Regulation of a ribosomal protein S6 kinase activity by the Rous sarcoma virus transforming protein, serum, or phorbol ester.

Protein kinase capable of phosphorylating 40S ribosomal protein S6 on serine residues has been detected in chicken embryo fibroblasts. This activity appears to be regulated in direct response to expression of pp60v-src in chicken embryo fibroblasts infected with a temperature-sensitive transformation mutant of Rous sarcoma virus. Partially purified S6 kinase was highly specific for S6 in 40S ribosomal subunits. The S6 kinase was not inhibited by calcium or by the heat-stable inhibitor of cAMP-dependent protein kinase, nor was it activated by phosphatidylserine, diacylglycerol, and calcium. Thus, it is distinct from protein kinase C and cAMP-dependent protein kinase, which are capable of phosphorylating S6 in vitro. The tumor-promoter phorbol 12-myristate 13-acetate also stimulated ribosomal protein S6 kinase activity in serum-starved chicken embryo fibroblasts, whereas phorbol, the inactive analog of phorbol 12-myristate 13-acetate, had no effect. S6 kinase activity stimulated by expression of pp60v-src, by phorbol 12-myristate 13-acetate, or by serum growth factors exhibited similar chromatographic properties upon ion-exchange chromatography. These results suggest that a common protein kinase may be activated by three diverse stimuli all involved in regulating cell proliferation.     

Insulin and growth factor effects on c-fos expression in normal and protein kinase C-deficient 3T3-L1 fibroblasts and adipocytes.

We investigated the expression of the protooncogene c-fos in 3T3-L1 fibroblasts and adipocytes in response to a variety of growth-promoting agents in normal cells and in cells preincubated with phorbol esters to deplete them of protein kinase C. There was a rapid accumulation of c-fos mRNA in fibroblasts and adipocytes treated with phorbol 12-myristate 13-acetate, platelet-derived growth factor, fibroblast growth factor, fetal calf serum, bombesin, and insulin, especially in the adipocytes. Phorbol 12-myristate 13-acetate pretreatment abolished the increase in c-fos mRNA due to additional phorbol 12-myristate 13-acetate treatment and decreased but did not eliminate the ability of platelet-derived growth factor, fibroblast growth factor, fetal calf serum, bombesin, and insulin to stimulate c-fos mRNA. These data suggested that c-fos mRNA could be induced in serum-deprived 3T3-L1 fibroblasts and adipocytes by at least two separate pathways, one involving protein kinase C and the other independent of protein kinase C. In the very insulin-sensitive 3T3-L1 adipocytes, insulin rapidly and transiently increased c-fos expression (c-fos mRNA appeared by 15 min and disappeared after 60 min) via interaction with its own cellular receptor, rather than by interacting with receptors for one of the insulin-like growth factors. Cycloheximide treatment in combination with insulin or phorbol 12-myristate 13-acetate resulted in superinduction of c-fos mRNA. We conclude that insulin can rapidly stimulate c-fos mRNA accumulation in 3T3-L1 adipocytes and that part of the growth factor-stimulated increase in this mRNA that occurs in protein kinase C-deficient cells may be due to activation of a pathway similar or identical to that activated by insulin.     

Both p21ras and pp60v-src are required, but neither alone is sufficient, to activate the Raf-1 kinase.

The raf genes encode a family of cytoplasmic proteins with intrinsic protein-serine/threonine kinase activity. The c-raf gene is the cellular homolog of v-raf, the transforming gene of murine sarcoma virus 3611. The constitutive kinase activity of the v-Raf protein has been implicated in transformation and mitogenesis. The activity of Raf-1, the protein product of the c-raf gene, is normally suppressed by a regulatory N-terminal domain. Activation of various tyrosine-kinase growth factor receptors results in activation of Raf-1 and its hyperphosphorylation. Further, Raf-1 has been shown to act either downstream or independently of the p21ras protein, as indicated by experiments involving microinjection of anti-Ras antibodies. To investigate the potential role of p21ras in the activation of Raf-1 by tyrosine kinases, we have used the baculovirus/Sf9 cell system to overproduce various wild-type and mutant forms of pp60src, p21ras, and Raf-1 proteins. We show that either pp60v-src or p21c-ras can independently activate the autokinase activity of Raf-1, but only to a limited extent. Surprisingly, both pp60v-src and p21c-ras are required to fully activate Raf-1. Analysis of the Raf-1 autokinase activity in vitro shows that Raf-1 autophosphorylation sites are distributed equally on serine and threonine residues. When Raf-1 is analyzed by immunoblotting, as previously reported for mammalian cell experiments, a marked increase in the apparent molecular weight of Raf-1 is seen only when it is coexpressed with both pp60v-src and p21ras.     

Interleukin 2 regulates Raf-1 kinase activity through a tyrosine phosphorylation-dependent mechanism in a T-cell line.

Previously we found that interleukin 2 (IL-2) induces tyrosine phosphorylation and activation of the serine/threonine-specific kinase encoded by the raf-1 protooncogene in a T-cell line, CTLL-2. Here we extended these findings by exploring the effects of selective removal of phosphate from tyrosines in p72-74-Raf-1 kinase that had been immunoprecipitated from IL-2-stimulated CTLL-2 cells. Treatment in vitro of IL-2-activated Raf-1 with the tyrosine-specific phosphatases CD45 and TCPTP (formerly called T-cell protein tyrosine phosphatase) reduced Raf kinase activity to nearly baseline levels. This effect was completely inhibited by the phosphatase inhibitor sodium orthovanadate. In contrast, treatment of Raf-1 with a serine/threonine-specific phosphatase, protein phosphatase 1 (PP-1), resulted in a more modest decrease in Raf in vitro kinase activity, and this effect was prevented by okadaic acid. Two-dimensional phosphoamino acid analysis confirmed the selective removal of phosphate from tyrosine by CD45 and from serine and threonine by PP-1. The immunoreactivity of p72-74-Raf-1 with anti-phosphotyrosine antibodies was also completely abolished by treatment with CD45 in the absence but not in the presence of sodium orthovanadate. These findings provide evidence that the IL-2-stimulated phosphorylation of Raf-1 on tyrosines plays an important role in upregulating the activity of this serine/threonine-specific kinase in CTLL-2 cells and, as such, provides a model system for studying the transfer of growth factor-initiated signals from protein tyrosine kinases to serine/threonine-specific kinases.     

Angiotensin II stimulates two myelin basic protein/microtubule-associated protein 2 kinases in cultured vascular smooth muscle cells.

In cultured vascular smooth muscle cells, angiotensin II (Ang II) stimulated a cytosolic protein kinase activity toward myelin basic protein (MBP) in a time- and dose-dependent manner. Phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate also increased the MBP kinase activity. Downregulation of protein kinase C by prolonged treatment of the cells with phorbol 12,13-dibutyrate markedly attenuated the Ang II- and PMA-induced MBP kinase activation. The Ang II- and PMA-stimulated MBP kinase activities were resolved almost equally into two distinct fractions on Mono-Q HR5/5 column chromatography (kinase 1 and kinase 2). The kinase assay in polyacrylamide gel revealed that apparent molecular masses of kinase 1 and kinase 2 were 40 and 45 kd, respectively. Microtubule-associated protein 2 also served as a substrate for both the kinases. Immunoblot analysis with an antiphosphotyrosine antibody suggested that both the kinases were tyrosine-phosphorylated during the action of Ang II. Phosphoamino acid analysis revealed that Ang II and PMA induced phosphorylation of both the kinases on serine/threonine as well as tyrosine residues. Phosphopeptide mapping patterns of kinase 1 and kinase 2 isolated from Ang II-stimulated cells were almost identical with those from PMA-stimulated cells. These results indicate that in vascular smooth muscle cells Ang II activates two species of MBP/microtubule-associated protein 2 kinases mainly through the protein kinase C-signaling pathway and suggest that tyrosine and serine/threonine phosphorylation may be involved in this process.     

Insulin- and glucose-induced phosphorylation of the Na(+),K(+)-adenosine triphosphatase alpha-subunits in rat skeletal muscle.

Phosphorylation of the alpha-subunits of Na(+),K(+)-adenosine triphosphatase in response to insulin, high extracellular glucose concentration, and phorbol 12-myristate 13-acetate was investigated in isolated rat soleus muscle. All three stimuli increased alpha-subunit phosphorylation approximately 3-fold. Phorbol 12-myristate 13-acetate- and high glucose-induced phosphorylation of the alpha-subunit was completely abolished by the PKC inhibitor GF109203X, whereas insulin-stimulated phosphorylation was only partially reduced. Notably, insulin stimulation resulted in phosphorylation of the alpha-subunit on serine, threonine, and tyrosine residues, whereas high extracellular glucose or phorbol 12-myristate 13-acetate stimulation mediated phosphorylation only on serine and threonine residues. Insulin stimulation resulted in translocation of Na(+),K(+)-adenosine triphosphatase alpha(2)-subunit to the plasma membrane and increased Na(+),K(+)-adenosine triphosphatase activity in the same membrane fraction. High glucose had no effect on alpha-subunits distribution. Immunoprecipitation with antiphosphotyrosine antibody and subsequent Western blot analysis with anti-alpha(1)- and -alpha(2)-subunit antibodies revealed that both alpha(1)- and alpha(2)-subunit isoforms underwent phosphorylation on tyrosine residues in response to insulin, although with different time course and magnitude. Thus, we show that insulin-stimulated phosphorylation of Na(+),K(+)-adenosine triphosphatase alpha-subunit occurs via a PKC- and tyrosine kinase-dependent mechanism, whereas high glucose-induced phosphorylation is only PKC-dependent. Phosphorylation of Na(+),K(+)-adenosine triphosphatase alpha-subunits may be involved in regulation of Na(+),K(+)-adenosine triphosphatase activity by insulin or high extracellular glucose in skeletal muscle.     

Induction of c-fos and c-myc mRNA by epidermal growth factor or calcium ionophore is cAMP dependent.

Phorbol esters activate protein kinase C and induce expression of the c-fos and c-myc protooncogenes in density-arrested BALB/c 3T3 (A31) cells; in contrast, epidermal growth factor (EGF) does not activate protein kinase C and is a poor inducer of c-fos and c-myc in these confluent cells. We show that, when A31 cells were subconfluent and made quiescent by serum deprivation, the phorbol ester phorbol 12-myristate 13-acetate induced c-fos and c-myc mRNA poorly, whereas EGF was a better inducer. Another platelet-derived growth factor-inducible gene, JE, did not show this differential regulation by phorbol 12-myristate 13-acetate and EGF. The ability of EGF to induce protooncogene mRNA was associated with elevated levels of intracellular cAMP. First, serum-deprived cells maintained cAMP at about 2-fold higher level than density-arrested cells. Second, induction was greatly enhanced by cholera toxin and 3-isobutyl-1-methylxanthine, which increased intracellular cAMP 3- to 10-fold. The calcium ionophore A23187 mimicked EGF in that it elevated c-fos and c-myc mRNA when administered with cholera toxin and isobutylmethylxanthine. Neither cholera toxin and isobutyl-methylxanthine nor A23187 appreciably induced these mRNAs when used alone. Our results suggest that c-fos and c-myc expression can be regulated by an EGF-directed pathway that utilizes calcium and cAMP as cooperating cytoplasmic messengers.     

Heparin suppresses the induction of c-fos and c-myc mRNA in murine fibroblasts by selective inhibition of a protein kinase C-dependent pathway.

Heparin is a complex glycosaminoglycan that inhibits the proliferation of several cell types in culture and in vivo. To begin to define the mechanism(s) by which heparin exerts its antiproliferative effects, we asked whether heparin interferes with the expression of the growth factor-inducible protooncogenes c-fos and c-myc. We show that heparin suppressed the induction of c-fos and c-myc mRNA by serum in murine (BALB/c) 3T3 fibroblasts. Using purified mitogens, we further show that suppression was most marked when protooncogene expression was induced by phorbol 12-myristate 13-acetate, an activator of protein kinase C. By contrast, there was little or no suppression when the cells were stimulated by epidermal growth factor, which, in these cells, utilizes a protein kinase C-independent pathway for the induction of gene expression. Heparin also inhibited the change in cell morphology induced by the phorbol ester but had no effect on the morphological change induced by epidermal growth factor and agents that raise intracellular cAMP. Heparin did not inhibit intracellular protein kinase C activity, phorbol ester-induced down-regulation of protein kinase C, or phosphorylation of the 80-kDa intracellular protein kinase C substrate. These results suggest that heparin inhibits a protein kinase C-dependent pathway for cell proliferation and suppresses the induction of c-fos and c-myc mRNA at a site distal to activation of the kinase.     

Persistent induction of cyclooxygenase in p60v-src-transformed 3T3 fibroblasts.

A BALB/c 3T3 cell line infected with the temperature-sensitive Rous sarcoma virus strain LA90 has been used to investigate early, p60v-src-dependent changes in gene expression (protein synthesis). Giant two-dimensional electrophoresis, which can resolve greater than 3000 polypeptides from [35S]methionine-labeled cell lysates, was used to detect the induction of a p72-74 (72-74 kDa) doublet (pI 7.5) after activation of p60v-src at 35 degrees C. Antiserum against cyclooxygenase (prostaglandin synthase or prostaglandin endoperoxide synthase) specifically immunoprecipitated the p72-74 doublet. The p72-74 doublet was also induced by platelet-derived growth factor and by phorbol 12-myristate 13-acetate and was elevated in an NIH 3T3 cell line transformed by wild-type src. Activation of p60v-src caused a persistent increase in p72-74, whereas the effect of the growth factor was transient. These dissimilar kinetics of induction were paralleled by changes in cyclooxygenase activity. Down-regulation of protein kinase C inhibited subsequent induction of cyclooxygenase by phorbol myristate acetate but did not block induction by p60v-src. The glucocorticoid agonist dexamethasone inhibited induction of cyclooxygenase by p60v-src. Although induction of this enzyme may not be directly involved in transformation, the data support the view that oncogenic transformation may result, not from expression of transformation-specific genes, but from persistent changes in the expression of genes normally induced only transiently during passage from the G0 stage of the cell cycle.     

Mitogen-activated protein kinase activation resulting from selective oncogene expression in NIH 3T3 and rat 1a cells.

Mitogen-activated protein kinases (MAPKs) are serine/threonine kinases that are rapidly activated in response to a variety of growth factors in many cell types. MAPKs are activated by phosphorylation of both tyrosine and threonine residues. They are proposed to be key integrators of growth factor receptor transduction systems involving conversion of tyrosine kinase signals to serine/threonine kinase activation. We have studied the influence of specific oncogenes on the regulation of MAPK activity in NIH 3T3 and Rat 1a fibroblasts. In NIH 3T3 cells, ras or raf oncogene expression, but not gip2 oncogene expression, induces a significant constitutive MAPK activation. In contrast, in Rat 1a cells, gip2, but not ras or raf oncogene expression, induces a strong constitutive MAPK activation. The findings indicate that, in a cell type-selective manner, different oncoproteins are capable of causing the constitutive activation of MAPK. However, the magnitude of oncogene-induced MAPK activation is not directly correlated with cellular transformation in either cell type. It appears that expression of only a subset of transforming oncogenes in a specific cell type is able to alter the regulation of the MAPK activation pathway. Thus, the network of cytoplasmic serine/threonine kinases will be differentially regulated when the same oncogene is expressed in different cell types.     

PKC activation by melatonin modulates vimentin intermediate filament organization in N1E-115 cells

Melatonin enters cells and causes cytoskeletal rearrangements in unicellular organisms, plants and vertebrates. This pineal secretory product causes microtubule enlargement and neurite outgrowth by a calmodulin antagonism in N1E-115 cells. Recently, direct in vitro activation of protein kinase C by melatonin was described. Vimentin intermediate filaments are attached to microtubules and their organization depends on both microtubule distribution and phosphorylation of specific proteins. Protein kinase C is a serine threonine kinase which phosphorylates vimentin and through this mechanism causes intermediate filament disassembly. In this work the effects of melatonin on protein kinase C activation, content, and subcellular distribution were studied in N1E-115 cells. Also, melatonin effects on vimentin phosphorylation and subcellular distribution were explored. The results show that melatonin both activates and increases protein kinase C content in the membrane cytoskeletal fraction. Melatonin protein kinase C activation was followed by an increase in both vimentin phosphorylation and by vimentin subcellular redistribution. Moreover, staurosporine, a serine threonine kinase inhibitor, prevented increased vimentin phosphorylation elicited by melatonin. Similar effects to those caused by melatonin were obtained with the protein kinase C activator phorbol 12-myristate 13-acetate. Data support the idea that melatonin modulates vimentin organization through protein kinase C activation.     

Interleukin 2 induces tyrosine phosphorylation and activation of p72-74 Raf-1 kinase in a T-cell line.

Interleukin 2 (IL-2) is a lymphokine, produced by T cells upon antigenic or mitogenic stimulation, that is a critical regulator of T-cell proliferation. Although the binding of IL-2 to its receptor has been well characterized, the molecular mechanisms by which IL-2 transmits its signal from the membrane to the interior of the cell are poorly understood. Like most other growth factors, IL-2 causes rapid phosphorylation of proteins within its target cells. Unlike many other growth factors, however, the known subunits of the IL-2 receptor lack tyrosine-specific kinase activity, and little is known about the kinases whose activities are regulated by IL-2. Here we show that IL-2 (but not IL-4) induces rapid phosphorylation of the p72-74 serine/threonine-specific kinase encoded by the c-Raf-1 protooncogene in an IL-2-dependent murine T-cell line, CTLL-2, and that this phosphorylation is associated with increased kinase activity in p72-74 Raf-1-containing immune complexes. The concentration dependence of IL-2-mediated elevations in Raf-1 kinase activity correlated well with IL-2-stimulated proliferation of CTLL-2 cells. Furthermore, much of the IL-2-stimulated phosphorylation of p72-74 Raf-1 occurred on tyrosines. To our knowledge, the Raf-1 kinase represents the first endogenous substrate of an IL-2-regulated tyrosine kinase to be identified.     

Characterization of the TPR-MET oncogene p65 and the MET protooncogene p140 protein-tyrosine kinases.

The proteins encoded by the human TPR-MET oncogene (p 65tpr-met) and the human MET protooncogene (p140met) have been identified. The p65tpr-met and p140met, as well as a truncated TPR-MET product expressed in Escherichia coli, p50met, are autophosphorylated in vitro on tyrosine residues. Using the immunocomplex kinase assay, p140met activity was detected in various human tumor epithelial cell lines. In vivo, p65tpr-met is phosphorylated on both serine and tyrosine residues, while p140met is phosphorylated on serine and threonine. p140met is labeled by cell-surface iodination procedures, suggesting that it is a receptor-like transmembrane protein-tyrosine kinase.     

Protein kinase C as a component of a nerve growth factor-sensitive phosphorylation system in PC12 cells.

The treatment of PC12 cells with either nerve growth factor or phorbol 12-myristate 13-acetate caused a decrease in the phosphorylation of a soluble 100-kDa protein (Nsp100). After treatment with nerve growth factor, the activity of Ca2+/phospholipid-dependent protein kinase (protein kinase C) in the cytosol was increased. When the cytosol from untreated PC12 cells was preincubated with purified protein kinase C and its cofactors, the phosphorylation of Nsp100 was decreased. The preincubation of cytosol from nerve growth factor-treated PC12 cells with protein kinase C did not decrease Nsp100 phosphorylation further. Moreover, preincubation of partially purified Nsp100 kinase with protein kinase C decreased its ability to phosphorylate Nsp100. These results suggest that the binding of nerve growth factor to its receptor on PC12 cells causes an increase in the activity of protein kinase C in the cytosol and phosphorylation of Nsp100 kinase, which in turn lowers its ability to phosphorylate Nsp100.     

Phorbol ester stimulates a protein-tyrosine/threonine kinase that phosphorylates and activates the Erk-1 gene product.

The regulation of the Erk (extracellular-signal-regulated kinase) gene-encoded protein kinase activity by reversible phosphorylation has been reported to involve either an activator of autophosphorylation or an upstream protein kinase. In this communication we describe assays utilizing the Erk-1 protein fused to glutathione S-transferase that permit the identification of protein kinase(s) that phosphorylate and activate the myelin basic protein kinase activity encoded by the Erk-1 gene. A phorbol ester-stimulated protein kinase activity was identified that phosphorylated a kinase-negative Erk-1 gene product on tyrosine and threonine. The protein kinase phosphorylated and activated wild-type protein expressed in bacteria from 20- to 50-fold. The activation of the Erk-1-encoded myelin basic protein kinase required ATP and correlated directly with the degree of phosphorylation on the same amino acid residues previously shown to be phosphorylated in vivo. Conversion of the tyrosine site of phosphorylation to phenylalanine yielded an Erk-1 gene product that could not be activated. Similar results were obtained when the threonine site was mutated to valine. It is likely that the phorbol ester-stimulated protein-tyrosine/threonine kinase(s) is an up-stream target for multiple extracellular signals.     

Recombinant human interleukin-9 induces protein tyrosine phosphorylation and synergizes with steel factor to stimulate proliferation of the human factor-dependent cell line, M07e.

Human interleukin-9 (IL-9) was originally identified and cloned based on its stimulatory effect on proliferation of human myeloid cell line, M07e. IL-9 synergized with Steel factor, the ligand for the c-kit product, to stimulate M07e cell proliferation. To investigate potential mechanisms for this, IL-9 was assessed for effects on protein tyrosine kinase activities in M07e cells by immunoblotting with anti-phosphotyrosine monoclonal antibody; results were compared with those of Steel factor alone and in combination with IL-9, and those of 12-0-tetradecanoyl phorbol-13-acetate (TPA). Recombinant human IL-9 (10 ng/mL) rapidly and transiently induced or enhanced at least four tyrosine phosphorylated protein bands with molecular weights of 105, 97, 85, and 81 Kd. This tyrosine phosphorylation pattern was different from that generated by recombinant murine Steel factor or TPA stimulation and the combination of IL-9 and Steel factor did not change the IL-9-induced pattern. IL-9-induced tyrosine phosphorylated bands were completely blocked by treatment of IL-9 with anti-IL-9 antibody under conditions that also neutralized the synergistic effect of IL-9 with Steel factor on M07e cell proliferation. Genistein, a tyrosine kinase inhibitor, blocked phosphorylation of IL-9 and Steel factor-induced bands. Unlike Steel factor or TPA, IL-9 did not appear to stimulate phosphorylation of 42-Kd mitogen-activated protein (MAP) kinase or Raf-1, or enhance MAP kinase activity. MAP kinase and Raf-1 are serine/threonine kinases that are phosphorylated and activated by many growth factors and by agonists for protein kinase C. While the combination of IL-9 plus SLF did not appear to induce phosphorylation of new bands not already seen with either IL-9 or SLF alone, or enhance the phosphorylation of those bands seen with either cytokine alone, the results suggest that IL-9 activates specific and unique signal transduction pathways.     

Granulocyte-macrophage colony-stimulating factor and interleukin-3 induce rapid phosphorylation and activation of the proto-oncogene Raf-1 in a human factor-dependent myeloid cell line.

The product of the c-raf-1 proto-oncogene, Raf-1, is a 74,000-dalton cytoplasmic serine/threonine protein kinase that has been implicated as an intermediate in signal transduction mechanisms. In the human factor-dependent myeloid cell line MO7, both granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-3 (IL-3) were found to induce rapid, dose-dependent phosphorylation of Raf-1, which resulted in altered Raf-1 mobility in sodium dodecyl sulfate-polyacrylamide gels. The increase in phosphorylation was due primarily to an increase in phosphoserine, with only a minor component (less than 2%) of phosphotyrosine. PMA (12-phorbol 13-myristic acid) also induced Raf-1 phosphorylation in MO7 cells, but the resulting alteration in electrophoretic mobility was different than that observed after GM-CSF or IL-3. GM-CSF and IL-3 rapidly and transiently increased Raf-1 kinase activity using Histone H1 as a substrate in an immune complex kinase assay in vitro. These results suggest that phosphorylation of Raf-1 could play a role in some aspect of GM-CSF and IL-3 signal transduction.     

Platelet-derived growth factor modulates epidermal growth factor receptors by a mechanism distinct from that of phorbol esters.

Platelet-derived growth factor (PDGF) and phorbol 12-myristate 13-acetate (PMA) decrease high affinity binding of 125I-labeled epidermal growth factor (EGF) and potentiate mitogenesis in BALB/c 3T3 cells, and both have been shown to induce the phosphorylation of the EGF receptor at threonine residues. These similarities suggest that the actions of PDGF on EGF binding may be mediated by protein kinase C, the cellular effector of PMA. We show that in density-arrested BALB/c 3T3 cells PDGF and PMA induce a rapid, transient, cycloheximide-independent loss of EGF binding activity. As has been previously shown for PDGF, the ability of PMA to reduce EGF binding was enhanced by cholera toxin, a potent activator of adenylate cyclase. In contrast to PMA, however, PDGF induced a further reduction in EGF binding that was strictly dependent upon continued protein synthesis. Furthermore, PDGF effectively reduced EGF binding in cells refractory to PMA. Cells desensitized to PMA, presumably due to the loss of protein kinase C activity, also remained mitogenically responsive to PDGF. These data suggest that the mechanism by which PDGF modulates EGF binding differs from that of PMA and thus, at least in part, is independent of protein kinase C.