The adhesion plaque protein, talin, is phosphorylated in vivo in chicken embryo fibroblasts exposed to a tumor-promoting phorbol ester.

Talin is a high molecular weight phosphoprotein that is localized at adhesion plaques. We have found that talin phosphorylation increases 3.0-fold upon exposure of chicken embryo fibroblasts to the tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate. Talin isolated from tumor promoter-treated cells is phosphorylated on serine and threonine residues. Vinculin, a 130 kDa talin-binding protein, also exhibits increased phosphorylation in vivo in response to tumor promoter, but to a lesser degree than does talin. Because tumor-promoting phorbol esters augment protein kinase C activity, we have compared the ability of purified protein kinase C to phosphorylate talin and vinculin in vitro. Both talin and vinculin were found to be substrates for protein kinase C; however, talin was phosphorylated to a greater extent than was vinculin. Cleavage of protein kinase C-phosphorylated talin by the calcium-dependent protease (Type II) revealed that while both the resulting 190-200 and 46 kDa proteolytic peptides were phosphorylated, the majority of label was contained within the 46-kDa fragment. Although incubation of chicken embryo fibroblasts with tumor-promoting phorbol ester induces a dramatic increase in talin phosphorylation, we detected no change in the organization of stress fibers and focal contacts in these cells. Exposure of the cells to tumor promoter did, however, result in a loss of actin and talin-rich cell surface elaborations that resemble focal contact precursor structures.     

Phorbol ester- and protein kinase C-mediated phosphorylation of the cellular Kirsten ras gene product

The effect of phorbol 12-myristate 13-acetate on the phosphorylation of the ras p21 protein was studied by metabolically labeling cultured cells with [32P]orthophosphate and using a monoclonal antibody to immunoprecipitate the protein. Phorbol 12-myristate 13-acetate (100 nM) induced phosphorylation of cKi-ras p21 in a mouse adrenocortical cell line (Yl) expressing high levels of cKi-ras with exon 4B. Phosphorylation was detected at 10 min and was maximal at 2 h. The ras protein was not phosphorylated in response to phorbol 12-myristate 13-acetate in NIH 3T3 cells expressing activated cHa-ras or vHa-ras. In vitro, protein kinase C phosphorylated cKi-ras in a phosphatidylserine and diolein-dependent manner. Both in intact cells and in vitro the amino acid phosphorylated was serine. Analysis of p21 from NIH 3T3 cells expressing a variety of ras proteins indicated that phosphorylation occurs within a domain encoded by exon 4B of cKi-ras. Phosphorylation affected neither the binding nor the GTPase activity of the ras protein. We conclude that cKi-ras is a substrate for protein kinase C and that the site of phosphorylation is likely to be serine 181 encoded by exon 4B.     

Vinculin, a cytoskeletal substrate of protein kinase C

Vinculin, a cytoskeletal protein localized at adhesion plaques, is a phosphoprotein containing phosphoserine, phosphothreonine, and phosphotyrosine. Vinculin has been previously shown to be a substrate for pp60src, a phosphotyrosine protein kinase, but the kinase(s) responsible for phosphorylation of the other amino acid residues is unknown. The present report examines the phosphorylation of vinculin by various serine- and threonine-specific protein kinases. Only protein kinase C, the calcium-activated phospholipid-dependent protein kinase, phosphorylates vinculin at a significant rate (24 nmol/min/mg) and displays marked specificity for vinculin. Both calcium and phosphatidylserine were required for vinculin phosphorylation by protein kinase C. In addition, both phorbol 12,13-dibutyrate (10 nM) and phorbol 12-myristate 13-acetate (10 nM) stimulated vinculin phosphorylation by protein kinase C at a limiting calcium concentration (10(-6) M). Tryptic peptide analysis revealed two major sites of phosphorylation. One site contained phosphoserine and the other contained phosphothreonine. When compared with tryptic maps of vinculin phosphorylated by src kinase, no overlapping phosphorylated peptides were found. The present findings coupled with the plasma membrane location of both these proteins suggest that vinculin may be a physiologic substrate for protein kinase C.     

The relationship between lysosomal enzyme release and protein phosphorylation in human monocytes stimulated by phorbol esters and opsonized zymosan

Since it was established that phorbol esters bind to and activate protein kinase C, a proposed mechanism of action for these compounds has been the phosphorylation of specific protein substrates (Niedel, J. E., Kuhn, L. J., and Vandenbark, G. R. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 36-40; Castagna, M., Takai, Y., Kaibuchi, K., Sano, K., Kikkawa, U., and Nishizuka, Y. (1982) J. Biol. Chem. 257, 7847-7851). To better understand this proposed relationship, we investigated the ability of a series of phorbol esters to elicit lysosomal enzyme release (LER) and specific substrate phosphorylation in human monocytes. In this system, phorbol 12-myristate 13-acetate stimulated the secretion of the lysosomal enzyme N-acetyl-beta-D-glucosaminidase in both a time- and concentration-dependent manner. Furthermore, the ability of a series of phorbol esters to stimulate LER was characterized and found to be in good agreement with the relative order of these compounds to stimulate the phosphorylation of four endogenous protein substrates. Phorbol ester-stimulated protein phosphorylation was examined in intact cell preparations and found to be concentration and structure-dependent. The phosphoproteins (pp) were designated pp28, pp55, pp61, and pp66 corresponding to their molecular masses in kilodaltons. These findings are consistent with the hypothesis that phorbol ester-mediated effects are the result of protein kinase C activation and subsequent protein phosphorylation. Finally, opsonized zymosan was found to elicit a concentration-dependent stimulation of N-acetyl-beta-D-glucosaminidase release similar in magnitude and time course to phorbol ester-stimulated LER. Opsonized zymosan also stimulated the phosphorylation of two phosphoproteins (pp61 and pp66) in a concentration-dependent manner. Specific phosphorylation of pp61 and pp66 by both secretagogues, phorbol 12-myristate 13-acetate and opsonized zymosan, suggests these two proteins may be key to the functional response of LER in human monocytes.     

Regulation of calcium channels in aortic muscle cells by protein kinase C activators (diacylglycerol and phorbol esters) and by peptides (vasopressin and bombesin) that stimulate phosphoinositide breakdown

Voltage-dependent Ca2+ channels of the aortic cell line A7r5 were studied using 45Ca2+ flux experiments. Ca2+ channels which have been studied belong to the L-type and are very sensitive to inhibitors and activators in the 1,4-dihydropyridine series as well as to (-)desmethoxyverapamil and d-cis-diltiazem. L-type Ca2+ channels in these smooth muscle cells are not affected by cyclic 8-bromo-AMP and dibutyryl cyclic AMP. However, the activity of these channels is strongly depressed after treatment with diacylglycerols (1-oleyl 2-acetylglycerol and 1,2-dioctanoylglycerol). Phorbol esters, which like diacylglycerols are well-known activators of protein kinase C (the Ca2+- and phospholipid-dependent enzyme), inhibit 70% of Ca2+ channel activity (K0.5 = 25 nM for phorbol 12-myristate 13-acetate and K0.5 = 200 nM for phorbol 12,13-dibutyrate). Phorbol esters that are inactive on kinase C are without effect on Ca2+ channel activity. [Arg8]Vasopressin and bombesin, two peptides that are well known for their action on polyphosphoinositide metabolism, inhibit Ca2+ channel activity to the same extent as active phorbol esters (65-70%). Oxytocin has the same type of effect presumably by acting at the V1-receptor. Both effects of [Arg8]vasopressin and oxytocin are suppressed by [1-(beta-mercapto-beta,beta-diethylpropionic acid)4-valine]arginine vasopressin, a specific vasopressin antagonist at the V1-receptor.     

Protein kinase C-stimulated phosphorylation in vitro of a Mr 80,000 protein phosphorylated in response to phorbol esters and growth factors in intact fibroblasts. Distinction from protein kinase C and prominence in brain

In previous studies in intact 3T3-L1 fibroblasts and adipocytes, we demonstrated that the phosphorylation state of an acidic, multicomponent Mr 80,000 protein appeared to be a specific and useful marker for the activation state of protein kinase C (Blackshear, P.J., Witters, L.A., Girard, P.R., Kuo, J.F., and Quamo, S.N. (1985) J. Biol. Chem. 260, 13304-13315). In the present studies, we demonstrate that the Mr 80,000 protein from rat adipose tissue was a substrate for protein kinase C in vitro, and co-migrated on two-dimensional gels with the analogous protein from murine 3T3-L1 adipocytes labeled by exposure of intact cells to 32Pi and phorbol 12-myristate 13-acetate. Partial proteolytic maps of the two 32P-proteins were nearly identical, supporting the postulate that the sites phosphorylated by protein kinase C in vitro, and in response to phorbol 12-myristate 13-acetate in vivo, were similar or identical. Despite their similar apparent molecular weights, we were able to distinguish between the Mr 80,000 protein and protein kinase C by several physical criteria. The Mr 80,000 protein kinase C substrate was found in fractions of all rat tissues examined, but was most prominent in rat brain. Phorbol 12-myristate 13-acetate also stimulated phosphorylation of the Mr 80,000 protein in several types of cultured neuronal cells, suggesting a possible role for this protein in cholinergic neurotransmission. The Mr 80,000 protein appears to be a useful marker for protein kinase C activation in a variety of cell types.     

Platelet protein phosphorylation and protein kinase C activation by phorbol esters with different biological activity and a novel synergistic response with Ca2+ ionophore

Phorbol esters with different biological activities have been tested for their ability to induce the phosphorylation of human platelet proteins. We have shown that only the potent platelet aggregatory phorbol esters were able to stimulate the phosphorylation of proteins of 76, 68, 47, 30 and 20 kDa in intact platelets. The ability of these esters to stimulate phosphorylation of the 47-kDa protein ('p47') correlated with their ability to cause platelet aggregation. When a non-platelet aggregatory deoxyphorbol (12-deoxyphorbol 13-phenylacetate 20-acetate) was combined with a subthreshold dose of the Ca2+ ionophore, A23187, a large increase in phosphorylation of p47 and a fourfold decrease in Ka was observed. This was in contrast to a barely detectable stimulation of phosphorylation at micromolar levels of this phorbol ester in the absence of the ionophore. This synergism was not evident for the potent platelet aggregatory derivatives. The Ka for DOPPA with a mixture of total platelet protein kinase C was 530 nM in the absence of calcium decreasing to 120 nM in the presence of calcium. In the presence of calcium, 12-deoxyphorbol 13-phenylacetate 20-acetate was shown to stimulate preferentially one of the isoforms of protein kinase C.     

Effect of phorbol esters and diacylglycerols on calcium transport by human platelet membranes

The effects of phorbol esters and diacylglycerols on Ca2+ transport in isolated human platelet membranes were determined. Phorbol 12-myristate 13-acetate (PMA) stimulated Ca2+-ATPase activity in crude and purified internal platelet membranes approximately 2-fold with half-maximal stimulation occurring at 10 nM. Dilauroylglycerol also stimulated Ca2+-ATPase activity half-maximally at a concentration of 7.5 microM, but dioctanoylglycerol was without effect at up to 30 microM. PMA also inhibited Ca2+ uptake when added before or after commencement of ATP-dependent transport. PMA (25 nM) doubled the rate of Ca2+ efflux from passively loaded membranes in the absence of ATP. No protein kinase C activity was detected in crude or purified membranes by histone phosphorylation or endogenous protein phosphorylation assays. These results suggest that PMA and dilauroylglycerol stimulate Ca2+-ATPase activity and inhibit ATP-dependent Ca2+ transport by increasing the permeability of the membranes to Ca2+.     

In vivo and in vitro phosphorylation of murine lymphocyte differentiation antigen CD5

Ly-1, the murine lymphocyte differentiation antigen CD5, is phosphorylated constitutively in vivo. This phosphorylation is enhanced by phorbol 12-myristate 13-acetate (PMA) treatment, but not by concanavalin A, Ca2+ ionophore or dibutyryl cAMP. Prolonged PMA treatment abolished PMA-induced Ly-1 phosphorylation but not constitutive phosphorylation, suggesting that protein kinase C (PKC) is responsible for this enhanced phosphorylation, but not the basal phosphorylation of Ly-1. Ly-1 is phosphorylated by PKC added to membranes, further supporting a role for protein kinase C in the in vivo phosphorylation of Ly-1.     

Effect of phorbol esters on the distribution and total activity of protein kinase C in the perfused rat heart

1. The perfused rat heart was treated with the tumour-promoter and protein kinase C activator, phorbol 12-myristate 13-acetate and the distribution of protein kinase C activity between cytosolic and particulate fractions determined. 2. Phorbol ester treatment led to a rapid loss of protein kinase C activity from the cytosol (t0.5 = 2 min) with a corresponding translocation into the particulate fraction. Translocated protein kinase C activity was tightly bound to the particulate fraction, could only be extracted with buffers containing 2% Triton X-100 and could therefore be misinterpreted as being down-regulated. 3. Claims of rapid down-regulation of protein kinase C activity by phorbol esters need to be supported by rigorous procedures for extraction of the particulate material.     

Na+-dependent elevation of the acidic cell surface pH (microclimate pH) of rat jejunal villus cells induced by cyclic nucleotides and phorbol ester: possible mediators of the regulation of the Na+/H+ antiporter

The effects of cyclic nucleotides and phorbol ester on the acidic cell surface pH of rat jejunal villi were studied by using single-barrelled pH-sensitive microelectrodes. Addition of dibutyryl cAMP (1 mM) to the mucosal bathing solution caused an elevation of the cell surface pH from 6.19 +/- 0.04 (n = 12 measurements from three animals) to 6.53 +/- 0.03 (12) in the presence of Na+ in the medium. However, dibutyryl cAMP had no significant effect in the absence of Na+ and presence of 1 mM amiloride. Dibutyryl cGMP (1 mM) also had an Na+-dependent inhibitory effect on the cell surface pH. A phorbol ester, phorbol 12-myristate 13-acetate, caused an elevation of the cell surface pH only in the presence of Na+ from 6.14 +/- 0.07 (12) to 6.46 +/- 0.08 (12). Phorbol and phorbol 13-acetate, which do not stimulate protein kinase C, were without significant effects. These results suggest that increased levels of the intracellular cyclic nucleotides and activation of protein kinase C raise the acidic cell surface pH by inhibiting the activity of the brush-border Na+/H+ antiporter in the rat jejunal villus cells.     

1,2-Diacylglycerols overcome cyclic AMP-mediated inhibition of phosphatidylcholine synthesis in GH3 pituitary cells.

Previous studies showed that phorbol esters and thyrotropin-releasing hormone (TRH) stimulated phosphatidylcholine synthesis via protein kinase C in GH3 pituitary cells [Kolesnick (1987) J. Biol. Chem. 262, 14525-14530]. In contrast, 1,2-diacylglycerol-stimulated phosphatidylcholine synthesis appeared independent of protein kinase C. The present studies compare phosphatidylcholine synthesis stimulated by these agents with inhibition via the cyclic AMP system. The potent phorbol ester phorbol 12-myristate 13-acetate (PMA, 10 nM) increased [32P]Pi incorporation into phosphatidylcholine at 30 min to 159 +/- 6% of control. The adenylate cyclase activator cholera toxin (CT; 10 nM) and the cyclic AMP analogue dibutyryl cyclic AMP (1 mM) abolished this effect. CT similarly abolished TRH-induced phosphatidylcholine, but not phosphatidylinositol, synthesis. This is the first report of inhibiton of receptor-mediated phosphatidylcholine synthesis by the cyclic AMP system. The 1,2-diacylglycerol 1,2-dioctanoylglycerol (diC8) also stimulated concentration-dependent phosphatidylcholine synthesis. DiC8 (3 micrograms/ml) induced an effect quantitatively similar to that of maximal concentrations of PMA and TRH, whereas a maximal diC8 concentration (30 micrograms/ml) stimulated an effect 3-4-fold greater than these other agents. CT decreased the effect of diC8 (3 micrograms/ml) by 80%. Higher diC8 concentrations overcame the CT inhibition. Similar results were obtained with dibutyryl cyclic AMP. Additional differences were found between low and high concentrations of diC8. Low concentrations of diC8 failed to induce additive phosphatidylcholine synthesis with maximal concentrations of PMA, whereas high concentrations were additive. Hence, low concentrations of 1,2-diacylglycerols appear to be regulated similarly to phorbol esters, and higher concentrations appear to act via a pathway unavailable to phorbol esters.     

Reversibility of phorbol diester-induced macrophage spreading

Phorbol 12-myristate 13-acetate and phorbol 12, 13-dibutyrate induced spreading of mouse macrophages with 50% effective concentrations of 3 nM and 35 nM, respectively. Macrophages treated with 100 or 1000 nM phorbol 12, 13- dibutyrate showed a time related decrease in spreading after washout. Spreading induced by 1, 10, or 100 nM phorbol 12-myristate 13-acetate was irreversible; however, washed phorbol 12,13-dibutyrate-treated cells respread after a second exposure to this compound. Washout of ³[H]phorbol diesters corroborated these observations in that 5% of ³H-phorbol 12-myristate 13-acetate and only 0.1% ³[H]phorbol, 12,13-dibutyrate remained associated with washed cells. Since phorbol 12-myristate 13 acetate is much more lipophilic than phorbol 12,13-dibutyrate, the reversibility of phorbol diester-induced macrophage spreading may depend upon the lipophilicity of the derivative utilized.Abbreviations DMEM Dulbecco's Minimal Essential Medium - PDA phorbol 12,13-diacetate - PDBu phorbol 12, 13-dibutyrate - PMA phorbol 12 myristate, 13 acetate - 4PDD phorbol 12, 13 didecanoate     

Phorbol esters inhibit inositol phosphate and diacylglycerol formation in proliferating HL60 cells. Relationship to differentiation

Phorbol 12-myristate 13-acetate (PMA) induces the differentiation of the human promyelocytic cell line, HL60, towards adherent macrophage-like cells within 2 days. We have examined the early effects of PMA on inositol phosphates and on diacylglycerol production, two second messengers derived from inositol lipids. In proliferating HL60 cells, PMA induced a time- and concentration-dependent decrease in inositol phosphate levels. Maximal effects were seen after 1 h at 10 nM PMA. PMA also induced the translocation of protein kinase C from the cytosol to the membrane. Comparison between the differentiating effects of several phorbol esters and of 1-oleoyl-2-acetylglycerol with their ability to inhibit inositol phosphate formation suggests that the two effects are correlated.     

Pertussis toxin and H-7 distinguish mechanisms involved in eicosanoid release from lipopolysaccharide-primed macrophages. Eicosanoid release from lipopolysaccharide-primed macrophages

Release of eicosanoids is an important response of macrophages to inflammation and bacterial infection. At low concentrations, bacterial lipopolysaccharide (1-2 micrograms/ml) fails to stimulate eicosanoid release in resident peritoneal macrophages but primes the macrophages for a greatly enhanced release of eicosanoids on stimulation with the calcium ionophore A23187 (0.1 microM) or with phorbol 12-myristate 13-acetate (50 nM), an activator of protein kinase C. Incubation of macrophages with Bordetella pertussis toxin, prior to priming with lipopolysaccharide, inhibited the release of both cyclooxygenase and lipoxygenase products upon A23187 stimulation. Pertussis toxin treatment of macrophages had no effect on eicosanoid release when the stimulus was phorbol 12-myristate 13-acetate. The presence of 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), an effective inhibitor of protein kinase C, during lipopolysaccharide priming and subsequent stimulation significantly inhibited eicosanoid release when phorbol 12-myristate 13-acetate was the stimulus, but did not affect eicosanoid release stimulated by A23187. Based on these results, at least two mechanisms, distinguished by apparent differences in sensitivity to pertussis-toxin-sensitive, guanine-nucleotide-binding proteins and protein kinase C, are involved in eicosanoid secretion by lipopolysaccharide-activated macrophages in response to A23187 and phorbol 12-myristate 13-acetate.     

Rapid modulation of tumor necrosis factor membrane receptors by activators of protein kinase C

Tumor necrosis factor membrane receptors are rapidly down-regulated upon treatment of activated T lymphocytes with various activators of protein kinase C. Loss of binding-capacity was half maximal after 2 min. incubation in 10 ng/ml of phorbol 12-myristate 13-acetate. A similar modulation could be induced with either the calcium ionophore A 23187 or the protein kinase C activator 1-oleyl-2-acetyl glycerol, whereas 1,2-diolein and dibutyryl cAMP were ineffective. Protein kinase C inhibitor H7 antagonizes the phorbol ester-induced TNF receptor modulation. These data suggest an important role of protein kinase C in the control of TNF responsiveness by regulation of TNF binding-capacity possibly via direct phosphorylation of specific receptor proteins.     

Effects of phorbol ester on tyrosine hydroxylase phosphorylation and activation in cultured bovine adrenal chromaffin cells

The phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) caused phosphorylation of phosphoproteins of 56-kDa which co-migrated with and had identical pI values to subunits of tyrosine hydroxylase. The phosphorylation was closely correlated with an increase of [3H]3,4-dihydroxyphenylalanine (DOPA) production which is a reflection of increased tyrosine hydroxylase activity. Only those phorbol esters which activate protein kinase C induced phosphorylation of the 56-kDa proteins and increased [3H]DOPA production. Neither TPA-induced phosphorylation of the 56-kDa proteins nor TPA-induced enhancement of [3H] DOPA production required extracellular Ca2+. TPA caused increases in phosphorylation of the 56-kDa proteins and increases in [3H]DOPA production over similar concentration ranges (10-1000 nM). TPA did not increase cellular cAMP. The data suggest that phorbol ester-induced phosphorylation of intracellular tyrosine hydroxylase, possibly by protein kinase C, results in increased tyrosine hydroxylase activity.     

Temporal integration of alpha 1-adrenergic responses in BC3H-1 muscle cells. Regulation of glycogen phosphorylase activity

Regulation of Ca2+-dependent glycogen phosphorylase activity by alpha 1-adrenergic and H1-histamine receptors has been examined in BC3H-1 muscle cells. Stimulation by either norepinephrine or histamine elevates the phosphorylase activity ratio within 5 s from a resting value of 0.37 +/- 0.03 to maximal values of 0.8-0.9. Phosphorylase activation by alpha-adrenergic agonists is sustained over 20-30 min of agonist exposure, whereas histamine exposure only transiently activates phosphorylase during the initial 5 min of stimulation. The initial activation of phosphorylase by either receptor is not attenuated by treated cells with Ca2+-deficient and [ethylenebis(oxyethylenenitrilo)]tetraacetic acid-supplemented buffer, whereas the response to sustained adrenergic stimulation depends largely, but not totally, upon extracellular Ca2+. The involvement of protein kinase C in agonist responses was tested by treating cells with phorbol 12-myristate 13-acetate. Phorbol 12-myristate 13-acetate inhibits receptor-mediated mobilization of intracellular Ca2+ (IC50 = 3.6 nM) yet activates phosphorylase independently of agonist. Phorbol 12-myristate 13-acetate has no effect on cellular 45Ca2+ fluxes in the absence of agonist. Thus, the two receptors coordinately regulate intracellular signaling through Ca2+- and protein kinase C-mediated pathways. alpha 1-Adrenergic receptors elicit sustained phosphorylase activation whereas H1-histaminergic receptors desensitize.     

The lipophilicity of phorbol esters as a critical factor in determining the pattern of translocation of protein kinase C delta fused to green fluorescent protein

Our previous study showed differential subcellular localization of protein kinase C (PKC) delta by phorbol esters and related ligands, using a green fluorescent protein-tagged construct in living cells. Here we compared the abilities of a series of symmetrically substituted phorbol 12,13-diesters to translocate PKC delta. In vitro, the derivatives bound to PKC with similar potencies but differed in rate of equilibration. In vivo, the phorbol diesters with short, intermediate, and long chain fatty acids induced distinct patterns of translocation. Phorbol 12,13-dioctanoate and phorbol 12,13-nonanoate, the intermediate derivatives and most potent tumor promoters, showed patterns of translocation typical of phorbol 12-myristate 13-acetate, with plasma membrane and subsequent nuclear membrane translocation. The more hydrophilic compounds (phorbol 12,13-dibutyrate and phorbol 12,13-dihexanoate) induced a patchy distribution in the cytoplasm, more prominent nuclear membrane translocation, and little plasma membrane localization at all concentrations examined (100 nM to 10 microM). The highly lipophilic derivatives, phorbol 12,13-didecanoate and phorbol 12, 13-diundecanoate, at 1 microM caused either plasma membrane translocation only or no translocation at incubation times up to 60 min. Our results indicate that lipophilicity of phorbol esters is a critical factor contributing to differential PKC delta localization and thereby potentially to their different biological activities.     

Growth factor-stimulated protein phosphorylation in 3T3-L1 cells. Evidence for protein kinase C-dependent and -independent pathways

Exposure of serum-deprived 3T3-L1 fibroblasts to phorbol 12-myristate 13-acetate (PMA), synthetic diacylglycerols, platelet-derived growth factor (PDGF), or pituitary fibroblast growth factor (FGF) resulted in stimulated phosphorylation of an acidic, multicomponent, soluble protein of Mr 80,000. Phosphorylation of this protein was promoted to a lesser extent by epidermal growth factor; however, neither insulin nor dibutyryl cAMP was effective. Phosphoamino acid analysis and peptide mapping of the Mr 80,000 32P-protein after exposure of fibroblasts to PDGF revealed identical patterns to those obtained with PMA or diacylglycerols. In contrast to the Mr 80,000 protein, proteins of Mr 22,000 (and pI 4.4) and Mr 31,000 were also phosphorylated in response to insulin as well as to PMA, diacylglycerols, epidermal growth factor, PDGF, and FGF in these cells. Similar findings were noted in fully differentiated 3T3-L1 adipocytes. Preincubation of the cells with high concentrations of active phorbol esters abolished specific [3H]phorbol 12,13-dibutyrate binding, protein kinase C activity, and immunoreactivity and also prevented stimulated phosphorylation of the Mr 80,000 protein by PMA, diacylglycerols, PDGF, or FGF, supporting the contention that this effect was mediated through protein kinase C. The stimulated phosphorylation of the Mr 22,000 and 31,000 proteins in response to PMA was also abolished by such pretreatment. In contrast, the ability of insulin, PDGF, and FGF to promote phosphorylation of the Mr 22,000 and 31,000 proteins was unaffected in the protein kinase C-deficient cells. We conclude that PDGF and FGF may exert some of their effects on these cells through at least two distinct pathways of protein phosphorylation, phorbol ester-like (P) activation of protein kinase C, and an insulin-like (I) pathway exemplified by phosphorylation of the Mr 22,000 and 31,000 proteins.