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.     

Evidence for the platelet-derived growth factor-stimulated tyrosine phosphorylation of the platelet-derived growth factor receptor in vivo. Immunopurification using a monoclonal antibody to phosphotyrosine

The addition of platelet-derived growth factor (PDGF) to intact BALB/c 3T3 cells results in the rapid (less than 1 min), dose-dependent phosphorylation of a number of proteins that could be isolated by a monoclonal antiphosphotyrosine antibody. The predominant tyrosinephosphorylated protein shared many characteristics with the PDGF receptor, including its molecular weight (170,000), isoelectric point (pI of about 4.2), its binding to DEAE-cellulose, and its pattern of binding to lectins. This 170-kDa protein, labeled with [35S] methionine, was substantially purified from PDGF-stimulated cells using the monoclonal anti-phosphotyrosine antibody but was not significantly immunopurified from unstimulated cells. At 37 degrees C, phosphorylation of the 170-kDa protein was maximal by 5-10 min of exposure to PDGF, and thereafter decreased rapidly. However, at 4 degrees C, the phosphorylation continued to increase after 3 h of exposure to PDGF. Subsequently, shifting the cells from 4 to 37 degrees C resulted in an additional rapid burst of tyrosine phosphorylation. Among the other PDGF-stimulated molecules, the most prominent and consistently observed was a cytosolic, acidic (pI of about 4.2), 74-kDa protein. These findings indicate that the action of PDGF in vivo is associated with the rapid and transient tyrosine phosphorylation of several membrane and cytosolic proteins; the most prominent of these proteins, isolated by monoclonal antibody to phosphotyrosine, is likely to be the PDGF receptor. The use of this antibody provides a new approach for purification of the PDGF receptor.     

Early phosphorylation events following the treatment of Swiss 3T3 cells with bombesin and the mammalian bombesin-related peptide, gastrin-releasing peptide.

Bombesin and the related mammalian peptides, such as gastrin-releasing peptide (GRP), are potent mitogens for some fibroblast cell lines. Here we have examined the bombesin- and GRP-mediated changes in the phosphorylation of proteins in Swiss 3T3 cells and compared these to the events observed after platelet-derived growth factor (PDGF), epidermal growth factor (EGF) and tumor promoter treatment. In agreement with previous reports, bombesin, GRP and PDGF, but not EGF, increased the activity of protein kinase C. This was assayed by an inhibition of [125I]EGF binding, stimulation in phosphorylation of pp60c-src on serine 12 and stimulation in phosphorylation of a group of 80 kd proteins. The different phosphorylated forms of the 80 kd proteins were examined by tryptic peptide mapping and shown to contain multiple phosphorylation sites. An investigation of the tyrosine phosphorylation events following mitogen treatment revealed a significant difference between PDGF and the bombesin peptides. PDGF treatment caused a marked increase in total cellular phosphotyrosine levels, and tyrosine phosphorylation both of known substrates and its own receptor. In contrast, bombesin and GRP treatments resulted in only a weak or undetectable increase in tyrosine phosphorylation of total cellular protein or known substrates. In this respect bombesin and GRP were more similar to EGF. The fact that the bombesin peptides do not induce a phosphorylation response identical with either PDGF or EGF suggests that there is not a single common signal pathway which is activated by all these mitogens.     

Platelet-derived growth factor induces rapid and sustained tyrosine phosphorylation of phospholipase C-gamma in quiescent BALB/c 3T3 cells.

Platelet-derived growth factor (PDGF) stimulates the proliferation of quiescent fibroblasts through a series of events initiated by activation of tyrosine kinase activity of the PDGF receptor at the cell surface. Physiologically significant substrates for this or other growth factor receptor or oncogene tyrosine kinases have been difficult to identify. Phospholipase C (PLC), a key enzyme of the phosphoinositide pathway, is believed to be an important site for hormonal regulation of the hydrolysis of phosphatidylinositol 4,5-bisphosphate, which produces the intracellular second-messenger molecules inositol 1,4,5-trisphosphate and 1,2-diacylglycerol. Treatment of BALB/c 3T3 cells with PDGF led to a rapid (within 1 min) and significant (greater than 50-fold) increase in PLC activity, as detected in eluates of proteins from a phosphotyrosine immunoaffinity matrix. This PDGF-stimulated increase in phosphotyrosine-immunopurified PLC activity occurred for up to 12 h after addition of growth factor to quiescent cells. Interestingly, the PDGF stimulation occurred at 3 as well as 37 degrees C and in the absence or presence of extracellular Ca2+. Immunoprecipitation of cellular proteins with monoclonal antibodies specific for three distinct cytosolic PLC isozymes demonstrated the presence of a 145-kilodalton isozyme, PLC-gamma (formerly PLC-II), in BALB/c 3T3 cells. Furthermore, these immunoprecipitation studies showed that PLC-gamma is rapidly phosphorylated on tyrosine residues after PDGF stimulation. The results suggest that mitogenic signaling by PDGF is coincident with tyrosine phosphorylation of PLC-gamma.     

Phospholipase C-gamma, a substrate for PDGF receptor kinase, is not phosphorylated on tyrosine during the mitogenic response to CSF-1.

Quiescent mouse NIH3T3 cells expressing a transduced human c-fms gene encoding the receptor for colony stimulating factor-1 (CSF-1) were stimulated with mitogenic concentrations of platelet-derived growth factor (PDGF) or CSF-1. Immunoprecipitated phospholipase C-gamma (PLC-gamma) was phosphorylated on tyrosine and calcium was mobilized following treatment of intact cells with PDGF. In contrast, only trace amounts of phosphotyrosine were incorporated into PLC-gamma and no intracellular calcium signal was detected after CSF-1 stimulation. Similarly, CSF-1 treatment did not stimulate phosphorylation of PLC-gamma on tyrosine in a CSF-1-dependent. SV40-immortalized mouse macrophage cell line that expresses high levels of the CSF-1 receptor. In fibroblasts, antiserum to PLC-gamma co-precipitated a fraction of the tyrosine phosphorylated form of the PDGF receptor (PDGF-R) after ligand stimulation, implying that phosphorylated PDGF-R and PLC-gamma were associated in a stable complex. Pre-treatment of cells with orthovanadate also led to tyrosine phosphorylation of PLC-gamma which was significantly enhanced by PDGF, but not by CSF-1. Thus, although the PDGF and CSF-1 receptors are structurally related and appear to be derived from a single ancestor gene, only PDGF-induced mitogenesis in fibroblasts correlated with tyrosine phosphorylation of PLC-gamma.     

Heterologous expression of the cytochrome P450cam hydroxylase operon and the repressor gene of Pseudomonas putida in Escherichia coli

Abstract Cytoplasmic proteins from the antarctic psychrotrophic bacterium Pseudomonas syringae showed two phosphorylated proteins of molecular mass 66 kDa and 62 kDa. The phosphorylation of 66 kDa protein was enhanced in the presence of Triton X-100 solubilised membrane proteins at a higher temperature (30°C) only. Western blot analysis and phosphoamino acid analysis indicated that the 66 kDa protein is phosphorylated at a tyrosine residue. Surprisingly, sodium orthovanadate, which is a known phosphotyrosine phosphatase (PTPase) inhibitor, inhibited the phosphorylation of the protein. The possible importance of this tyrosine phosphorylated protein to growth at low temperature is suggested.     

Characterization of a high-molecular-weight protein immunoprecipitated by platelet-derived growth factor antisera

We previously demonstrated that a high-molecular-weight glycoprotein could be immunoprecipitated from metabolically labeled U-2 OS cells with platelet-derived growth factor (PDGF) antiserum and that it appears to be derived from a different precursor than is the 30 kD PDGF-like mitogen produced by these cells. These findings were unexpected, since the molecular weight of this glycoprotein is too large to be encoded by the PDGF structural genes. From experiments with metabolically labeled U-2 OS human osteosarcoma, fibroblasts, and NRK cells, we report here that a 185 kD protein immunoprecipitated with PDGF antiserum has the following characteristics. 1) It is a PDGF binding protein that is unrelated to alpha 2-macroglobulin. 2) It is phosphorylated in response to PDGF stimulation. 3) It is immunoprecipitated by phosphotyrosine antibodies. 4) It is not a substrate of epidermal growth factor-induced tyrosine kinase activity. These studies indicate that high-molecular-weight proteins immunoprecipitated by antiserum to PDGF represent a complex between PDGF and a binding protein capable of being phosphorylated by a PDGF-induced tyrosine kinase. These characteristics are identical to those of the PDGF receptor.     

Tyrosine phosphorylation of the asialoglycoprotein receptor

The asialoglycoprotein (ASGP) receptor undergoes constitutive endocytosis through the coated pit/coated vesicle pathway in hepatocytes. Studies on HepG2 cells have shown that the receptor is phosphorylated at serine under control conditions and following protein kinase C stimulation. This study examined whether the ASGP receptor could also serve as a substrate for a tyrosine kinase in HepG2 cells. 32P labeling was performed in membrane preparations, in permeabilized cells at 4 degrees C, and in intact cells at 37 degrees C. The phosphorylated ASGP receptor was isolated by immunoprecipitation, hydrolyzed in 6 N HCl at 110 degrees C, and analyzed by two-dimensional high voltage electrophoresis. The receptor isolated from a membrane preparation incubated in vitro with [gamma-32P]ATP incorporated radiolabel predominantly (greater than 90%) into phosphotyrosine. ASGP receptor phosphorylation at both tyrosine and serine was detected in intact cells incubated with phosphatase inhibitors for 60 min at 37 degrees C. The presence of both phenylarsine oxide (20 microM) and sodium orthovanadate (200 microM) was required for tyrosine phosphorylation. Use of these inhibitors together resulted in a 16.4-fold increase in phosphorylation of the immunoprecipitated ASGP receptor, whereas phosphorylation of total HepG2 membrane proteins was not significantly augmented by this procedure. Selective proteolytic digestion of ASGP receptors in isolated vesicles demonstrated that the phosphorylation site identified in these studies is located at tyrosine 5 in the cytoplasmic tail.     

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.     

Temperature-dependent tyrosine phosphorylation of microtubule-associated protein kinase in epidermal growth factor-stimulated human fibroblasts.

Treatment of normal human fibroblasts with epidermal growth factor (EGF) results in the rapid (0.5 min) and simultaneous tyrosine phosphorylation of the EGF receptor (EGFr) and several other proteins. An exception to this tyrosine phosphorylation wave was a protein (42 kDa) that became phosphorylated on tyrosine only after a short lag time (5 min). We identified this p42 kDa substrate as the microtubule-associated protein (MAP) kinase using a monoclonal antibody to a peptide corresponding to the C-terminus of the predicted protein (Science 249, 64-67, 1990). EGF treatment of human fibroblasts at 37 degrees C for 5 min resulted in the tyrosine phosphorylation of 60-70% of MAP kinase as determined by the percent that was immunoprecipitated with antiphosphotyrosine antibodies. Like other tyrosine kinase growth factor receptors, the EGFr is activated and phosphorylated at 4 degrees C but is not internalized. Whereas most other substrates were readily tyrosine phosphorylated at 4 degrees C, MAP kinase was not. When cells were first stimulated with EGF at 4 degrees C and then warmed to 37 degrees C without EGF, tyrosine phosphorylation of MAP kinase was again observed. Treatment of cells with the protein kinase C activator phorbol myristate acetate (PMA) also resulted in the tyrosine phosphorylation of MAP kinase, and again only at 37 degrees C. Tryptic phosphopeptide maps demonstrated that EGF and PMA both induced the phosphorylation of the same peptide on tyrosine and threonine. This temperature and PMA sensitivity distinguishes MAP kinase from most other tyrosine kinase substrates in activated human fibroblasts.     

Kinetics and regulation of the tyrosine phosphorylation of epidermal growth factor receptor in intact A431 cells.

We have previously reported that antibodies to phosphotyrosine recognize the phosphorylated forms of platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) receptors (Zippel et al., Biochim. Biophys. Acta 881:54-61, 1986, and Sturani et al., Biochem. Biophys. Res. Commun. 137:343-350, 1986). In this report, the time course of receptor phosphorylation is investigated. In normal human fibroblasts, ligand-induced phosphorylation of PDGF and EGF receptors is followed by rapid dephosphorylation. However, in A431 cells the tyrosine-phosphorylated form of EGF receptor persists for many hours after EGF stimulation, allowing a detailed analysis of the conditions affecting receptor phosphorylation and dephosphorylation. In A431 cells, the number of receptor molecules phosphorylated on tyrosine was quantitated and found to be about 10% of total EGF receptors. The phosphorylated receptor molecules are localized on the cell surface, and they are rapidly dephosphorylated upon removal of EGF from binding sites by a short acid wash of intact cells and upon a mild treatment with trypsin. ATP depletion also results in rapid dephosphorylation, indicating that continuous phosphorylation-dephosphorylation reactions occur in the ligand-receptor complex at steady state. Phorbol 12-myristate 13-acetate added shortly before EGF reduces the rate and the final extent of receptor phosphorylation. Moreover, it also reduces the amount of phosphorylated receptors if it is added after EGF. Down-regulation of protein kinase C by chronic treatment with phorbol dibutyrate increases the receptor phosphorylation induced by EGF, suggesting a homologous feedback regulation of EGF receptor functions.     

Thrombin and collagen induce rapid phosphorylation of a common set of cellular proteins on tyrosine in human platelets

The ability of thrombin and collagen to induce protein-tyrosine phosphorylation in intact human platelets was assessed by using antibodies to phosphotyrosine in conjugation with immunoblots. Upon stimulation by thrombin there was an increase in the amount of protein-tyrosine phosphorylation of three bands with molecular masses of 135, 124, and 76 kDa in a time-dependent manner. The tyrosine phosphorylation in these three proteins increased in a concurrent fashion and reached a maximum level in 10 s and then a plateau or a slight decrease. Stimulation by collagen was also followed by an increase in tyrosine phosphorylation of 135- and 124-kDa proteins. Unlike stimulation by thrombin, collagen induced no obvious tyrosine phosphorylation of 76-kDa protein. The time courses for thrombin- or collagen-induced protein-tyrosine phosphorylation were similar to that for [14C] serotonin release. These results suggest that 135- and 124-kDa proteins are a common set of proteins that become phosphorylated on tyrosine residue during platelet activation.     

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.     

Distinct protein tyrosine phosphorylation during mitogenesis induced in quiescent sv40-transformed 3T3 T cells by insulin or vanadate

Insulin and vanadate selectively induce mitogenesis in quiescent SV40 large T antigen-transformed 3T3 T cells (CSV3–1) but not in quiescent nontransformed 3T3 T cells. Insulin and vanadate mediate this effect in CSV3–1 cells by distinct signal transduction mechanisms that involve protein tyrosine kinase activity. To further study these processes, changes in protein tyrosine phosphorylation induced by insulin and vanadate were investigated. Using immunoprecipitation and Western blotting techniques with antiphosphotyrosine antibodies, we report distinct protein phosphorylation characteristics in insulin- and vanadate-stimulated CSV3–1 cells. The insulin receptor β-subunit is phosphorylated within 2 min after insulin stimulation of transformed CSV3–1 cells. Insulin also stimulates a rapid increase in tyrosine phosphorylation of the 170 kDa insulin receptor substrate-1 and complex formation between the phosphorylated insulin receptor substrate-1 and the 85 kDa subunit of phosphatidylinositol 3'-kinase. In contrast, vanadate does not initially increase detectable phosphorylation of any proteins, including neither the insulin receptor nor the insulin receptor substrate-1. After 60 min, however, a marked increase in tyrosine phosphorylation of 55 and 64 kDa proteins is observed in vanadate-treated CSV3–1 cells. Furthermore, treatment of CSV3–1 cells with genistein abolishes the effects of vanadate on protein tyrosine phosphorylation but only minimally inhibits the effects of insulin. Finally, insulin stimulates the phosphorytion of a 33 kDa protein, whereas vanadate does not. By comparison, in nontransformed 3T3 T cells, insulin induces a delayed and weaker tyrosine phosphorylation of the insulin receptor β-subunit and vanadate does not enhance the tyrosine phosphorylation of the 55 and 64 kDa proteins. These data together indicate that the mitogenic effects of insulin and vanadate are associated with distinct protein phosphorylation patterns that appear to be differentially regulated in SV40-transformed and nontransformed 3T3 T cells. © 1994 Wiley-Liss, Inc.     

A new function for a phosphotyrosine phosphatase: linking GRB2-Sos to a receptor tyrosine kinase.

Autophosphorylated growth factor receptors provide binding sites for the src homology 2 domains of intracellular signaling molecules. In response to epidermal growth factor (EGF), the activated EGF receptor binds to a complex containing the signaling protein GRB2 and the Ras guanine nucleotide-releasing factor Sos, leading to activation of the Ras signaling pathway. We have investigated whether the platelet-derived growth factor (PDGF) receptor binds GRB2-Sos. In contrast with the EGF receptor, the GRB2 does not bind to the PDGF receptor directly. Instead, PDGF stimulation induces the formation of a complex containing GRB2; 70-, 80-, and 110-kDa tyrosine-phosphorylated proteins; and the PDGF receptor. Moreover, GRB2 binds directly to the 70-kDa protein but not to the PDGF receptor. Using a panel of PDGF beta-receptor mutants with altered tyrosine phosphorylation sites, we identified Tyr-1009 in the PDGF receptor as required for GRB2 binding. Binding is inhibited by a phosphopeptide containing a YXNX motif. The protein tyrosine phosphatase Syp/PTP1D/SHPTP2/PTP2C is approximately 70 kDa, binds to the PDGF receptor via Tyr-1009, and contains several YXNX sequences. We found that the 70-kDa protein that binds to the PDGF receptor and to GRB2 comigrates with Syp and is recognized by anti-Syp antibodies. Furthermore, both GRB2 and Sos coimmunoprecipitate with Syp from lysates of PDGF-stimulated cells, and GRB2 binds directly to tyrosine-phosphorylated Syp in vitro. These results indicate that GRB2 interacts with different growth factor receptors by different mechanisms and the cytoplasmic phosphotyrosine phosphatase Syp acts as an adapter between the PDGF receptor and the GRB2-Sos complex.     

Stimulation of factor-dependent myeloid cell lines with interleukin 3 induces tyrosine phosphorylation of several cellular substrates

Interleukin 3 (IL-3) is required for the proliferation of growth factor-dependent myeloid cell lines. To determine the possible signal transduction mechanisms involved in IL-3 growth regulation, we have examined the effects of IL-3 on tyrosine phosphorylation. Using a monoclonal antibody against phosphotyrosine, IL-3 was found to specifically and rapidly induce tyrosine phosphorylation of cytoplasmic proteins of 70, 56, and 38 kDa and a membrane-associated glycoprotein of 140 kDa. Minor and/or variable detected phosphoproteins of 120, 85, 51, and 28 kDa were also seen. Oncogenes encoding tyrosine protein kinases abrogate the requirement of factor-dependent myeloid cells for IL-3. We therefore compared the phosphoprotein profiles of a transformed, IL-3-independent cell line with the IL-3-induced profile. In cells transformed with trk, the 56-, 51-, and 38-kDa cytoplasmic phosphoproteins were constitutively phosphorylated, whereas the 140-kDa phosphoprotein was only phosphorylated in the presence of IL-3. Taken together, these results support a role for tyrosine phosphorylation in the IL-3 signal transduction pathway and suggest that growth factor abrogation by oncogenes encoding tyrosine protein kinases may be due to the phosphorylation of substrates which are normally phosphorylated in response to IL-3.     

Functional proteomics analysis of signal transduction pathways of the platelet-derived growth factor beta receptor

We report efficient methods for using functional proteomics to study signal transduction pathways in mouse fibroblasts following stimulation with PDGF. After stimulation, complete cellular proteins were separated using two-dimensional electrophoresis and phosphorylated proteins were detected with anti-phosphotyrosine and anti-phosphoserine antibodies. About 260 and 300 phosphorylated proteins were detected with the anti-phosphotyrosine and anti-phosphoserine antibodies, respectively, at least 100 of which showed prominent changes in phosphorylation as a function of time after stimulation. Proteins showing major time-dependent changes in phosphorylation were subjected to in-gel digestion with trypsin and identified by mass spectroscopy using MALDI-TOF mass fingerprinting and ESI peptide sequencing. We have observed phosphorylated proteins known to be part of the PDGF signal transduction pathway such as ERK 1, serine/threonine protein kinase akt and protein tyrosine phosphatase syp, proteins such as proto-oncogene tyrosine kinase fgr previously known to participate in other signal transduction pathways, and some proteins such as plexin-like protein with no previously known function in signal transduction. Information about the phosphorylation site was obtained for proto-oncogene tyrosine kinase fgr and for cardiac alpha-actin. The methods used here have proven to be suitable for the identification of time-dependent changes in large numbers of proteins involved in signal transduction pathways.     

Similar effects of platelet-derived growth factor and epidermal growth factor on the phosphorylation of tyrosine in cellular proteins

Platelet-derived growth factor (PDGF) stimulates the phosphorylation of proteins at tyrosine when added to quiescent 3T3 cells, as evidenced by the increase in the amount of phosphotyrosine, relative to phosphoserine and phosphothreonine, in cellular proteins. The increase was detected within 1 min of adding PDGF and was maximal by 5 min. This effect showed the same dependence on PDGF concentration as did association of 125I-PDGF with the cells. In different 3T3 cell lines the magnitude of the increase was approximately proportional to the number of PDGF receptors per cell. A number of proteins phosphorylated at tyrosine in response to PDGF have been detected by two-dimensional gel electrophoresis. They include a pair of related 45 kilodalton phosphoproteins, a pair of related 43 kilodalton phosphoproteins and a 42 kilodalton phosphoprotein. Similar changes were noted when quiescent 3T3 cells were incubated with epidermal growth factor. Possibly, these phosphoproteins are primary substrates of the tyrosine protein kinases activated by the receptors for PDGF and epidermal growth factor, and are involved in physiological effects common to the two growth factors.     

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.     

Platelet-derived growth factor (PDGF)-dependent association of phospholipase C-gamma with the PDGF receptor signaling complex.

We investigated the interaction of phospholipase C-gamma (PLC-gamma) with wild-type and mutant forms of the platelet-derived growth factor (PDGF) beta-receptor both in vivo and in vitro. After PDGF treatment of CHO cell lines expressing wild-type or either of two mutant (delta Ki and Y825F) PDGF receptors, PLC-gamma became tyrosine phosphorylated and associated with the receptor proteins. The receptor association and tyrosine phosphorylation of PLC-gamma correlated with the ability of these receptors to mediate ligand-induced phosphatidylinositol turnover. However, both the delta Ki and Y825F mutant receptors were deficient in transmitting mitogenic signals, suggesting that the PDGF-induced tyrosine phosphorylation and receptor association of PLC-gamma are not sufficient to account for the growth-stimulatory activity of PDGF. Wild-type and delta Ki mutant PDGF receptor proteins expressed with recombinant baculovirus vectors also associated in vitro with mammalian PLC-gamma. However, baculovirus-expressed c-fms, v-fms, c-src, and Raf-1 proteins failed to associate with PLC-gamma under similar conditions. Phosphatase treatment of the baculovirus-expressed PDGF receptor greatly decreased its association with PLC-gamma. This requirement for receptor phosphorylation was also observed in vivo, where PLC-gamma could not associate with a mutant PDGF receptor (K602A) defective in autophosphorylation. PLC-gamma also coimmunoprecipitated with two other putative receptor substrates, the serine-threonine kinase Raf-1 and the 85-kilodalton phosphatidylinositol-3' kinase, presumably through its association with the ligand-activated receptor. Furthermore, baculovirus-expressed Raf-1 phosphorylated purified PLC-gamma in vitro at sites which showed increased serine phosphorylation in vivo in response to PDGF. These results suggest that PDGF directly influences PLC activity by inducing the association of PLC-gamma with a receptor signaling complex, resulting in increased tyrosine and serine phosphorylation of PLC-gamma.