Characterization and use of monoclonal antibodies for isolation of phosphotyrosyl proteins from retrovirus-transformed cells and growth factor-stimulated cells.

Protein kinases that phosphorylate the hydroxyl group of tyrosine residues of proteins have been implicated in cell transformation by some retroviruses and in regulation of normal cell growth by some polypeptide growth factors. To facilitate the identification of tyrosine kinase substrates, we developed monoclonal antibodies to the hapten azobenzylphosphonate. One of these antibodies, MA-2G8, proved to be especially attractive in that it bound a derivative of aminophenylphosphate, a close phosphotyrosine analog, with higher affinity than it bound the corresponding derivative of aminobenzylphosphonate; however, its affinity for phosphoserine was negligible. In this paper we describe the optimal conditions for using this antibody to isolate phosphotyrosine proteins, emphasizing particularly that its interaction with phosphotyrosyl proteins is sensitive to ionic detergents and to antibody density on the immunosorbent matrix. The antibody also bound ATP citrate lyase; this enzyme lacks phosphotyrosine but contains phosphohistidine, which is similar structurally to phosphotyrosine. By attaching the antibody at high density to Sepharose beads and omitting ionic detergents from the buffers, it was possible by microbatch immunoadsorption (followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) to isolate the 120,000-dalton transforming protein and several other phosphotyrosyl proteins from cells transformed by Abelson murine leukemia virus. Under the same conditions, phosphotyrosyl proteins were also isolated from human epidermal carcinoma cells (A431) that had been stimulated with epidermal growth factor; most prominent among these proteins was the 170,000-dalton receptor for epidermal growth factor.     

Phosphotyrosine-containing proteins in bovine chromaffin cells: Effects of insulin-like growth factor I (IGF-I)

1. Antiphosphotyrosine antibodies were used to detect phosphotyrosine-containing proteins in immunoblots of bovine chromaffin cell proteins. 2. Unstimulated cells exhibited two major phosphotyrosine-containing proteins, which had Mr's of 121,000 and 70,000. Insulin-like growth factor I (IGF-I) had little effect on the phosphotyrosine content of these two proteins but greatly increased the phosphotyrosine content of three other proteins of Mr 185,000, 170,000, and 96,000. These proteins were found predominantly in the particulate fraction of cell homogenates. 3. The effects of the IGF-I were time and concentration dependent, with maximal increases in phosphorylation occurring after 1 min of treatment with 10 nM IGF-I. Na3VO4, an inhibitor of phosphotyrosine phosphatases, potentiated the effects of IGF-I. 4. Thus, the IGF-I receptor appears to function as an IGF-I-activated protein tyrosine kinase in chromaffin cells. The tyrosine kinase activity of the IGF-I receptor presumably mediates the effects of IGF-I on chromaffin cell function.     

Preparation of monoclonal antibodies to phosphotyrosine and their use for identification of phosphotyrosine-containing proteins

Protein kinases phosphorylating proteins at tyrosine residues play an essential role in the cell growth regulation and neoplastic transformation. However, the functions of the majority of tyrosine protein kinases are still obscure, thus creating hindrances in the identification and isolation of phosphotyrosine-containing proteins. The use of the phosphotyrosine structural analog, aminobenzyl phosphonate, as a hapten group enabled the preparation of monoclonal antibodies capable of reacting to phosphotyrosine. The phosphotyrosine specificity of six clones of monoclonal antibodies was tested by a competitive solid phase immunoenzymatic assay. Using fluorescence quenching, the values of constants of binding for antibodies of four clones to phosphotyrosine (2.5-4.0 x 10(6) M-1) were determined. Using two independent methods, it was shown that clone B4 antibodies reveal the highest specificity towards phosphotyrosine. An immunoadsorbent based on clone B4 antibodies was obtained; this immunoadsorbent possessed an ability to selectively interact with an EFR receptor phosphorylated at tyrosine residue. Using eluate acid hydrolysis from the immunoadsorbent, it was demonstrated that clone B4 antibodies interact only with the phosphotyrosine-containing proteins. The experimental results are suggestive of clone B4 monoclonal antibody specificity to phosphotyrosine and of the feasibility of their application for the isolation and identification of tyrosine protein kinases and their substrates.     

Inhibition of phosphotyrosine phosphatases reveals candidate substrates of the PDGF receptor kinase

In normal fibroblasts stimulated by platelet derived growth factor (PDGF), PDGF receptors are transiently phosphorylated on tyrosine and represent the major phosphotyrosine containing protein. The phosphate of the phosphotyrosine groups turns over rapidly, and extensive evidence indicates a dynamic balance between phosphorylation and dephosphorylation reactions. Thus, the effect of an inhibitor of phosphatases, orthovanadate, on the pattern of the tyrosine phosphorylations induced by PDGF in Swiss 3T3 fibroblasts was investigated. Western blot analysis with antibodies against phosphotyrosine indicated that whereas in unstimulated cells no phosphotyrosine containing proteins were detected, treatment of cells with orthovanadate alone elicited the slow phosphorylation of several proteins including a 170 kDa component that was recognized to be the phosphorylated PDGF receptor. Addition of PDGF to cells shortly pretreated with vanadate highly increased the intensity of the 170 kDa band corresponding to the phosphorylated receptor and caused its stabilization during time. In addition, the phosphorylation on tyrosine of other proteins (molecular mass 116, 80, 73, 60, 50 and 39 kDa) was also induced. Both the receptor and the other tyrosine phosphorylated proteins appeared to be associated with the detergent insoluble matrix.     

Phenylarsine oxide stimulates hexose transport in 3T3-L1 adipocytes by a mechanism other than an increase in surface transporters

Phenylarsine oxide (PAO) has been shown to exert a biphasic effect on glucose transport in 3T3-L1 adipocytes. At 10 microM, PAO activates transport threefold, but at higher concentrations an inhibition of transport is observed. In this paper we report a procedure for the subcellular fractionation of these cells which we use to examine the distribution of glucose transporters following PAO challenge. Quantitative immunoblotting showed that the glucose transporter content of the plasma membrane fraction increased with increasing PAO concentrations; a parallel increase in another insulin-responsive protein, the transferrin receptor, also occurred. However, cell-surface labeling procedures for the glucose transporter and transferrin receptor showed that PAO actually decreased the cell-surface concentrations of these proteins; the basis of this discrepancy may be that in the presence of PAO, intracellular vesicles containing these proteins associate with the plasma membrane, but do not fuse with it. The possibility that PAO modulated transport by direct interaction with the glucose transporter was investigated by examining the effects of PAO on transport in both erythrocytes and a reconstituted system of purified erythrocyte transporter in lipid vesicles. PAO was without effect on the rate of transport in these systems. The hypothesis that the stimulatory effect of PAO on transport might be due to the activation of the insulin receptor kinase activity was examined by assessing the phosphotyrosine content of the receptor and other proteins using anti-phosphotyrosine antibodies. PAO alone caused no detectable increase in receptor phosphotyrosine content. However, the combination of PAO and insulin led to the tyrosine phosphorylation of two proteins of Mr 68,000 and 57,000 which were not detected in cells treated with either PAO or insulin, and an increased phosphotyrosine content of proteins of Mr 95,000 and 165,000 when compared to cells treated with insulin alone.     

In vivo phosphorylation and dephosphorylation of the platelet-derived growth factor receptor studied by immunoblot analysis with phosphotyrosine antibodies

Antibodies against the synthetic hapten azobenzyl phosphonate which specifically crossreact with phosphotyrosine have been produced and used to detect the proteins phosphorylated in tyrosine following exposure of intact quiescent Swiss 3T3 fibroblasts to the platelet-derived growth factor (PDGF). Western blotting of sodium dodecyl sulfate-polyacrylamide gel electrophoresis-fractionated proteins followed by decoration with phosphotyrosine antibodies and 125I-labeled protein A have been used. The major tyrosine-phosphorylated component was a 170 kDa protein. The following lines of evidence suggest that this protein is the PDGF receptor in its tyrosine-phosphorylated form: (a) both proteins have the same (170 kDa) molecular weight; (b) the phosphorylated 170 kDa protein was detectable only in cell lines bearing the PDGF receptor; (c) the phosphorylation of the 170 kDa protein required PDGF and was dose-dependent. Kinetic studies showed that the phosphorylation of the receptor was maximal after 5-10 min at 37 degrees C and was followed by a rapid decrement of the band. The loss of the 170 kDa component was not prevented by inhibitors of membrane internalization and of lysosomal proteinases, while it was inhibited by lowering the temperature to 5 degrees C. In PDGF-stimulated cells, phosphotyrosine antibodies detected also a minor 36 kDa component phosphorylated at tyrosine.     

Tyrosine phosphorylation in human neutrophil

Protein tyrosine phosphorylation in human neutrophils was examined by immunoblotting with antibodies specific for phosphotyrosine. The addition of the human hormone granulocyte-macrophage colony stimulating factor to human neutrophils caused an increase in the tyrosine phosphorylation levels of several proteins. The increases in at least two of these proteins having molecular masses of 40 kDa (p40) and 54 kDa (p54) were rapid and were inhibited in pertussis toxin treated cells. The newly synthesized tyrosine kinase inhibitor ST 638 inhibited the increases in the levels of the tyrosine phosphorylation in p92, p78, p54 and p40 proteins. The epidermal growth factor receptor tyrosine kinase inhibitors were less effective. The addition of the chemotactic factor fMet-Leu-Phe to human neutrophils also caused an increase in tyrosine phosphorylation in some of these proteins. The pattern of the fMet-Leu-Phe-induced tyrosine phosphorylation was different from that produced by GM-CSF. The increases were also inhibited by ST 638. In addition, ST 638 inhibited superoxide production but not actin polymerization in control and GM-CSF-treated cells stimulated with fMet-Leu-Phe. Moreover, the active but not inactive phorbol esters increase the tyrosine phosphorylation only in the 40 kDa protein. These results suggest several points: (a) some of the responses produced by GM-CSF and fMet-Leu-Phe are mediated through tyrosine phosphorylation, (b) the GM-CSF receptor is coupled to a pertussis toxin sensitive G-protein, (c) the 40 kDa protein is probably the Gi alpha 2, and (d) the 78 or the 92 kDa protein is most likely the receptor for GM-CSF, which indicates that the receptor may have a tyrosine kinase domain.     

Ratiometric assay of epidermal growth factor receptor tyrosine kinase activation

Activation of cells is frequently followed by tyrosine phosphorylation of proteins. To quantify this process, we developed a ratiometric enzyme-linked immunosorbent assay (ELISA) using epidermal growth factor receptors (EGFR) as a model. Microtiter dishes were coated with anti-EGFR monoclonal antibodies to capture the receptor followed by parallel detection of receptor and phosphotyrosine content with secondary antibodies. The ratio of these two parameters was found to directly reflect EGFR activation and was insensitive to the effect of receptor downregulation. Our assay could resolve differences in EGFR activation due to small changes (less than 1 ng/ml) in ligand. We found that phosphotyrosine detection by ELISA was 8- to 32-fold more sensitive than Western blot detection and could be reliably detected using as little as 4 ng of cellular lysate. Detection of EGFR levels by ELISA was 30 times more sensitive than Western blot analysis and was reliable for as low as 8 ng of cellular lysate per well. Because of the wide linear range of the ELISA, we could directly compare receptor activation in cell types with different EGFR expression levels. Our assay provides a rapid and sensitive method of determining EGFR activation status and could be easily modified to evaluate any tyrosine-phosphorylated protein.     

Comparative study of the tyrosine phosphorylation of proteins in Swiss 3T3 fibroblasts stimulated by a variety of mitogenic agents

We have carried out a comparative study of the protein tyrosine phosphorylation induced by a wide range of mitogenic stimuli on a single cell type, Swiss 3T3 mouse fibroblasts. For this purpose we have used high-affinity antibodies directed to phosphotyrosine residues on proteins (Wang: Mol. Cell. Biol. 5:3640-3643, 1985) in immunoblotting and immunofluorescence microscopy experiments. Immunoblotting experiments showed that all of the mitogens tested, including epidermal growth factor, platelet-derived growth factor, basic fibroblast growth factor, insulin, fetal calf serum, trypsin, and 12-O-tetradecanoylphorbol-13-acetate, increased the phosphorylation on tyrosine of a number of proteins. Most of the increase in tyrosine phosphorylation induced by each factor involved a small set of proteins with apparent molecular weights (Mr) above 50,000. Following stimulation with epidermal growth factor, platelet-derived growth factor, and basic fibroblast growth factor, increased phosphotyrosine modification of proteins with molecular weights corresponding to those of the respective receptors was observed. A protein band of apparent Mr 160,000 contained substantially increased levels of phosphotyrosine following insulin treatment, but tyrosine phosphorylation of the insulin receptor was apparently below the level of detectability. The phosphotyrosine content of proteins with apparent Mr of 220,000, 120,000, and 70,000 was increased by all the agents tested. Phosphorylation on tyrosine of most of the proteins increased within a few minutes of the mitogenic stimulation, reached a peak, and returned more slowly to basal levels. Immunofluorescence labeling with the antibodies specific for phosphotyrosine showed a substantial increase in the amount of phosphotyrosine containing proteins only in the presence of platelet-derived growth factor and fetal calf serum. This finding suggests that most of the proteins phosphorylated on tyrosine in Swiss 3T3 fibroblasts are not concentrated in specific subcellular structures, but rather are diffusely distributed throughout the cell and are therefore not detectable by immunofluorescence microscopy.     

Isolation of antibodies for phosphotyrosine by immunization with a v-abl oncogene-encoded protein.

Immunization of rabbits with a tyrosine-phosphorylated v-abl protein resulted in the production of antibodies for the v-abl protein and for phosphotyrosine. The antiphosphotyrosine antibodies could be purified by affinity chromatography with O-phosphotyramine coupled to Sepharose. These antibodies detected a variety of tyrosine-phosphorylated proteins, including receptors for peptide growth factors. The usefulness of these antibodies was demonstrated by the detection of previously unidentified tyrosine-phosphorylated proteins in v-src-, v-abl-, and v-erbB-transformed cell lines.     

Ligand-induced tyrosine kinase activity of the colony-stimulating factor 1 receptor in a murine macrophage cell line.

Metabolic labeling of simian virus 40-immortalized murine macrophages with 32Pi and immunoblotting with antibodies to phosphotyrosine demonstrated that the c-fms proto-oncogene product (colony-stimulating factor 1 [CSF-1] receptor) was phosphorylated on tyrosine in vivo and rapidly degraded in response to CSF-1. Stimulation of the CSF-1 receptor also induced immediate phosphorylation of several other cellular proteins on tyrosine. By contrast, the mature cell surface glycoprotein encoded by the v-fms oncogene was phosphorylated on tyrosine in the absence of CSF-1, suggesting that it functions as a ligand-independent kinase.     

Effect of brassinolide on tyrosine phosphorylation of pea leaf proteins

Brassinosteroid-induced phosphorylation of tyrosine residues in proteins was studied. Proteins of crude extract of pea leaves were analyzed by one- and two-dimensional electrophoresis followed by Western blotting with monoclonal antibodies PY20 to phosphotyrosine proteins. One- and two-dimensional electrophoresis revealed 7 and 13 tyrosine-phosphorylated proteins, respectively. Brassinolide increased the phosphorylation level of most of these proteins. With inhibitors of tyrosine protein phosphatases, such as phenylarsine oxide and orthovanadate, the level of tyrosine phosphorylation of these proteins increased.     

Changes associated with tyrosine phosphorylation during short-term hypoxia in retinal microvascular endothelial cells in vitro

The occlusion of capillary vessels results in low oxygen tension in adjacent tissues which triggers a signaling cascade that culminates in neovascularization. Using bovine retinal capillary endothelial cells (BRCEC), we investigated the effects of short-term hypoxia on DNA synthesis, phosphotyrosine induction, changes in the expression of basic fibroblast growth factor receptor (bFGFR), protein kinase C (PKCα), heat shock protein 70 (HSP70), and SH2-containing protein (SHC). The effect of protein tyrosine kinase (PTK) and phosphatase inhibitors on hypoxia-induced phosphotyrosine was also studied. Capillary endothelial cells cultured in standard normoxic (pO₂ = 20%) conditions were quiesced in low serum containing medium and then exposed to low oxygen tension or hypoxia (pO₂ = 3%) in humidified, 5% CO₂, 37°C, tissue culture chambers, on a time-course of up to 24 h. DNA synthesis was potentiated by hypoxia in a time-dependent manner. This response positively correlated with the cumulative induction of phosphotyrosine and the downregulation of bFGFR (M_r ～ 85 kDa). Protein tyrosine kinase inhibitors, herbimycin-A, and methyl 2,5-dihydroxycinnamate, unlike genistein, markedly blocked hypoxia-induced phosphotyrosine. Prolonged exposure of cells to phosphatase inhibitor, sodium orthovanadate, also blocked hypoxia-induced phosphotyrosine. The expression of HSP70, PKCα, and SHC were not markedly altered by hypoxia. Taken together, these data suggest that short-term hypoxia activates endothelial cell proliferation in part via tyrosine phosphorylation of cellular proteins and changes in the expression of the FGF receptor. Thus, endothelial cell mitogenesis and neovascularization associated with low oxygen tension may be controlled by abrogating signaling pathways mediated by protein tyrosine kinase and phosphatases. © 1995 Wiley-Liss, Inc.     

Antiphosphotyrosine antibodies modulate insulin receptor kinase activity and insulin action

Insulin elicits the autophosphorylation of the beta-subunit of its receptor on tyrosine residues: this effect appears to be the earliest post-binding event involved in insulin action. In the present study we have raised highly specific antibodies to phosphotyrosine residues, and we have taken advantage of these antibodies to further evaluate the role of the insulin receptor tyrosine kinase in the generation of insulin's biological responses. Using a cell-free phosphorylation assay, we show here that these antibodies increase the tyrosine kinase activity of the receptor, and its phosphorylation on tyrosine residues. In contrast, the antibodies do not interfere with dephosphorylation of the insulin receptor. Introduction of the same antibodies in living Fao hepatoma cells enhances the effect of insulin on both glucose transport and aminoacid uptake. As a whole our data indicate that the insulin receptor kinase is involved in the generation of an early (glucose transport) and late (aminoacid uptake) response to insulin. Further, conformational changes in phosphotyrosine containing domains of the insulin receptor appear to modulate insulin's biological effects. Finally, the injection of antibodies in intact cells provides us with a novel and promising tool to search for cellular substrates for the insulin receptor tyrosine kinase.     

Novel tyrosine kinase substrates from Rous sarcoma virus-transformed cells are present in the membrane skeleton

We have previously reported the production of monoclonal antibodies directed against phosphotyrosine, which is the modification induced by many oncogene products and growth factor receptors. One of these antiphosphotyrosine antibodies (py20) was used in affinity chromatography to isolate phosphotyrosine (PY)-containing proteins from Rous sarcoma virus-transformed chick embryo fibroblasts (RSV-CEFs). Mice were immunized with these PY-proteins for the production of monoclonal antibodies to individual substrates. Fifteen antibodies were generated in this way to antigens with molecular masses of 215, 76, 60, and 22 kD. Antibodies to individual substrates were used to analyze the subcellular location in normal and RSV-CEFs. Antibodies to the 215- and 76-kD antigen stained focal contacts when used in immunofluorescence microscopy while anti-22-kD protein antibodies resulted in punctate staining concentrated in the margins of cells and in parallel arrays. Both distributions were altered in transformed cells. When cells were extracted with nonionic detergent under conditions that stabilize the cytoskeleton, 50% of the 76-kD protein and greater than 90% of the 22-kD protein fractionated with the cytoskeleton. This study offers a new approach to both the identification of membrane skeletal proteins in fibroblasts and changes that occur upon transformation by an activated tyrosine kinase.     

Proteomic analysis reveals novel molecules involved in insulin signaling pathway

The binding of insulin to its receptor triggers a signaling cascade regulated by protein complexes via tyrosine phosphorylation events on a multitude of associated proteins. To search novel phosphotyrosine proteins or associated proteins involved in insulin signaling pathway, we employed a method in which Rat1 cells stably expressing the human insulin receptor were stimulated with or without insulin and sub-fractionated prior to enrichment of phosphotyrosine proteins by immunoprecipitation and analysis by LC-MS/MS. Bioinformatic analysis and manual confirmation of peptide phosphorylation site assignments led to identification of 35 phosphotyrosine sites derived from 31 protein groups. Over 50% of these proteins were reported for the first time as tyrosine phosphorylated, including gigaxonin, XIAP and CDK10. In addition, we also found that calcium/calmodulin-dependent protein serine kinase (CASK), a key protein in protein-targeting and vesicle transport in neurons, forms a complex with two unidentified phosphotyrosine proteins pp100 and pp95 in response to insulin-stimulation, though CASK is not itself tyrosine phosphorylated. Furthermore, insulin was able to decrease CASK nuclear location, as well as down-regulate the expression of CASK targeted genes. Our results imply CASK as a novel joint knot connecting CASK-mediated pathways with the insulin signaling. Our data provide a wealth of information potentially paving the way to identify new components in the insulin signaling network.     

Phosphotyrosine antibodies identify the p210c-abl tyrosine kinase and proteins phosphorylated on tyrosine in human chronic myelogenous leukemia cells.

Antibodies against phosphotyrosine are a powerful tool with which to identify proteins phosphorylated on tyrosine residues, such as viral oncogene-encoded transforming proteins and their cellular protein substrates. Probed on human leukemia cell lines, phosphotyrosine antibodies recognized a 210,000-molecular-weight protein (p210) in K562 cells, a cell line derived from a Philadelphia (Ph)'-positive chronic myelogenous leukemia (CML), but recognized no protein in control Ph'-negative non-CML leukemia cells. The p210 protein was also recognized by antisera against v-abl-encoded polypeptides and displayed kinase activity, phosphorylating itself on tyrosine, in an immunocomplex kinase assay. These data are consistent with reported findings of the expression of a recombined bcr-abl gene in Ph'-positive CML cells, leading to the synthesis of an altered p210c-abl protein endowed with tyrosine kinase activity. Phosphotyrosine antibodies also detected the expression of the p210c-abl protein in fresh bone marrow cells harvested from CML patients in blast crisis. Besides the p210c-abl protein kinase, phosphotyrosine antibodies recognized other proteins with molecular weights of 110,000, 68,000, and 36,000 (p110, p68, and p36) in K562 cells. When [gamma-32P]ATP was added to nonionic detergent-extracted cells, these proteins became phosphorylated on tyrosine, as confirmed by phosphoamino acid analysis. A comparison with fibroblasts transformed by the v-abl, v-src, and v-fps oncogenes suggested the identity of the p36 protein with the common 36-kilodalton protein substrate of viral oncogene-encoded tyrosine kinases. Enhanced tyrosine phosphorylation of cellular proteins is thus a feature shared by cells transformed by v-abl and cells expressing a rearranged bcr-abl gene.     

Immunodetection of biotinylated lymphocyte-surface proteins by enhanced chemiluminescence: a nonradioactive method for cell-surface protein analysis.

Biotinylation and radioiodination have been compared for labeling lymphocyte-surface proteins and the labeled proteins symmetrically immunoprecipitated with antibodies recognizing major lymphocyte markers such as the murine Thy-1, CD25 (the alpha subunit of the interleukin-2 receptor), CD45, and human CD2 glycoproteins. The detection of biotinylated proteins by enhanced chemiluminescence after transfer to nitrocellulose was found to be fast and as efficient as the detection of iodinated proteins by autoradiography. The vectoriality of cell-surface biotinylation was ascertained by two-dimensional electrophoresis of the cellular extract in which the major cytoplasmic proteins were not found biotinylated. This nonradioactive labeling procedure offers a convenient and efficient alternative to radiolabeling of cell surfaces for the biochemical analysis of extracellular domains of membrane proteins.     

Paxillin is a major phosphotyrosine-containing protein during embryonic development

Phosphotyrosine-containing proteins were immunoprecipitated from embryonic chicken tissue extracts using anti-phosphotyrosine antibody coupled to agarose beads. Major phosphotyrosine-containing proteins of 110, 70, and 50 kD were observed following blotting with anti-phosphotyrosine antibody. The 70-kD band was selectively removed from the samples by precipitation with antibodies to the focal adhesion protein paxillin, therefore identifying paxillin as one of the major tyrosine kinase substrates during chick embryonic organogenesis. The tyrosine phosphorylation of paxillin is regulated developmentally: during embryogenesis, a marked decrease in its phosphotyrosine content was observed, although the total level of paxillin remained essentially constant. Approximately 20% of the paxillin was phosphorylated on tyrosine in the early embryo. In contrast, tyrosine phosphorylation of paxillin was undetectable in the adult. A similar profile of phosphotyrosine-containing proteins was identified in rat embryos. Paxillin was also found to be a major phosphotyrosine-containing protein in the rat embryo. These data suggest that the regulated phosphorylation of tyrosine residues on paxillin may perform a critical role in controlling cell and tissue cytoarchitecture rearrangement during vertebrate development.     

Epidermal growth factor receptor metabolism and protein kinase activity in human A431 cells infected with Snyder-Theilen feline sarcoma virus or harvey or Kirsten murine sarcoma virus.

When human A431 cells, which carry high numbers of epidermal growth factor (EGF) receptors, are exposed to EGF, the total content of phosphotyrosine in cell protein is increased, the EGF receptor becomes phosphorylated at tyrosine, and new phosphotyrosine-containing 36,000- and 81,000-dalton proteins are detected. We examined the properties of A431 cells infected with Snyder-Theilen feline sarcoma virus, whose transforming protein has associated tyrosine protein kinase activity, and Harvey and Kirsten sarcoma viruses, whose transforming proteins do not. In all cases, the infected cells were more rounded and more capable of anchorage-independent growth than the uninfected cells. EGF receptors were assayed functionally by measuring EGF binding and structurally by metabolic labeling and immunoprecipitation. In no case did infection appear to alter the rate of EGF receptor synthesis, but infection reduced EGF receptor stability by about 50% for cloned Harvey sarcoma virus-infected cells and by 80% for cloned feline sarcoma virus-infected cells. The corresponding reductions in EGF binding were 70 and 90%, respectively. The proteins of feline sarcoma virus-infected A431 cells contained an increased amount of phosphotyrosine, and the 36,000- and 81,000-dalton phosphoproteins were detected. The EGF receptor was not detectably phosphorylated at tyrosine, however, unless the cells were exposed to EGF. The Harvey and Kirsten sarcoma virus-infected cells did not exhibit elevated levels of phosphotyrosine either in the total cell proteins or in the EGF receptor, nor were the 36,000- and 81,000-dalton proteins detectable. However, these phosphoproteins were found in the infected cells after EGF treatment. Thus, all of the infected A431 cells exhibited reduced EGF binding and increased degradation of EGF receptors, yet their patterns of protein phosphorylation were distinct from those of EGF-treated A431 cells.