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The role of Src family tyrosine kinases in cellular proliferation is well established; however, their role in cellular differentiation is less well understood. In this study we have investigated the role played by Src in the differentiation of squamous epithelial cells. Transfection of activated Src into A431 cells resulted in morphological changes that resembled epidermal differentiation. When we used Src mutants to characterize the observed phenotypic changes, we found that protein tyrosine kinase activity, correct membrane localization and the activity of the SH2 domain were required, but the SH3 domain was not. Furthermore, downstream activity of Ras was not required for the Src-mediated changes in A431 cells.
We recently have shown that activated Ras, but not Raf, causes transformation of intestinal (RIE-1, IEC-6) epithelial cells, whereas both activated Ras and Raf transform NIH 3T3 fibroblasts (Oldham, S. M., Clark, G. J., Gangarosa, L. M., Coffey, R. J., and Der, C. J. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 6924-6928). The observations that conditioned medium from Ras-, but not Raf-, transfected RIE-1 cells, as well as exogenous transforming growth factor alpha (TGFalpha), promoted morphological transformation of parental RIE-1 cells prompted us to identify epidermal growth factor (EGF) receptor (EGFR) ligands produced by Ras-transformed RIE-1 cells responsible for this autocrine effect. Since studies in fibroblasts have shown that v-Src is transforming, we also determined if v-Src could transform RIE-1 cells. H- or K-Ras-transformed cells secreted significant amounts of TGFalpha protein, and mRNA transcripts for TGFalpha, amphiregulin (AR), and heparin-binding EGF-like growth factor (HB-EGF) were induced. Like Ras, v-Src caused morphological and growth transformation of parental RIE-1 cells. However, TGFalpha protein was not secreted by RIE-1 cells stably expressing v-Src or activated Raf, and only minor increases in EGFR ligand mRNA expression were detected in these cells. A selective EGFR tyrosine kinase inhibitor PD153035 attenuated the Ras-, but not Src-, transformed phenotype. Taken together, these observations provide a mechanistic and biochemical basis for the ability of activated Ras, but not activated Raf, to cause transformation of RIE-1 cells. Finally, we suggest that an EGFR-dependent mechanism is necessary for Ras, but not Src, transformation of these intestinal epithelial cells.
Transformation of chicken embryo cells by oncogenic forms of pp60src (e.g., pp60v-src or pp60527F) is linked with a concomitant increase in the steady-state levels of tyrosine-phosphorylated cellular proteins. Activated forms of the Src protein-tyrosine kinase stably associate with tyrosine-phosphorylated proteins, including a protein of 110 kDa, pp110. Previous reports have established that stable complex formation between pp110 and pp60src requires the structural integrity of the Src SH2 and SH3 domains, whereas tyrosine phosphorylation of pp110 requires only the structural integrity of the SH3 domain. In normal chicken embryo cells, pp110 colocalizes with actin stress filaments, and in Src-transformed cells, pp110 is found associated with podosomes (rosettes). Here, we report the identification and characterization of cDNAs encoding pp110. The predicted open reading frame encodes a polypeptide of 635 amino acids which exhibits little sequence similarity with other protein sequences present in the available sequence data bases. Thus, pp110 is a distinctive cytoskeleton-associated protein. On the basis of its association with actin stress filaments, we propose the term AFAP-110, for actin filament-associated protein of 110 kDa. In vitro analysis of AFAP-110 binding to bacterium-encoded glutathione S-transferase (GST) fusion proteins revealed that AFAP-110 present in normal cell extracts binds efficiently to Src SH3/SH2-containing fusion proteins, less efficiently to Src SH3-containing proteins, and poorly to SH2-containing fusion proteins. In contrast, AFAP-110 in Src-transformed cell extracts bound to GST-SH3/SH2 and GST-SH2 fusion proteins. Analysis of AFAP-110 cDNA sequences revealed the presence of sequence motifs predicted to bind to SH2 and SH3 domains, respectively. We suggest that AFAP-110 may represent a cellular protein capable of interacting with SH3-containing proteins and, upon tyrosine phosphorylation, binds tightly to SH2-containing proteins, such as pp60src or pp59fyn. The potential roles of AFAP-110 as an SH3/SH2 cytoskeletal binding protein are discussed.
The transforming protein of Rous sarcoma virus (pp60v-src) and its normal cellular homolog (pp60c-src) are demonstrated to be phosphorylated at serine 12 in vivo under certain conditions. We propose that protein kinase C is responsible for this modification based on the following evidence. First, the tumor promoters, 12-O-tetradecanoylphorbol-13-acetate and teleocidin, and synthetic diacylglycerol, known activators of protein kinase C in vivo, cause nearly complete phosphorylation of pp60src at serine 12. Second, among five purified serine/threonine-specific protein kinases tested, only protein kinase C phosphorylates pp60c-src and pp60v-src in vitro at serine 12. Third, purified protein kinase C phosphorylates a synthetic peptide corresponding to the N-terminal 20 amino acids of pp60c-src at serine 12. The physiological significance of this novel phosphorylation is discussed.
The role of tyrosine phosphorylation in the regulation of tyrosine protein kinase activity was investigated using site-directed mutagenesis to alter the structure and environment of the three tyrosine residues present in the C terminus of avian pp60c-src. Mutations that change Tyr 527 to Phe or Ser activate in vivo tyrosine protein kinase activity and induce cellular transformation of chicken cells in culture. In contrast, alterations of tyrosine residues present at positions 511 or 519 in c-src do not induce transformation or in vivo tyrosine protein kinase activity. Amber mutations, which alter the structure of the pp60c-src C terminus by inducing premature termination of the c-src protein at either residue 518 or 523 also induce morphological transformation and increase in vivo tyrosine phosphorylation, whereas removal of the last four residues of c-src by chain termination at residue 530 does not alter the kinase activity or the biological activity of the resultant c-src protein. We conclude from these studies that C-terminal alterations which either remove or replace Tyr 527 serve to activate the c-src protein resulting in cellular transformation and increased in vivo tyrosine protein kinase activity.
We have identified two phosphotyrosine-containing cellular proteins with relative molecular masses of 130,000 (pp130) and 110,000 (pp110) daltons in chicken embryo cells that coimmunoprecipitated with pp60v-src and activated forms of chicken pp60c-src (pp60(527)F). Most if not all of the tyrosine-phosphorylated forms of pp130 and pp110 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, pp130 and pp110 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 pp130 and pp110 or with pp110 alone. Mutant src proteins, defective for myristylation, showed increased tyrosine phosphorylation of and association with pp110. Expression of src variants with mutations in the A box (pp60dl92/527F) or B box (pp60dl155/527F) of the src homology region induced differences in phosphorylation of pp130 and pp110, 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 pp110 and may be involved in the interaction of activated src proteins with cellular substrates.
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 pp60src, 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 pp60src were also detected by several of the monoclonal antibodies.
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 by activated pp60c-src has been correlated by genetic analysis with the tyrosine phosphorylation of a 120 kilodalton (kDa) protein, p120. We now demonstrate tyrosine phosphorylation of p120 following stimulation of cells by growth factors whose receptors have intrinsic tyrosine-specific protein kinase activity. Stimulation of quiescent NIH3T3 cells with platelet-derived growth factor (PDGF) resulted in the tyrosine phosphorylation of p120 that was maximal by 5 min and returned to background levels by 30 min. p120 was also phosphorylated on tyrosine after addition of colony-stimulating factor 1 (CSF-1) or epidermal growth factor (EGF) to NIH3T3 cells engineered to express high levels of their respective receptors. Two additional src substrates, p110 and p85, were analysed under identical assay conditions. PDGF, CSF-1, and EGF induced only a minimal increase in the tyrosine phosphorylation of p85 and no change in the phosphorylation of p110. Thus, the marked ligand-induced tyrosine phosphorylation of p120 was a property not shared by the other src substrates examined. Immunoblotting with antibodies to p120 and the ras GTPase activating protein, GAP, suggests that p120 and GAP are unrelated. In addition, the amino acid sequences of four cyanogen bromide peptides derived from p120 showed no homology to GAP or to sequences in either the PIR or Swiss-Prot databases. These data suggest that tyrosine phosphorylation of p120 may contribute to both signal transduction through growth factor receptors and pp60src induced transformation.
A novel protein tyrosine kinase (PTK) substrate, p120, has been previously implicated in ligand-induced signaling through the epidermal growth factor, platelet-derived growth factor and colony-stimulating factor 1 receptors, and in cell transformation by p60v-src. We have isolated a near full-length cDNA encoding murine p120. The encoded protein lacks significant homology with any reported protein, but it contains four copies of an imperfect 42 amino acid repeat that occurs 12.5 times in the protein encoded by Drosophila armadillo (arm), and its direct homologs, human plakoglobin (plak) and Xenopus laevis beta-catenin (beta-cat). The presence of this motif implies that p120 may share at least one aspect of its function with the arm protein and its homologs.