Protein kinase C of human erythrocytes phosphorylates bands 4.1 and 4.9

Addition of 10 nM 12-O-tetradecanoylphorbol 13-acetate (TPA) to intact human erythrocytes results in rapid phosphorylation of two cytoskeletal components, bands 4.1 and 4.9. The synthetic diacylglycerol, 1-oleoyl-2-acetylglycerol, shows a similar effect, while the biologically inactive phorbol ester, 4 alpha-phorbol didecanoate, fails to enhance phosphorylation. That TPA and 1-oleoyl-2-acetylglycerol stimulate this phosphorylation suggests that protein kinase C is being activated. In the presence of TPA, bands 4.1 and 4.9 incorporate 1.5 mol Pi/mol protein and 1.2 mol Pi/mol protein, respectively. The pattern and extent of phosphorylation shows that it is not due to cAMP-dependent protein kinases, which also phosphorylate bands 4.1 and 4.9. Ca2+-phospholipid-dependent protein kinase activity is demonstrable in the soluble fraction of erythrocytes, and has been partially purified (2200-fold) from the hemolysate by affinity chromatography (Uchida and Filburn, 1984. J. Biol. Chem. 259, 12311-12314). The affinity purified erythrocyte kinase has a 42 A Stokes' radius and phosphorylates purified bands 4.1 and 4.9 in vitro in a Ca2+- and phospholipid-dependent manner. These results show that human erythrocytes contain protein kinase C, and that band 4.1 and 4.9 are the major endogenous substrates for this kinase.     

Protein kinase C phosphorylates a recently identified membrane skeleton-associated calmodulin-binding protein in human erythrocytes

A membrane skeleton-associated protein with calmodulin-binding activity recently has been purified and characterized from human erythrocytes (Gardner, K. and Bennett, V. (1986) J. Biol. Chem. 261, 1339-1348). This new protein (CaM-BP103/97) has now been identified as a major substrate for protein kinase C in erythrocytes since phosphorylation of both of its subunits (Mr = 103,000 and 97,000) is elevated 3-15-fold in the presence of the phorbol ester, 12-O-tetradecanoylphorbol beta-acetate (TPA), under the following conditions: ghost membranes incubated with protein kinase C purified from rat brain, ghost membranes from erythrocytes pretreated with TPA, and intact erythrocytes metabolically labeled with 32PO4 and stimulated by TPA. The sites of phosphorylation of this protein by exogenous and endogenous protein kinase C are identical since two-dimensional 32P-peptide maps of both subunits labeled by either endogenous or exogenous enzyme are indistinguishable. Each subunit of CaM-BP103/97 accepts up to 3 mol of phosphate/polypeptide chain. In the presence of low calcium concentrations and in the absence of cytosol, the phosphorylation of CaM-BP103/97 is, on a molar basis, equal to or greater than that of proteins 4.1 and 4.9. As a target for both calmodulin and protein kinase C, CaM-BP103/97 is likely to play a key role in the effect of calcium on erythrocyte membrane shape and stability.     

Differential phosphorylation of multiple sites in protein 4.1 and protein 4.9 by phorbol ester-activated and cyclic AMP-dependent protein kinases

The phosphorylation of the membrane skeleton components protein 4.1 and protein 4.9 in intact erythrocytes is shown to increase in the presence of either 1 microM 12-O-tetradecanoyl phorbol 13-acetate or 2 mM dibutyryl cAMP. The phosphorylation induced by these protein kinase activators is compared by two-dimensional tryptic peptide mapping. In both proteins, the pattern of peptides phosphorylated in the presence of 12-O-tetradecanoyl phorbol 13-acetate differs from the pattern of peptides phosphorylated in the presence of dibutyryl cAMP. The relative locations of the phosphorylated sites on protein 4.1 have been determined using limited proteolysis by alpha-chymotrypsin.     

The second messenger pathway for germ cell-mediated stimulation of Sertoli cells.

Treatment of cultured rat Sertoli cells with FSH or dibutyryl cAMP for 30 min resulted in phosphorylation of the same Sertoli cell proteins. Different Sertoli cell proteins were phosphorylated after calcium ionophore A23187 and 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment. A23187 stimulated the phosphorylation of hsp27, while TPA alone had no effect. TPA plus A23187 resulted in phosphorylation of a 14 kDa protein, in addition to hsp27. The effect of TPA plus A23187 was identical to that of germ cells on Sertoli cell protein phosphorylation. FSH-stimulated cAMP production by Sertoli cells was reduced by prior exposure of Sertoli cells to germ cells. The results indicate that germ cells stimulate Sertoli cells by the inositol trisphosphate/diacylglycerol mediated second messenger pathway. The results also suggest that the germ cell-activated pathway interacts within Sertoli cells to modulate Sertoli cell response to FSH.     

Cyclic AMP-like effects of polyamines on phosphatidylcholine synthesis and protein phosphorylation in human promyelocytic leukemia HL60 cells. Comparison with the effects of phorbol ester

Spermine or putrescine increased cAMP levels through a catalase-sensitive mechanism, resulting in, most notably, a dephosphorylation of protein A (Mr 45,000, pI 5.15) and protein B (Mr 45,000, pI 4.9) and slightly increased phosphatidylcholine (PC) synthesis in HL60 cells. Exogenous dibutyryl cAMP mimicked the polyamine effects. 12-O-Tetradecanoyl phorbol-13-acetate (TPA) also promoted the protein dephosphorylation and PC synthesis, the effects augmented by R59022 and mimicked by exogenous 1-oleoyl-2-acetylglycerol. The effects of spermine (or dibutyryl cAMP) and TPA on PC synthesis were synergistic. It was suggested that cAMP-dependent protein kinase and protein kinase C might mediate, in an independent but inter-related manner, the effects of polyamines and TPA.     

Nerve Growth Factor-Induced Transient Increase in the Phosphorylation of Ribosomal Protein S6 Mediated Through a Mechanism Independent of Cyclic AMP-Dependent Protein Kinase and Protein Kinase C

Treatment of PC12h cells with nerve growth factor (NGF) induced a transient increase in the phosphorylation of a 35,000-dalton protein. This transient increase was observed also when extracts of NGF-treated cells were incubated with [gamma-32P]ATP. In the intact-cell phosphorylation system, treatment with N,2'-dibutyryladenosine 3',5'-cyclic monophosphate (dBcAMP) or 12-O-tetradecanoylphorbol 13-acetate (TPA) also induced a transient increase in the phosphorylation of the 35,000-dalton protein, but the effect was less than that of NGF. An effect comparable to that of NGF was obtained by the combination of dBcAMP and TPA. Pretreatment of PC12h cells with dBcAMP plus TPA for 3 days, which deprived the cells of their ability to respond to a rechallenge with dBcAMP, TPA, or dBcAMP plus TPA by increasing the rate of 35,000-dalton protein phosphorylation, caused only a slight attenuation of the NGF effect, directly indicating a minimal role of cyclic AMP (cAMP)-dependent protein kinase and protein kinase C in the mechanism of the NGF action. Pretreatment of the cells with K-252a, a protein kinase inhibitor, at a concentration of 300 nM almost completely blocked the action of NGF, but scarcely affected the action of dBcAMP, TPA, or dBcAMP plus TPA in intact-cell phosphorylation experiments. This NGF-sensitive 35,000-dalton protein was a ribosomal protein and identified as ribosomal protein S6. The results lead us to conclude that NGF activates some NGF-sensitive component(s), probably some specific protein kinase(s) other than cAMP-dependent protein kinase or protein kinase C, which is suppressed by K-252a and directly or indirectly activates a 35,000-dalton protein kinase(s) [S6 kinase(s)] to increase the rate of phosphorylation of the 35,000-dalton ribosomal protein (S6).     

Muscarinic receptor-linked elevation of cAMP in SH-SY5Y neuroblastoma cells is mediated by Ca2+ and protein kinase C.

The mechanisms of muscarinic receptor-linked increase in cAMP accumulation in SH-SY5Y human neuroblastoma cells has been investigated. The dose-response relations of carbachol-induced cAMP synthesis and carbachol-induced rise in intracellular free Ca2+ were similar. The stimulated cAMP synthesis was inhibited by about 50% when cells were entrapped with the Ca2+ chelator BAPTA or in the presence of the protein kinase C (PKC) inhibitor staurosporine. Production of cAMP could be induced also by the Ca2+ ionophore, ionomycin and by TPA, an activator of PKC. When added together TPA and ionomycin had a synergistic effect. When cAMP synthesis was activated with cholera toxin, PGE1 or PGE1 + pertussis toxin carbachol stimulated cAMP production to the same extent as in control cells. Ca2+ and protein kinase C thus seem to be the mediators of muscarinic-receptor linked cAMP synthesis by a direct action on adenylate cyclase.     

Stimulation of polyphosphoinositide turnover upon activation of protein kinases in human erythrocytes

Activation of protein kinase C in erythrocytes by 4-beta-phorbol 12-myristate 13-acetate (PMA) resulted in a parallel stimulation (time course and dose response) of the phosphorylation of both membrane proteins (heterodimers of 107 kDa and 97 kDa, protein 4.1 and 4.9, respectively) and of phosphatidylinositol 4-phosphate (PIP) and, to a lesser extent, of phosphatidylinositol 4,5-bisphosphate (PIP2). Evidence that the effect on lipid was mediated by protein kinase C activation and not by a direct action of PMA was provided by (1) the lack of effect of a phorbol ester that did not activate protein kinase C or of PMA addition on isolated membranes from control erythrocytes, (2) the reversal of the effect in the presence of protein kinase C inhibitors (alpha-cobrotoxin, H-7 (1-(5-isoquinolinesulfonyl)-2-methylpiperazine) or trifluoperazine). PMA treatment did not change the specific activity of ATP or the content of PIP2, but increased the content of PIP and decreased that of PI, indicating that the phosphorylation or dephosphorylation reactions linking PI and PIP were the target for the action of PMA. PMA treatment had no effect on the Ca2+-dependent PIP/PIP2 phospholipase C activity measured in isolated membranes. Mezerein, another protein kinase activator, had similar effects on both protein and lipid phosphorylation, when added with alpha-cobrotoxin. Activation of protein kinase A by cAMP also produced increases in phosphorylation, although quantitatively different from those induced by protein kinase C, in proteins and PIP. Simultaneous addition of PMA and cAMP at maximal doses resulted in only a partially additive effect on PIP labelling. These results show that inositol lipid turnover can be modulated by a protein kinase C and protein kinase A-dependent process involving the phosphorylation of a common protein. This could be PI kinase or PIP phosphatase or another protein regulating the activity of these enzymes.     

Effects of protein kinase C activators on germinal vesicle breakdown and polar body emission of mouse oocytes

Protein phosphorylation mediated by cAMP-dependent protein kinase is instrumental in maintaining meiotic arrest of mouse oocytes. To assess whether protein phosphorylation mediated by calcium/phospholipid-dependent protein kinase (protein kinase C) might also inhibit the resumption of meiosis, we treated oocytes with activators of this enzyme. The active phorbol esters 12-O-tetra-decanoyl phorbol-13-acetate (TPA) and 4 beta-phorbol 12,13-didecanoate (4 beta-PDD) inhibited germinal vesicle breakdown (GVBD), as did a more natural activator of protein kinase, C, sn-1,2-dioctanoylglycerol (diC8). An inactive phorbol ester, 4 alpha-phorbol 12,13-didecanoate (4 alpha-PDD), did not inhibit GVBD. We then examined whether protein kinase C activators inhibit a step in the cAMP-modulated pathway that regulates resumption of meiosis. TPA did not inhibit the maturation-associated decrease in oocyte cAMP. Microinjected heat-stable protein inhibitor of cAMP-dependent protein kinase failed to induce GVBD in the presence of TPA. Both TPA and diC8 partially inhibited specific changes in oocyte phosphoprotein metabolism that are tightly correlated with resumption of meiosis; these agents also induced the apparent phosphorylation of specific oocyte proteins. These results suggest that protein kinase C activators may inhibit resumption of meiosis by acting distal to a decrease in cAMP-dependent protein kinase activity, but prior to changes in oocyte phosphoprotein metabolism that are presumably required for resumption of meiosis. Finally, we compared the effects of db-cAMP and protein kinase C activators on polar body emission following GVBD. TPA, 4 beta-PDD or diC8, but not 4 alpha-PDD or db-cAMP, inhibited polar body emission in a dose-dependent manner. The morphology and cytology of oocytes in which polar body emission was inhibited by TPA or 4 beta-PDD differed from that of oocytes treated with diC8. Thirty to 60% of the former were round in shape and exhibited a clump of chromosomes but no spindle; the remainder were distended in shape and exhibited a metaphase I spindle. All oocytes treated with diC8, however, were round, had dispersed chromosomes, and no spindle. These results suggest that, in contrast to resumption of meiosis, polar body emission is inhibited by activation of protein kinase C but not cAMP-dependent protein kinase.     

Phorbol ester stimulation of insulin release and secretory-granule protein phosphorylation in a transplantable rat insulinoma.

The effects of tumour-promoting phorbol esters on protein-phosphorylation reactions and secretion in rat insulinoma tissue were investigated with the objective of assessing the possible role of Ca2+- and phospholipid-dependent protein kinases (protein kinase C) in insulin release. 4 beta-Phorbol 12-myristate 13-acetate (TPA) was a potent secretagogue at concentrations above 0.1 microM. TPA-induced release was inhibited by adrenaline or omission of Ca2+ from the extracellular medium and was augmented by theophylline. These findings suggested that TPA activated an exocytotic process. TPA enhanced the Ca2+- and phospholipid-dependent phosphorylation of histone III-S by a soluble protein fraction of the tissue. Endogenous phosphorylation reactions involving soluble and secretory-granule membrane proteins were also stimulated by TPA in tissue homogenates and reconstituted subcellular fractions. Histone phosphorylation and the granule-protein phosphorylation reactions showed similar concentration-dependencies for activation by both Ca2+ and TPA, thus indicating that the same enzyme was involved. It is concluded that the phosphorylation of cytosolic and membrane protein substrates by protein kinase C may be important in the stimulus-secretion coupling mechanism of insulin release.     

Effects of phorbol esters on cytoskeletal proteins in cultured bovine chromaffin cells: induction of neurofilament phosphorylation and reorganization of actin

Bovine chromaffin cells normally express mostly nonphosphorylated neurofilaments (NFs) in primary culture, and thus provide a unique model for examining the kinase capable of phosphorylating these proteins in situ. The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) which activates protein kinase C induced NF phosphorylation both in the perikaryon and in neuritic extensions of neurite-bearing cells as judged by immunofluorescence using monoclonal anti-NF antibodies which distinguish between phosphorylated and nonphosphorylated epitopes. NF phosphorylation was suppressed by pretreating the cells with sphingosine, an inhibitor of protein kinase C, and was not observed in the presence of the phorbol ester. 4 alpha-phorbol-12,13-didecanoate (PDD) which does not activate protein kinase C, arguing that protein kinase C was responsible for the observed phosphorylation. Immunochemical analysis of cytoskeletal extracts indicated that TPA induced a 3 to 6-fold increase in NF phosphorylation and showed that the 150,000 dalton NF subunit was the principal protein kinase C substrate. In addition to the TPA effect on NF phosphorylation, TPA provoked a reversible membrane ruffling, which eventually resulted in a flattening of chromaffin cells. These morphological alterations were linked with actin patching and the development of stress fibers, respectively. Sphingosine blocked the TPA-induced membrane ruffling and actin patching, and these phenomena were correlated with increased protein kinase C activity. In contrast, there was no change in the localization of microtubules and NFs. The actin reorganization and NF phosphorylation induced by TPA suggest that at least two distinct proteins of the neuronal cytoskeleton are susceptible to the influence of protein kinase C activation. It remains to be established whether protein kinase C plays a role in the regulatory mechanism controlling actin organization and neurofilament phosphorylation during neuronal differentiation.     

Phorbol ester inhibits phosphatidylserine synthesis in human promyelocytic leukaemia HL60 cells. Possible involvement of free radicals and correlation with phosphorylation of nuclear protein 1b.

Treatment of human promyelocytic leukaemia HL60 cells in conditioned medium with 12-O-tetradecanoylphorbol 13-acetate (TPA) for 4 h resulted in 25-30% inhibition of labelling of phosphatidylserine (PS) with [U-14C]serine. PS labelling was 40% lower, and no inhibitory TPA effect was observed when the experiments were performed in fresh medium. Cycloheximide or puromycin also inhibited PS labelling by 38-44%; their inhibitory effects were non-additive with that of TPA and occurred only in conditioned medium. Catalase (CAT) and superoxide dismutase (SOD), both free-radical scavengers, and H7, a protein kinase C inhibitor, reversed to various extents the inhibitory effect of TPA on PS synthesis. On the other hand, chlorobenzoic acid, a free-radical-generating agent, also inhibited PS synthesis by 22% after 4 h treatment when conditioned medium was used. When ethanolamine was added to cells in conditioned medium to quench PS formation through the exchange of free serine with the ethanolamine moiety of phosphatidylethanolamine (PE), PS labelling was decreased by 33% and the inhibitory TPA effect was significantly decreased. On the other hand, ethanolamine had marginal quenching effect on PS labelling when added to cells in fresh medium. TPA increased the phosphorylation of various proteins in the cells, including protein lb (Mr 80,000; pI 5.5) shown to be localized mainly in the nuclear fraction. Chlorobenzoic acid selectively stimulated the phosphorylation of protein lb, whereas CAT and SOD specifically attenuated the TPA-stimulated phosphorylation of this protein. All these agents affected phosphorylation of protein lb only if conditioned medium was used. The findings suggested that net synthesis of PS through the base-exchange mechanism was stimulated in HL60 cells by cell products present in the conditioned medium. TPA inhibited this stimulated PS synthesis by a mechanism which appeared to involve active oxygen species and protein synthesis and might be related to the phosphorylation of protein lb.     

The cAMP responsive element and CREB partially mediate the response of the tyrosine hydroxylase gene to phorbol ester

Tyrosine hydroxylase (TH) gene promoter activity is increased in PC12 cells that are treated with the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA). Mutagenesis of either the cAMP responsive element (CRE) or the activator protein-1 element (AP1) within the TH gene proximal promoter leads to a dramatic inhibition of the TPA response. The TH CRE and TH AP1 sites are also independently responsive to TPA in minimal promoter constructs. TPA treatment results in phosphorylation of cAMP responsive element binding protein (CREB) and activation of cAMP-dependent protein kinase (PKA) in PC12 cells; hence, we tested whether CREB and/or PKA are essential for the TPA response. In CREB-deficient cells, the response of the full TH gene proximal promoter or the independent response of the TH CRE by itself to TPA is inhibited. The TPA-inducibility of TH mRNA is also blocked in CREB-deficient cells. Expression of the PKA inhibitor protein, PKI, also inhibits the independent response of the TH CRE to TPA. Our results support the hypothesis that TPA stimulates the TH gene promoter via signaling pathways that activate either the TH AP1 or TH CRE sites. Both signaling pathways are dependent on CREB and the TH CRE-mediated pathway is dependent on PKA.     

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.     

Topography of protein kinase C substrates analyzed by membrane splitting

We have used the methods of planar cell and membrane monolayer formation and monolayer splitting to study structural details of the transmembrane signaling process mediated by protein kinase C. We analyzed human red cell membrane proteins phosphorylated by phorbol ester activation of protein kinase C. Planar single membrane preparations, extraction procedures, and gel electrophoresis coupled with silver staining and autoradiography confirmed that two bands in the 100 kDa region, and bands 4.1, and 4.9, were peripheral and phosphorylated by treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA). TPA also stimulated minor incorporation of [32 P]Pi into most integral membrane proteins, including band 3, glycophorin A, the band 4.5 region (glucose transporter) and band 7. Planar cell and membrane-splitting methods revealed that neither integral nor peripheral phosphorylated polypeptides were cleaved by freeze fracture, that all phosphorylated peripheral proteins partitioned intact with the cytoplasmic side of the membrane, and that the percentages of [32P]Pi-labeled peripheral proteins were the same in split membrane cytoplasmic leaflets as in intact membranes. As a unique approach to examining protein topographies membrane splitting provides strong evidence that the major phosphorylated products of the polyphosphatidylinositide pathway are topographically associated with the cytoplasmic leaflet of the human erythrocyte plasma membrane. We further conclude that TPA-induced phosphorylation of red cell peripheral proteins does not significantly alter their transbilayer partitioning patterns after membrane splitting.     

Ribosomal protein S6 phosphorylation and morphological changes in response to the tumour promoter 12-O-tetradecanoylphorbol 13-acetate in primary human tumour cells, established and transformed cell lines

The phosphorylation of ribosomal protein S6 in fibroblasts, primary human tumour cells, established and SV40-transformed human cell lines was compared after the addition of 12-O-tetradecanoylphorbol 13-acetate (TPA). In fibroblasts and primary tumour cell cultures, stimulation of S6 phosphorylation was about 4-6-fold. Established and transformed cell lines showed enhanced S6 phosphorylation which was not further stimulated by the addition of TPA. These findings indicated that the influence of TPA on the metabolic pathway, that finally leads to the phosphorylation of protein S6 in cells with a limited lifespan (fibroblasts, primary human tumour cells) can be mimicked by unknown steps also associated with immortalization (establishment function) and the transformed state of the tumour cells. Another interesting observation were morphological changes of the established and SV40-transformed cells which were visible as early as 20 min after the addition of TPA. In fibroblasts and primary tumour cells no changes in morphology were observed, even after 8h incubation.     

Early changes in phosphoprotein patterns of Friend erythroleukaemia cells induced by dimethylsulphoxide or phorbol esters

Immediate changes in protein phosphorylation in Friend erythroleukaemia cells (FELC) were examined in response to stimulation with dimethylsulphoxide (DMSO), which triggers cell differentiation, 12-O-tetra-decanoyl-phorbol 13-acetate (TPA), a tumour promoter, and the Ca++ ionophore A23187. The effects of the cyclic nucleotides, cAMP and cGMP, on the patterns of phosphoproteins were also analysed. Autoradiographs of two-dimensional gels reveal that within 30 min of treatment by the various agents differences in the patterns of protein phosphorylation can be seen. Treatment with DMSO and TPA altered the phosphoprotein patterns in different ways. These patterns were distinct from those observed in untreated and cyclic nucleotide treated cells. Treatment with the calcium ionophore caused a unique phosphorylation pattern unlike any of the others. These data suggest that the very early changes seen in the patterns of protein phosphorylation in FELC may be related to the induction of differentiation.     

Protein kinase C activity in erythrocytes in primary hypertension: regulation of cell shape and cation transport

Protein kinase C activity in the lysate of erythrocytes of patients with essential hypertension (EH) and spontaneously hypertensive rats (SHR) was found to be increased by 1.6-2.0 times as compared with normotensive controls. Membrane cytoskeleton alterations observed in the erythrocytes of patients with EH and SHR were revealed in decreased average erythrocyte volume, increase of cup-shaped cell formation, and increase of basal phosphorylation of band 4.9 protein. In addition, the rate of Na(+)-H+ exchange in erythrocytes of EH patients and SHR was increased by 1.9-fold. In vitro treatment of erythrocytes of healthy donors and Wistar-Kyoto rats (WKY) with protein kinase C activator (12-O-tetradecanoylphorbol-13-acetate) leads to similar changes of cell shape, cell volume, band 4.9 protein phosphorylation and Na(+)-H+ exchange, as well as to an increase of diS-C3-(5) fluorescence. It may be assumed that alterations of these parameters revealed in primary hypertension are caused by increased activity of protein kinase C.     

Mitogenic, melanogenic, and cAMP responses of cultured neonatal human melanocytes to commonly used mitogens.

The following studies have been undertaken to compare and correlate the effects of 12-O-tetradecanoylphorbol acetate (TPA), basic fibroblast growth factor (bFGF), cholera toxin (CT), and isobutyl methylxanthine (IBMX) on neonatal human melanocyte (NHM) proliferation, tyrosinase activity, and cyclic adenosine monophosphate (cAMP) concentration. NHM proliferated at a maximal rate in medium containing 8 nM TPA, 200 ng/ml CT, and 10(-4) M IBMX. TPA alone did not result in optimal melanocyte proliferation, and, as previously shown, its mitogenic effect was greatly enhanced by the addition of CT and IBMX individually or concomitantly. Human recombinant (hr) bFGF could replace TPA in the NHM growth medium. Maximal proliferation was achieved using 3 ng/ml hrbFGF, 20 ng/ml CT, and 10(-4) M IBMX. The mitogenic effect of 1.2 ng/ml hrbFGF was potentiated in the concomitant but not individual presence of CT and IBMX. TPA alone in the absence of CT and IBMX caused a dose-dependent stimulation of tyrosinase activity. Maximal tyrosinase activity was obtained in the presence of 0.8 nM TPA, 20 ng/ml CT, and 10(-4) M IBMX. Unlike TPA, hrbFGF alone resulted in inhibition of tyrosinase activity. In the presence of hrbFGF, tyrosinase activity was potentiated by CT and IBMX, but not by CT alone. Neither TPA nor hrbFGF alone could increase intracellular cAMP levels. The effects of CT and IBMX on intracellular cAMP concentration were enhanced to a greater extent by TPA than by hrbFGF. Under our experimental conditions, in the presence of hrbFGF, CT but not IBMX resulted in a dose-dependent increase in cAMP concentration. Further studies on NHM will be aimed at determining the exact role of protein kinase C (PKC) in regulating proliferation and melanogenesis and the mechanism(s) activated by hrbFGF.     

Antileukemic agent alkyllysophospholipid regulates phosphorylation of distinct proteins in HL60 and K562 cells and differentiation of HL60 cells promoted by phorbol ester

The tumor-promoting 12-0-tetradecanoylphorbol-13-acetate (TPA) stimulated phosphorylation of several proteins in block I (including protein Ia) and protein 3 in HL60 cells. The antileukemic agent alkyllysophospholipid (ALP) inhibited the TPA-stimulated phosphorylation of these proteins and the TPA-induced differentiation of the cells. In comparison, TPA only stimulated phosphorylation of protein 3 in K562 cells which, in contrast, were not induced to differentiate by TPA and lacked protein Ia and had a very high basal phosphorylation of protein B. ALP inhibited phosphorylation of protein 3 as well as protein B in K562 cells. The data suggest that the presence of distinct phosphoproteins and regulation of their phosphorylation may be related to the selective susceptibility of the two leukemia cell lines to the maturating effect of TPA and cytotoxicity of ALP.