Nuclear pp64 is phosphorylated in both serine/threonine and tyrosine through complex pathways regulated by 12-O-tetradecanoylphorbol-13-acetate and platelet-derived growth factor

Platelet-derived growth factor (PDGF) induces the time and dose dependent serine/threonine phosphorylation of pp64, a nuclear protein in normal rat kidney (NRK) cells. pp64 is phosphorylated additionally on tyrosine in SSV-transformed NRK cells. To further characterize the regulation of phosphorylation of pp64, other mitogens and inhibitors were studied. 12-O-tetradecanoylphorbol-13-acetate (TPA) but not epidermal growth factor (EGF) or insulin induced the phosphorylation of nuclear pp64. Addition of the inhibitor H7 to TPA-treated NRK cells resulted in a striking further increase in phosphorylation of pp64 and, to a lesser extent, in NRK cells treated with PDGF and H7. When cells were treated with PDGF and H7, pp64 was recognized by anti-phosphotyrosine antisera. The increased phosphorylation induced by H7 was inhibited when forskolin was included. This loss of phosphorylation in pp64 with forskolin treatment paralleled a loss of immunoreactivity of pp64 to anti-phosphosphotyrosine. Complex and independent pathways thus appear to signal the growth factor dependent nuclear phosphorylation of pp64, involving phosphorylations both on serine/threonine and on tyrosine.     

Cytolytic T cells recognize the two amino-terminal domains of H-2 K antigens in tandem in influenza A infected cells

Simian sarcoma virus-transformed NIH 3T3 (SSV-NIH 3T3) and SSV-NRK cells secrete a potent growth-promoting activity identical with the platelet-derived growth factor (PDGF) in mitogenic assays. The secreted activity is blocked by anti-PDGF antisera and competes with 125I-PDGF for receptor binding, suggesting that the secreted protein is the transforming protein of SSV, p28v-sis, or its processed product. Secreted p28v-sis appears to stimulate autocrine cell growth of SSV-transformed cells because anti-PDGF antisera block 3H-thymidine incorporation into growing SSV-NIH 3T3 and SSV-NRK cells. SSV-transformed cells have reduced numbers of high-affinity 125I-PDGF receptors; PDGF/p28v-sis receptor was purified from SSV-NIH 3T3 cells and retained active protein tyrosine kinase activity stimulated by PDGF. The rate of tumor growth in athymic nude mice injected with SSV-transformed cells was compared with levels of secreted growth factor activity. The rate of tumor growth in nude mice correlated directly with levels of p28v-sis secreted by SSV-transformed cells.     

Simian sarcoma virus transformation of normal rat kidney fibroblasts is associated with markedly increased basic fibroblast growth factor expression.

Transformation of normal rat kidney fibroblasts (NRK) by the simian sarcoma virus (SSV) occurs as a result of expression of p28v-sis, a homologue of platelet-derived growth factor-B chain. Chromatographic separation revealed that the bulk (85%) of the mitogenic activity in SSV-transformed NRK cells was not due to p28v-sis but rather two distinct endothelial cell growth factors that eluted off heparin-Sepharose between 1 and 2 M NaCl. Protein purification and Northern blot analysis revealed that one of these growth factors was the 18 kd form of bFGF, the expression of which was found to increase 15-fold with SSV-transformation of NRK cells. The pure 18 Kd bFGF had no effect on NRK cell growth but was a potent neurotrophic agent for fetal rat cortical neurones and a potent growth factor for fetal bovine heart endothelial cells, suggesting a paracrine but not autocrine role for this protein. The second endothelial cell growth factor activity in SSV-transformed NRK cells was due to an 18 Kd protein which could be distinguished immunologically, biochemically, and mitogenically from bFGF.     

The phenotypic characteristics of simian sarcoma virus-transformed human fibroblasts suggest that the v-sis gene product acts solely as a PDGF receptor agonist in cell transformation.

Previous studies have indicated that the oncogene v-sis of simian sarcoma virus (SSV) encodes a growth factor that is structurally and functionally similar to platelet-derived growth factor (PDGF). In the present investigation we have analysed the phenotypic characteristics of human foreskin fibroblasts transformed by SSV. It was found that the PDGF receptors were extensively down-regulated. This finding is consistent with a high, local, extracellular concentration of a PDGF-like factor, synthesized by the transformed cell. The receptors were up-regulated by suramin, a drug that is known to dissociate PDGF and the v-sis product from the PDGF receptors. A cell-associated v-sis product of mol. wt 24,000 was identified by immunoprecipitation with PDGF antibodies; release of this component was induced by a high concentration of exogenous PDGF, indicating that a fraction of the product is associated with the PDGF receptors. SSV was not found to be an immortalizing virus; when serially passaged, SSV-transformed cells had essentially the same life-span as their non-transformed counterparts. Moreover, SSV did not induce growth in soft agar beyond the level afforded by exogenously added PDGF. Thus, the present study favors the notion that SSV transformation is mediated by a growth factor that mimics PDGF but has no further cellular effects.     

Basic fibroblast-like growth factor is present in the conditioned medium of simian sarcoma virus transformed NRK cells

The conditioned medium of Simian sarcoma virus (SSV)-transformed NRK cells contains at least two activities that down regulate the epidermal growth factor receptor. To identify these activities, we analyzed the medium for the presence of factors both related to and distinct from the v-sis oncogene product. Fractionation of the conditioned medium from SSV-transformed NRK cells by chromatography on heparin-Sepharose yielded two active fractions capable of inhibiting EGF binding. The first component, which eluted at 0.8 M NaCl, is able to induce autophosphorylation of the platelet-derived growth factor (PDGF) receptor, is a mitogen for Swiss 3T3 cells and corresponds to the PDGF B chain product of the v-sis oncogene. The second component requires 2 M NaCl for elution, is mitogenic for Swiss 3T3 cells and inhibits high affinity EGF binding through a protein kinase C-independent pathway, all properties of basic FGF. These results suggest that the conditioned medium of v-sis-transformed cells contains at least two factors that can act in an autocrine capacity, one derived from v-sis and one corresponding to basic FGF.     

Virus-specific phosphoproteins in simian sarcoma virus-transformed primate cells.

Cells transformed by simian sarcoma virus (SSV) express a 115000-dalton protein ( p115 ) that is precipitated by a goat antiserum to disrupted SSV/SSAV-infected and transformed cells but not by antibodies directed against the viral gag protein, p30, or envelope proteins. The protein is detected in productively as well as in nonproductively infected, transformed cells. It is not present in untransformed cells infected with helper virus (SSAV). The protein can be phosphorylated in vivo and in vitro at the tyrosine residue and SSV-transformed cells contain elevated levels of phosphotyrosine.     

Normal rat kidney cells secrete both phosphorylated and nonphosphorylated forms of osteopontin showing different physiological properties

We have reported previously that the 69-kDa major phosphoprotein, secreted by normal rat kidney (NRK) cells, is osteopontin, a glycosylated bone matrix protein. Here we show that this 69-kDa osteopontin is secreted by NRK cells in both phosphorylated (pp69) and nonphosphorylated (np69) forms, with estimated isoelectric points of 3.8 and 4.5, respectively. Electrophoretic analysis of radioiodinated cell surface proteins immunoprecipitated with an anti-69-kDa osteopontin serum, demonstrates that the 69-kDa osteopontin is also present on the cell surface, but only its phosphorylated form (pp69) shows such cell surface association. Because osteopontin mediates cell adhesion and spreading, and contains an Arg-Gly-Asp-Ser cell-binding sequence, our observations strongly suggest that the cell surface localization of pp69 osteopontin is receptor-mediated, and the modification by phosphorylation may be crucial for its receptor binding activity. We also report that antisera directed against either fibronectin or 69-kDa osteopontin co-immunoprecipitate both np69 osteopontin and fibronectin as a heat-dissociable complex. In contrast, pp69 osteopontin does not co-precipitate with fibronectin. These observations demonstrate an interactive relationship between np69 and soluble fibronectin. Furthermore, compared to NRK cells, vanadyl sulfate-treated NRK cells which acquire a reversible transformed phenotype, including anchorage-independent growth, show increased levels of pp69 on the cell surface, concomitant with significantly decreased levels of pp69 and elevated levels of np69 in the conditioned media. The data presented here establish transformation sensitivity of NRK cell-secreted osteopontin with respect to its secretion and cell surface localization, and demonstrate that phosphorylated and nonphosphorylated forms of osteopontin have different physiological properties, which may regulate the functional roles of this extracellular matrix protein.     

PDGF-Independent Activation of PDGF-β Receptors in NIH-3T3 Cells Transformed by c-met Protooncogene

We have previously reported that c-met protooncogene, a member of a new class of receptor tyrosine-kinase gene family, is transforming when overexpressed in NIH-3T3 cells. In this paper, we report that the c-met protooncogene-transformed cells proliferate in a serum- and growth factor-free medium and exhibit constitutive tyrosine phosphorylation of several cellular proteins including the met protooncogene-encoded p145 and p185. Further investigations revealed platelet-derived growth factor (PDGF)-independent phosphorylation of PDGF-β receptors in the transformed cells. Phosphoamino acid analysis revealed phosphorylation of PDGF receptors at tyrosine and serine residues. The PDGF receptor phosphorylation is unlikely to occur via autocrine production of PDGF since we could not detect PDGF activity both at the RNA level and at a functional protein level. Additionally, phospholipase C-γ (PLC-γ) a substrate of activated PDGF receptors, was found to be physically associated with PDGF receptors in the absence of PDGF stimulation in (transformed cells. Furthermore, PDGF receptors coimmunoprecipitated along with PLC-γ. Taken together, our results demonstrate a PDGF-independent phosphorylation and activation of PDGF-β receptor in NIH-3T3 cells transformed by c-met protooncogene.     

Altered response to growth factors or retinoic acid in phenotypic transformation of normal rat kidney cells expressing human c-fos gene.

Anchorage-independent growth of normal rat kidney (NRK) fibroblast in soft agar depends on both transforming growth factor beta (TGF beta) and epidermal growth factor (EGF). To examine whether c-fos protein is involved in phenotypic transformation of NRK cells, we have transfected and isolated several NRK cell lines that carry the human c-fos gene fused to the metallothionein IIA promoter. A transfectant, Nf-1, had constitutive levels of the human c-fos expression. Anchorage-independent growth of Nf-1 was already stimulated by EGF alone, and the colony sizes of Nf-1 were comparable to those of the parental NRK in the presence of both EGF and TGF beta. Anchorage-independent growth of NRK could be observed in the presence of TGF beta or retinoic acid or platelet derived growth factor (PDGF) and EGF. No growth of NRK in soft agar appeared when basic fibroblast growth factor (bFGF) and EGF were present. By contrast, anchorage-independent growth of Nf-1 was surprisingly enhanced by EGF and TGF beta or retinoic acid or PDGF or bFGF. Expression of the human c-fos gene may compensate the signal to phenotypic transformation induced by TGF beta as well as retinoic acid or PDGF or bFGF.     

Modulation of Prostaglandin G/H Synthase Expression in Mesangial Cells Transfected by pp60Proto-oncogene

The role of the protein tyrosine kinase pp60^c-srcin the expression of prostaglandin G/H synthase (PGHS), the key enzyme of prostaglandin synthesis, was investigated in rat renal mesangial cells. Transfection of mesangial cells with the proto-oncogene c-src resulted in nontransformed cells with constitutively enhanced pp60^c-srckinase activity. As a control, mesangial cells were transfected with inactive pp60^c-src, mutated in position 295 (lysine replaced by methionine). Expression of the constitutive isoform PGHS-1 was enhanced in cells overexpressing wild-type c-src compared to cells transfected with the kinase negative c-src mutant. Levels of other constitutively expressed proteins such as GAPDH and β-actin were also enhanced. PGHS-2 was barely detectable in resting cells but was inducible by PDGF-AB, PDGF-BB, serotonin, FCS, and calcium ionophore A23187. Induction was diminished in pp60^c-srckinase-overexpressing cells, independent of the stimulus used, suggesting interference at a late step in the signaling cascade. No induction of PGHS-2 mRNA was observed in mesangial cells transformed by the oncogene v-src. An increase in intracellular calcium levels is an early step in signal transduction by PDGF and serotonin in mesangial cells. c-src kinase overexpression reduced PDGF- and serotonin-mediated changes in Ca²⁺signaling, indicating multiple targets of pp60^c-srcaction. Overexpression of pp60^c-srcin mesangial cells thus affected basal protein expression, reflected by the enhanced PGHS-1 mRNA and protein expression. With regard to induction of PGHS-2, overexpression of pp60^c-srcreduced induction by stimuli coupled to different types of signaling pathways.     

Loss in transformed cells of cell cycle regulation of expression of a nuclear protein recognized by SLE patient antisera

The identification of cellular proteins involved in the control of cell proliferation in normal cells is essential for understanding the mechanism underlying growth regulation and cellular transformation. A nuclear protein termed Ki antigen with a relative mobility of 32,000 (Mr 32K) and which is recognized by SLE patient antisera has been identified in cells of human, bovine, and murine origin. Recently, cDNA clones for the bovine and human Ki antigens have been isolated using SLE patient antisera (T. Nikaido, et al., in preparation). The nucleotide sequence predicted a protein of 239 amino acids with a possible nuclear localization signal resembling that identified in SV40 T antigen and other nuclear proteins. Here we show that the expression of Ki antigen is regulated in the normal cell, but not in the transformed cell. Furthermore, in the K-ras temperature-sensitive mutant cell line, ts 371 normal rat kidney (NRK), Ki antigen expression increases several-fold at the permissive temperature relative to the nonpermissive temperature. These results suggest that expression of Ki antigen might be correlated with cellular transformation as well as with cell growth regulation.     

Epidermal growth factor (EGF)-nonresponsive variants of normal rat kidney cell line: response to EGF and transforming growth factor-beta

Anchorage-independent growth in soft agar of normal rat kidney (NRK) fibroblasts depends on both transforming growth factor-beta (TGF-beta) and epidermal growth factor (EGF) (or TGF-alpha). We have isolated two EGF-nonresponsive cell lines, N-3 and N-9, from chemically mutagenized NRK cells, after selection of mitogen-specific nonproliferative variants in the presence of EGF and colchicine. Saturation binding kinetics with 125I-EGF showed one-half or fewer EGF receptors in N-3 and N-9 than in their parental NRK. Cellular uptake of 2-deoxy-D-glucose was enhanced in all NRK, N-3, and N-9 cell lines by TGF-beta treatment, whereas treatment with EGF significantly enhanced the cellular uptake of the glucose analog in NRK cells, but not in N-3 and N-9 cells. DNA synthesis of NRK during the quiescent state, but not that of N-3 and N-9, was stimulated by EGF. Anchorage-independent growth of N-9 could not be observed even in the presence of both EGF and TGF-beta, whereas that of N-3 was significantly enhanced by TGF-beta alone. EGF stimulated phosphorylation of a membrane protein with molecular size 170 kDa of NRK, but not of N-3, when immunoprecipitates reacting with anti-phosphotyrosine antibody were analyzed. Exposure of NRK cells to EGF increased cellular levels of TGF-beta mRNA, but there appeared little expression of TGF-beta mRNA in N-3 and N-9 cells. Exposure of N-3 cells to EGF or TGF-beta enhanced the secretion of EGF into culture medium, but exposure of NRK or N-9 cells did not. Altered response to EGF of N-3 or N-9 might be related to their aberrant growth behaviors.     

Platelet-derived growth factor stimulation of GTPase-activating protein tyrosine phosphorylation in control and c-H-ras-expressing NIH 3T3 cells correlates with p21ras activation.

Platelet-derived growth factor (PDGF) stimulation of NIH 3T3 cells leads to the rapid tyrosine phosphorylation of the GTPase-activating protein (GAP) and an associated 64- to 62-kDa tyrosine-phosphorylated protein (p64/62). To assess the functions of these proteins, we evaluated their phosphorylation state in normal NIH 3T3 cells as well as in cells transformed by oncogenically activated v-H-ras or overexpression of c-H-ras genes. No significant GAP tyrosine phosphorylation was observed in unstimulated cultures, while PDGF-BB induced rapid tyrosine phosphorylation of GAP in all cell lines analyzed. In NIH 3T3 cells, we found that PDGF stimulation led to the recovery of between 37 and 52% of GAP molecules by immunoprecipitation with monoclonal antiphosphotyrosine antibodies. Furthermore, PDGF exposure led to a rapid and sustained increase in the levels of p21ras bound to GTP, with kinetics similar to those observed for GAP tyrosine phosphorylation. The PDGF-induced increases in GTP-bound p21ras in NIH 3T3 cells were comparable to the steady-state level observed in serum-starved c-H-ras-overexpressing transformants, conditions in which these cells maintained high rates of DNA synthesis. These results imply that the level of p21ras activation following PDGF stimulation of NIH 3T3 cells is sufficient to support mitogenic stimulation. Addition of PDGF to c-H-ras-overexpressing cells also resulted in a rapid and sustained increase in GTP-bound p21ras. In these cells GAP, but not p64/62, showed increased tyrosine phosphorylation, with kinetics similar to those observed for increased GTP-bound p21ras. All of these findings support a role for GAP tyrosine phosphorylation in p21ras activation and mitogenic signaling.     

Erythropoietin induces cytosolic protein phosphorylation and dephosphorylation in erythroid cells.

Erythropoietin, the prime regulator of red blood cell growth and differentiation, causes rapid changes in the phosphorylation of several integral plasma membrane proteins (Choi, H-S., Wojchowski, D. M., and Sytkowski, A. J. (1987) J. Biol. Chem. 262, 2933-2936; Choi, H-S., Bailey, S. C., Donahue, K. A., Vanasse, G. J., and Sytkowski, A. J. (1990) J. Biol. Chem. 265, 4143-4148). In the present study we have demonstrated that erythropoietin's signal is transduced rapidly to the cytosol resulting in specific phosphorylation/dephosphorylation events. Erythropoietin treatment of Rauscher murine erythroleukemia cells previously labeled with [32P]orthophosphate results in a rapid increase in phosphorylation of two cytosolic proteins, designated pp96 and pp80, and a decrease in phosphorylation of another protein, designated pp90. The relative molecular mass and pI of pp80 are virtually identical to those reported for the protein kinase C substrate p80, or "MARCKS protein." Treatment of the cells with 12-O-tetradecanoylphorbol-13-acetate also increases pp80 but not pp96 phosphorylation, suggesting that erythropoietin triggers a protein kinase C-dependent pathway to pp80 and a protein kinase C-independent pathway to pp96. The effect of erythropoietin on pp96 phosphorylation was also shown in nontransformed erythroid cells isolated from the spleens of phenylhydrazine-treated mice. In contrast, almost no 32P labeling of pp80 or pp90 was detected, and pp80 and pp90 protein were nearly absent from these normal cells. These differences in expression and phosphorylation of erythropoietin-sensitive phosphoproteins may be related to the growth factor independence or dependence of the erythroid cells.     

Insulin and insulin-like growth factor I (IGF I) stimulate phosphorylation of a Mr 175,000 cytoskeleton-associated protein in intact FRTL5 cells

FRTL5 rat thyroid cells possess separate high affinity receptors for insulin and insulin-like growth factor I (IGF I) that undergo beta-subunit phosphorylation upon interaction with the specific ligand. Phosphorylation is rapid and dose-dependent and occurs primarily on tyrosine residues. Within 2 min, both insulin and IGF I also give rise to a Mr 175,000 phosphoprotein (pp175) that can be immunoprecipitated by anti-phosphotyrosine antibody (alpha-Tyr(P]. Phosphorylation of pp175 occurs on serine and threonine as well as tyrosine residues. When FRTL5 cells are solubilized with 1% Triton X-100, alpha-Tyr(P) immunoprecipitates phosphorylated insulin and IGF I receptors but little pp175 from the Triton-soluble fraction. After treatment of the Triton-insoluble portion with 1% sodium dodecyl sulfate at 100 degrees C, pp175 can be identified by immunoprecipitation with alpha-Tyr(P). The fraction of FRTL5 cells that remains after extraction of an attached monolayer with 1% Triton for 5 min at 22 degrees C contains most of the cytoskeleton and also nuclei. Extraction of this 32P-labeled cytoskeleton preparation with sodium dodecyl sulfate followed by alpha-Tyr(P) immunoprecipitation results in almost complete recovery of the pp175 content of the cells. When a nuclear fraction was prepared from FRTL5 cells by differential centrifugation, pp175 was not found in the nuclear pellet from labeled cells, but greater than 80% of pp175 was recovered in the supernatant. We conclude that pp175 is a common substrate for insulin and IGF I receptor kinases which, in FRTL5 cells, is associated with the cytoskeleton. It is suggested that phosphorylation of proteins associated with cytoskeletal elements could be involved in insulin and IGF I action in cells.     

Common elements in growth factor stimulation and oncogenic transformation: 85 kd phosphoprotein and phosphatidylinositol kinase activity

The phosphorylation of proteins on tyrosine in vivo and in vitro was examined in 3T3 cells stimulated by platelet-derived growth factor (PDGF) and transformed by polyoma middle T antigen (MTAg) by using an antibody directed against phosphotyrosine (P-tyr). Two common events were observed upon PDGF stimulation or MTAg transformation of cells: the appearance in the immunoprecipitates of an 85 kd phosphoprotein, and increased phosphatidylinositol (PI) kinase activity. In PDGF-stimulated cells, the 85 kd phosphoprotein and PI kinase activity appeared rapidly, within 1 min of growth factor addition. The PI kinase activity and 85 kd phosphorylation were also increased in anti-P-tyr immunoprecipitates from cells transformed by v-fms and v-sis, but not by SV40 T antigen. The presence of the tyrosine-phosphorylated 85 kd protein correlated with PI kinase activity during several purification steps. These results suggest that the 85 kd phosphoprotein, a putative PI kinase, is a substrate for both the PDGF receptor and MTAg/pp60c-src tyrosine kinase activities.     

Phosphorylation and nuclear processing of the androgen receptor.

Although transformed androgen receptor (AR) complexes derived from cytosol and nuclear AR complexes have been shown to bind with high affinity to nuclei and DNA, we have shown that the binding characteristics of the two receptor populations to rat ventral prostate nuclei are different. To account for these differences, we investigated the possibility that the two receptor populations differed in phosphorylation status. Significantly, an anti-phosphotyrosine antibody immunoprecipitated androgen binding from the nuclear AR preparation but not from the transformed cytosolic receptor preparation. These studies suggest that (i) further processing of the AR complex takes place after it has become transformed, and (ii) phosphorylation of the complex is one modification which occurs during the processing of the nuclear receptor.     

Phosphorylated 350 kD protein in the nucleus as it is associated with cell transformation

Protein kinases are thought to play a key role in signal transduction and oncogenesis, but little is known about the intranuclear phosphorylation events associated with transformation. Here we report on cell cycle-dependent phosphorylation of cytoskeleton-associated 350 kD protein and the regular interchange in its location between the nucleus and cytoplasm of normal cells. Persistent intranuclear location of the phosphorylated 350 kD protein was also found throughout the cell cycle in transformed cells, as detected by immunoprecipitation of 32P-phosphorylated 350 kD protein from isolated nuclei and immunofluorescent staining with a monoclonal antibody that recognized phosphorylated site of 350 kD protein. A conditional transformed phenotype induced by a temperature-sensitive (ts) viral oncogene or a transforming growth factor was also associated with the intranuclear presence of the phosphorylated 350 kD protein. Thus the 350 kD protein seems to be a target molecule of protein kinases that are stimulated directly or indirectly by growth factors or by oncogene products in the nucleus, and appears to be a new transformation-related nuclear antigen.     

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.