoprecipitation, and their presence in the immunoprecipitates appears to have been due to a direct interaction with activated src proteins. src protein variants that induce different morphological phenotypes were altered in their ability to form detergent-stable complexes with ppl30 and ppllO or with ppllO alone. Mutant src proteins, defective for myristylation, showed increased tyrosine phosphorylation of and association with ppllO. Expression of src variants with mutations in the A box (pp60dl921527F) or B box (pp6OdIl55/527F) of the src homology region induced differences in phosphorylation of ppl30 and pplO, as well as changes in their association with variant src proteins. Sequences within the B-box region appeared to be necessary for stable complex formation with pp130 and ppllO and may be involved in the interaction of activated src proteins with cellular substrates.The transforming gene of Rous sarcoma virus (RSV), v-src, encodes a 60-kilodalton (kDa) protein tyrosine kinase (pp60vsr() whose expression is both necessary and sufficient to initiate and maintain cellular transformation (1, 2, 19-21, 23, 35, 56; J. A. Cooper, in B. Kemp and P. F. Alewood, ed., Peptides and Protein PhosphoylWation, in press). Its normal cellular counterpart, c-src, encodes a similar protein (pp6Oc--') which is not oncogenic, even when expressed at high levels (22,41,57). Although there are numerous amino acid differences between the two proteins, the major oncogenic difference occurs at the C-terminal end, where 19 C-terminal amino acids in pp6Ocsr( have been replaced with 12 unrelated amino acids in pp60v-r'c (55,60). The kinase activity of pp60c--"' is restricted by phosphorylation of a C-terminal tyrosine residue, Tyr-527, which is not present in pp60vs"" (10,11,13,34). Mutations within this C-terminal regulatory region, e.g., Tyr-527 to Phe or Ser or the truncation of the c-src protein within the C-terminal 17 residues, activate the tyrosine protein kinase activity of pp6Oc-''' and concomitantly, the transforming potential of the c-src gene product (8,30,31,47,52).Other structural domains within pp60''' have been shown to influence transforming activity. Myristylation is required for stable membrane association and transformation (6,7,14,16,54). Substitution of Ala or Glu for Gly-2 yields a soluble src protein with unaltered tyrosine kina...

The monoclonal antibody 2B12 is directed toward p120, a 120-kDa cellular protein originaUly identified as a protein tyrosine kinase substrate in cells expressing membrane-associated oncogenic variants of pp6(Yrc. In this report, we show that p120 was tyrosine phosphorylated in avian cells expressing membrane-associated, enzymatically activated variants of c-src, including variants having structural alterations in the src homology regions 2 and 3. In contrast, p120 was not tyrosine phosphorylated in cells expressing enzymatically activated, nonmyristylated pp60'. Furthermore, p120 was tyrosine phosphorylated in avian ceUls expressing middle T antigen, the transforming protein of polyomavirus, as weUl as in rodent cells stimulated with either epidermal growth factor (EGF) or platelet-derived growth factor. Analysis of the time course of p120 tyrosine phosphorylation in EGF-stimulated cells revealed a rapid onset of tyrosine phosphorylation. In addition, both the extent and duration of p120 phosphorylation increased when cells overexpressing the EGF receptor were stimulated with EGF. Biochemical analysis showed that p120 (in both normal and src-transformed cells) was membrane associated, was myristylated, and was phosphorylated on serine and threonine residues. Hence, p120 appears to be a substrate of both nonreceptor-and ligand-activated transmembrane receptor tyrosine kinases and of serine/threonine kinases and is perhaps a component of both mitogen-stimulated and tyrosine kinase oncogene-induced signaling pathways.