Chelerythrine is a potent and specific inhibitor of protein kinase C

The benzophenanthridine alkaloid chelerythrine is a potent, selective antagonist of the Ca++/phospholopid-dependent protein kinase (Protein kinase C: PKC) from the rat brain. Half-maximal inhibition of the kinase occurs at 0.66 microM. Chelerythrine interacted with the catalytic domain of PKC, was a competitive inhibitor with respect to the phosphate acceptor (histone IIIS) (Ki = 0.7 microM) and a non-competitive inhibitor with respect to ATP. This effect was further evidenced by the fact that chelerythrine inhibited native PKC and its catalytic fragment identically and did not affect [3H]- phorbol 12,13 dibutyrate binding to PKC. Chelerythrine selectively inhibited PKC compared to tyrosine protein kinase, cAMP-dependent protein kinase and calcium/calmodulin-dependent protein kinase. The potent antitumoral activity of celerythrine measured in vitro might be due at least in part to inhibition of PKC and thus suggests that PKC may be a model for rational design of antitumor drugs.     

Galanin reduces PDBu-induced protein phosphorylation in rat ventral hippocampus.

The effect of galanin (GAL) on basal and phorbol-12,13-dibutyrate (PDBu) induced protein phosphorylation in rat ventral hippocampal miniprisms was investigated. GAL (0.5, 1 and 2 microM) inhibited PDBu stimulation in a concentration-dependent manner without altering basal protein phosphorylation. This inhibitory effect was prevented by the GAL antagonist galantide. GAL did not affect either the activity of protein kinase C (PKC) from rat brain or basal phosphorylation in ventral hippocampal hippogenates, suggesting that it did not directly modulate PKC activity. Depolarization of miniprisms from ventral hippocampi by 18 mM K+ prevented the effect of GAL on PDBu-induced phosphorylation. The results indicate that GAL indirectly regulates neuronal protein phosphorylation by a GAL receptor-mediated action.     

Protein kinase C mediates phosphorylation, desensitization, and trafficking of the D2 dopamine receptor

Previously, D2 dopamine receptors (D2 DARs) have been shown to undergo G-protein-coupled receptor kinase phosphorylation in an agonist-specific fashion. We have now investigated the ability of the second messenger-activated protein kinases, protein kinase A (PKA) and protein kinase C (PKC), to mediate phosphorylation and desensitization of the D2 DAR. HEK293T cells were transiently transfected with the D2 DAR and then treated with intracellular activators and inhibitors of PKA or PKC. Treatment with agents that increase cAMP, and activate PKA, had no effect on the phosphorylation state of the D2 DAR, suggesting that PKA does not phosphorylate the D2 DAR in HEK293T cells. In contrast, cellular treatment with phorbol 12-myristate 13-acetate (PMA), a PKC activator, resulted in an approximately 3-fold increase in D2 DAR phosphorylation. The phosphorylation was specific for PKC as the PMA effect was mimicked by phorbol 12,13-dibutyrate, but not by 4alpha-phorbol 12,13-didecanoate, active and inactive, phorbol diesters, respectively. The PMA-mediated D2 DAR phosphorylation was completely blocked by co-treatment with the PKC inhibitor, bisindolylmaleimide II, and augmented by co-transfection with PKCbetaI. In contrast, PKC inhibition had no effect on agonist-promoted phosphorylation, suggesting that PKC is not involved in this response. PKC phosphorylation of the D2 DAR was found to promote receptor desensitization as reflected by a decrease in agonist potency for inhibiting cAMP accumulation. Most interestingly, PKC phosphorylation also promoted internalization of the D2 DAR through a beta-arrestin- and dynamin-dependent pathway, a response not usually associated with PKC phosphorylation of G-protein-coupled receptors. Site-directed mutagenesis experiments resulted in the identification of two domains of PKC phosphorylation sites within the third intracellular loop of the receptor. Both of these domains are involved in regulating sequestration of the D2 DAR, whereas only one domain is involved in receptor desensitization. These results indicate that PKC can mediate phosphorylation of the D2 DAR, resulting in both functional desensitization and receptor internalization.     

Propranolol stimulates histone phosphorylation by a putative PK-C in partially purified homogenate of rat testicular interstitial cells. A possible mechanism for increased testosterone secretion by propranolol.

The beta-adrenoceptor blocker propranolol stimulated testosterone secretion by rat testicular interstitial cells (Leydig cell-enriched preparation) in vitro at concentrations ranging from 10(-5) M to 10(-4) M. Treatment of these cells with H7 (20 microM), an inhibitor of protein kinase C, reduced the stimulatory effect of L-propranolol on testosterone secretion by about 5-fold. At concentrations ranging from 31.25 microM to 1000 microM, L-propranolol reduced [3H]phorbol 12,13-dibutyrate binding (IC50 = 75 microM) to rat testicular interstitial cells. At similar concentrations, L-propranolol displaced the binding of [3H]phorbol 12,13-dibutyrate to the homogenate of these cells by only 5%. These findings suggest that the effect of L-propranolol on [3H]phorbol 12,13-dibutyrate binding could be indirect, possibly by increasing the concentration of a chemical mediator interacting with the regulatory domain of protein kinase C. At even lower concentrations (10(-9) M to 10(-7) M), propranolol added directly to the reaction mixture with protein kinase C partially purified from rat testicular interstitial cells increases the phosphorylation of histone. This phosphorylation was comparable to that obtained with (25 microg/ml) phosphatidylserine. The D- and L-stereoisomers of propranolol were equally active. A complete reversal of this propranolol effect on histone phosphorylation was achieved with (20 microM) H-7. In the absence of Ca2+, propranolol was not able to phosphorylate the histone. Taken together, these results suggest that protein kinase C could be the putative kinase involved in this reaction and that its activation by propranolol may be due to interaction of the drug with the regulatory domain of the enzyme at a site differing from the site of interaction with phorbol 12,13-dibutyrate. The ability of propranolol to activate the putative protein kinase C could be related to its stimulatory effect on testosterone secretion by Leydig cells.     

The protein kinase inhibitor staurosporine, like phorbol esters, induces the association of protein kinase C with membranes

Staurosporine induced the association of purified protein kinase C (PKC) with inside-out vesicles from erythrocyte membranes. This effect was Ca2+ and concentration dependent, and maximum PKC translocation was observed at 50 nM staurosporine and 0.5 microM Ca2+, or higher. A significant effect of staurosporine was already obtained at free Ca2+ concentrations in the range found in resting cells. Under these conditions, the PKC activator 4-phorbol 12,13-dibutyrate was by itself inactive, but enhanced translocation by staurosporine. Protein phosphorylation by staurosporine-translocated PKC was inhibited in the presence or absence of phorbol esters. Translocation and inhibition of PKC occurred in the same staurosporine concentration range.     

Dexmedetomidine-induced Contraction Involves Phosphorylation of Caldesmon by JNK in Endothelium-denuded Rat Aortas

Caldesmon, an inhibitory actin binding protein, binds to actin and inhibits actin-myosin interactions, whereas caldesmon phosphorylation reverses the inhibitory effect of caldesmon on actin-myosin interactions, potentially leading to enhanced contraction. The goal of this study was to investigate the cellular signaling pathway responsible for caldesmon phosphorylation, which is involved in the regulation of the contraction induced by dexmedetomidine (DMT), an alpha-2 adrenoceptor agonist, in endothelium-denuded rat aortas. SP600125 (a c-Jun NH₂-terminal kinase [JNK] inhibitor) dose-response curves were generated in aortas that were pre-contracted with DMT or phorbol 12,13-dibutyrate (PDBu), a protein kinase C (PKC) activator. Dose-response curves to the PKC inhibitor chelerythrine were generated in rat aortas pre-contracted with DMT. The effects of SP600125 and rauwolscine (an alpha-2 adrenoceptor inhibitor) on DMT-induced caldesmon phosphorylation in rat aortic vascular smooth muscle cells (VSMCs) were investigated by western blot analysis. PDBu-induced caldesmon and DMT-induced PKC phosphorylation in rat aortic VSMCs was investigated by western blot analysis. The effects of GF109203X (a PKC inhibitor) on DMT- or PDBu-induced JNK phosphorylation in VSMCs were assessed. SP600125 resulted in the relaxation of aortas that were pre-contracted with DMT or PDBu, whereas rauwolscine attenuated DMT-induced contraction. Chelerythrine resulted in the vasodilation of aortas pre-contracted with DMT. SP600125 and rauwolscine inhibited DMT-induced caldesmon phosphorylation. Additionally, PDBu induced caldesmon phosphorylation, and GF109203X attenuated the JNK phosphorylation induced by DMT or PDBu. DMT induced PKC phosphorylation in rat aortic VSMCs. These results suggest that alpha-2 adrenoceptor-mediated, DMT-induced contraction involves caldesmon phosphorylation that is mediated by JNK phosphorylation by PKC.     

Ethanol-induced phosphorylation and potentiation of the activity of type 7 adenylyl cyclase. Involvement of protein kinase C delta

Ethanol can enhance G(salpha)-stimulated adenylyl cyclase (AC) activity. Of the nine isoforms of AC, type 7 (AC7) is the most sensitive to ethanol. The potentiation of AC7 by ethanol is dependent on protein kinase C (PKC). We designed studies to determine which PKC isotype(s) are involved in the potentiation of Galpha(s)-activated AC7 activity by ethanol and to investigate the direct phosphorylation of AC7 by PKC. AC7 was phosphorylated in vitro by the catalytic subunits of PKCs. The addition of ethanol to AC7-transfected HEK 293 cells increased the endogenous phosphorylation of AC7, as indicated by a decreased "back-phosphorylation" of AC7 by PKC in vitro. The potentiation of Galpha(s)-stimulated AC7 activity by either phorbol 12,13-dibutyrate or ethanol, in HEL cells endogenously expressing AC7, was not through the Ca(2+)-sensitive conventional PKCs. However, the potentiation of AC7 activity by ethanol or phorbol 12,13-dibutyrate was found to be reduced by the selective inhibitor of PKCdelta (rottlerin), a PKCdelta-specific inhibitory peptide (deltaV1-1), and the expression of the dominant negative form of PKCdelta. Immunoprecipitation data indicated that PKCdelta could bind and directly phosphorylate AC7. The results indicate that the potentiation of AC7 activity by ethanol involves phosphorylation of AC7 that is mediated by PKCdelta.     

Involvement of functional protein kinase C in the mitogenic response to the H-ras oncogene product.

Microinjection of purified protein kinase C (PKC) into Swiss 3T3 fibroblasts pretreated with the phorbol ester phorbol-12,13-dibutyrate restores the mitogenic response of the cells to phorbol-12,13-dibutyrate (G. Pasti, J.C. Lacal, B.S. Warren, S.A. Aaronson, and P.M. Blumberg, Nature [London] 324:375-377, 1986). Our present studies demonstrate that the mitogenic activity of the H-ras oncogene in H-ras p21-microinjected quiescent cells is markedly reduced under conditions in which PKC is downregulated by chronic phorbol ester treatment. The ability to reconstitute the mitogenic response upon microinjection of both H-ras p21 and PKC implies involvement of functional PKC in the mitogenic activity of the H-ras oncogene product.     

Ca2+-stimulated Ca2+ oscillations produced by the Ca2+-sensing receptor require negative feedback by protein kinase C

We examined the role of protein kinase C (PKC) in the mechanism and regulation of intracellular Ca(2+) concentration ([Ca(2+)](i)) oscillations elicited by an increase in the extracellular concentration of Ca(2+) ([Ca(2+)](e)) in human embryonic kidney 293 cells expressing the Ca(2+)-sensing receptor (CaR). Exposure to the PKC inhibitors bisindolylmaleimide I (GF I) or Ro-31-8220 converted oscillatory responses to transient, non-oscillatory responses, significantly reducing the percentage of cells that showed [Ca(2+)](i) oscillations but without decreasing the overall response to increase in [Ca(2+)](e). Exposure to 100 nm phorbol 12,13-dibutyrate, a direct activator of PKC, eliminated [Ca(2+)](i) oscillations. Addition of phorbol 12,13-dibutyrate at lower concentrations (3 and 10 nm) did not eliminate the oscillations but greatly reduced their frequency in a dose-dependent manner. Co-expression of CaR with constitutively active mutants of PKC (either epsilon or beta(1) isoforms) also reduced [Ca(2+)](i) oscillation frequency. Expression of a mutant CaR in which the major PKC phosphorylation site is altered by substitution of alanine for threonine (T888A) eliminated oscillatory behavior, producing [Ca(2+)](i) responses almost identical to those produced by the wild type CaR exposed to PKC inhibitors. These results support a model in which phosphorylation of the CaR at the inhibitory threonine 888 by PKC provides the negative feedback needed to cause [Ca(2+)](i) oscillations mediated by this receptor.     

Vasopressin rapidly stimulates protein kinase C in quiescent Swiss 3T3 cells

Addition of vasopressin to quiescent cultures of Swiss 3T3 cells caused a rapid increase in the phosphorylation of an acidic molecular weight 80,000 cellular protein (termed 80K). The effect was concentration- and time-dependent; enhancement in 80K phosphorylation could be detected as early as 30 sec after the addition of the hormone. Recently, a rapid increase in the phosphorylation of an 80K cellular protein following treatment with phorbol esters or diacylglycerol has been shown to reflect the activation of protein kinase C in intact Swiss 3T3 cells. Here we show that the 80K phosphoproteins generated in response to vasopressin and phorbol 12,13-dibutyrate (PBt2) were identical as judged by one- and two-dimensional polyacrylamide gel electrophoresis (PAGE) and peptide mapping following partial proteolysis with Staphylococcus aureus V8 protease. In addition, prolonged pretreatment of 3T3 cells with PBt2 which leads to the disappearance of protein kinase C activity blocked the ability of vasopressin to stimulate the phosphorylation of 80K. The effect of vasopressin on 80K phosphorylation and mitogenesis was selectively blocked by the vasopressin antagonist (Pmp1-O-Me-Tyr2-Arg8) vasopressin suggesting that these responses are mediated by its specific receptor in these cells. The removal of vasopressin leads to dephosphorylation (within minutes) of the 80K phosphoprotein. We conclude that vasopressin rapidly stimulates protein kinase C activity in intact 3T3 cells.     

Alpha1-adrenoceptor-mediated phosphorylation of MYPT-1 and CPI-17 in the uterine artery: role of ERK/PKC

We previously demonstrated that ERK/PKC signaling pathways play a key role in regulation of Ca(2+) sensitivity and contractility of the uterine artery. The present study tested the hypothesis that ERK and PKC differentially regulated myosin light chain phosphatase activity by phosphorylation of myosin phosphatase target protein-1 (MYPT-1) and CPI-17. Agonist-induced contractions and phosphorylation of MYPT-1/Thr(696), MYPT-1/Thr(850), and CPI-17/Thr(38) were measured simultaneously in the same tissues of isolated near-term pregnant ovine uterine arteries. Phenylephrine produced time-dependent concurrent increases in the phosphorylation of ERK(44/42) and MYPT-1/Thr(850) that preceded contractions. In addition, phenylephrine induced phosphorylation of CPI-17/Thr(38) that was concurrent with the contractions. In contrast, phenylephrine did not induce phosphorylation of MYPT-1/Thr(696) in the uterine artery. PD-098059 inhibited phosphorylation of ERK(44/42) and the initial peak phosphorylation of MYPT-1/Thr(850) but did not affect CPI-17/Thr(38) phosphorylation. Activation of PKC by phorbol 12,13-dibutyrate induced a time-dependent phosphorylation of CPI-17/Thr(38) that preceded contractions of the uterine artery. In addition, phorbol 12,13-dibutyrate activated PKC-alpha and induced a coimmunoprecipitation of PKC-alpha with caldesmon. The results suggest that phosphorylation of MYPT-1/Thr(850) and CPI-17/Thr(38) play important roles in regulation of agonist-mediated Ca(2+) sensitivity in the uterine artery, in part by ERK and PKC, respectively. In addition, phosphorylated CPI-17 may regulate Ca(2+) sensitivity by interacting with caldesmon and reversing its inhibitory effect on myosin ATPase.     

Effect of phorbol esters on Ca2+ sensitivity and myosin light-chain phosphorylation in airway smooth muscle

We studied in -escin-permeabilized canine tracheal smoothmuscle (CTSM) the effect of the protein kinase C (PKC) agonist phorbol12,13-dibutyrate (PDBu) on isometric force at a constant submaximalCa²⁺ concentration (i.e., theeffect on Ca²⁺ sensitivity) andregulatory myosin light-chain (rMLC) phosphorylation. PDBuincreased Ca²⁺sensitivity, an increase associated with a concentration-dependent, sustained increase in rMLC phosphorylation. PDBu altered therelationship between rMLC phosphorylation and isometric force such thatthe increase in isometric force was less than that expected for the increase in rMLC phosphorylation observed. The effect of four PKCinhibitors [calphostin C, chelerythrine chloride, apseudosubstrate inhibitor for PKC, PKC peptide-(1931) (PSSI), andstaurosporine] on PDBu-inducedCa²⁺ sensitization as well as theeffect of calphostin C and PSSI on rMLC phosphorylation weredetermined. Whereas none of these compounds prevented or reversed thePDBu-induced increase in Ca²⁺sensitivity, the PDBu-induced increase in rMLC phosphorylation wasinhibited. We conclude that PDBu increases rMLC phosphorylation byactivation of PKC but that the associated PDBu-induced increases inCa²⁺ sensitivity are mediated bymechanisms other than activation of PKC in permeabilized airway smoothmuscle.

     

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.     

Two distinct mechanisms underlie the stimulation of neurotransmitter release by phorbol esters in clonal rat pheochromocytoma PC12 cells

Phorbol ester treatment induces the phosphorylation of SNAP-25 at Ser(187) and the potentiation of Ca(2+)-induced dopamine (DA) and acetylcholine (Ach) release from PC12 cells. In order to evaluate the functional consequences of phosphorylation, quantitative analysis was carried out using an anti-phosphopeptide antibody that specifically recognizes SNAP-25 phosphorylated at Ser(187). DA and ACh release, assayed in low-K(+) as well as high-K(+) solution, increased by treating the cells with phorbol-12-myristate-13-acetate (PMA); however, the stimulation of high-K(+)-dependent release occurred at lower concentrations and with shorter exposures to PMA than that of the basal release in low-K(+)-solution. The PMA-induced phosphorylation of SNAP-25 did not correlate with the potentiation of high-K(+)-dependent neurotransmitter release. The potentiation of high-K(+)-dependent DA release by phorbol 12,13-diacetate (PDA), a water soluble phorbol ester, almost completely disappeared within 1 min after washing PDA in the presence of okadaic acid, conditions under which the phosphorylation of SNAP-25 persisted for at least 15 min. PMA-induced phosphorylation of SNAP-25 was inhibited by staurosporine, however, the potentiation of high-K(+)-dependent DA release was suppressed only partially. These results indicate that protein kinase activation does not account for a large fraction of the phorbol ester-induced potentiation of depolarization-dependent neurotransmitter release from PC12 cells.     

Protein kinase C-alpha produces reciprocal effects on the phorbol ester stimulated tyrosine phosphorylation of a 50 kDa kinase in Jurkat cells.

A detergent extract isolated from the enriched fraction of integral membrane proteins of Jurkat cells showed an enhanced tyrosine phosphate level when phosphorylated in the presence of phorbol 12-myristate 13-acetate (TPA) and phorbol 12,13-dibutyrate (PDBu). The enhanced tyrosine phosphorylation was observed when the reaction time exceeded 6 min; at shorter incubation times, however, TPA inhibited tyrosine phosphorylation. When the reaction proceeded for a constant time period longer than 6 min and phorbol esters were added at different times after the start of the reaction, two phases of an enhanced tyrosine phosphorylation of a 50 kDa protein were observed. An increased phosphorylation of the 50 kDa protein was correlated with an enhanced phosphorylation of poly(Glu4,Tyr1). The two phases of enhanced phosphorylation differed in their TPA and PDBu requirement and in the proteins that were tyrosine phosphorylated. Studies with protein kinase C (PKC) inhibitors showed a negatively correlated effect on the enhanced tyrosine phosphorylation in phase I; tyrosine phosphorylation was further augmented. In phase II the regulation of tyrosine phosphorylation correlated with the efficiency of the PKC inhibitors on the alpha-isoform of PKC which was found in the cell extract. Separation of the proteins present in the investigated cell extract by gel filtration revealed a co-migration of the alpha-PKC and the 50 kDa protein. The metabolic labeling of intact Jurkat cells with 32Pi indicated that phorbol esters are also able to induce tyrosine phosphorylation of the 50 kDa protein underin vivo conditions. These data suggest an activation of two different tyrosine phosphorylation pathways by phorbol esters involving tyrosine phosphorylation/autophosphorylation of a 50 kDa kinase, as confirmed by 5'-p-fluorosulfonylbenzoyladenosine (FSBA) labeling, that are accurately regulated by alpha-PKC.     

Synthesis and tumor-promoting activities of 12-Epi-phorbol-12,13-dibutyrate

12-Epi-phorbol-12,13-dibutyrate (1), the C12-epimer of the most frequently used phorbol ester probe, phorbol-12,13-dibutyrate (PDBu), has been synthesized from phorbol in 9 steps in order to investigate the structural requirements for tumor-promoting activity. Compound 1 showed about 100-fold weaker in vitro biological activities related to in vivo tumor promotion, Epstein-Barr virus early antigen (EBV-EA)-inducing ability, superoxide (O2-) generation-inducing ability, and binding to the protein kinase C (PKC) regulatory domain surrogate peptides. The results indicated that the beta-stereochemistry at position 12 of the phorbol skeleton is important for optimal activity. Binding selectivity to each PKC C1 domain of 1 was almost equal to that of PDBu.     

Molecular cloning and characterization of the human protein kinase D2. A novel member of the protein kinase D family of serine threonine kinases

We have isolated the full-length cDNA of a novel human serine threonine protein kinase gene. The deduced protein sequence contains two cysteine-rich motifs at the N terminus, a pleckstrin homology domain, and a catalytic domain containing all the characteristic sequence motifs of serine protein kinases. It exhibits the strongest homology to the serine threonine protein kinases PKD/PKCmicro and PKCnu, particularly in the duplex zinc finger-like cysteine-rich motif, in the pleckstrin homology domain and in the protein kinase domain. In contrast, it shows only a low degree of sequence similarity to other members of the PKC family. Therefore, the new protein has been termed protein kinase D2 (PKD2). The mRNA of PKD2 is widely expressed in human and murine tissues. It encodes a protein with a molecular mass of 105 kDa in SDS-polyacrylamide gel electrophoresis, which is expressed in various human cell lines, including HL60 cells, which do not express PKCmicro. In vivo phorbol ester binding studies demonstrated a concentration-dependent binding of [(3)H]phorbol 12,13-dibutyrate to PKD2. The addition of phorbol 12,13-dibutyrate in the presence of dioleoylphosphatidylserine stimulated the autophosphorylation of PKD2 in a synergistic fashion. Phorbol esters also stimulated autophosphorylation of PKD2 in intact cells. PKD2 activated by phorbol esters efficiently phosphorylated the exogenous substrate histone H1. In addition, we could identify the C-terminal Ser(876) residue as an in vivo phosphorylation site within PKD2. Phosphorylation of Ser(876) of PKD2 correlated with the activation status of the kinase. Finally, gastrin was found to be a physiological activator of PKD2 in human AGS-B cells stably transfected with the CCK(B)/gastrin receptor. Thus, PKD2 is a novel phorbol ester- and growth factor-stimulated protein kinase.     

Differential effects of phorbol ester and diacylglycerols on inositol phosphate formation in C62B glioma cells

Application of acetylcholine (ACh) to C62B glioma cells results in a rapid release of inositol phosphates. Since this response is transient, we evaluated the possible role of protein kinase C (PKC) in its desensitization. Pretreatment with 100 nM phorbol 12,13-dibutyrate (PDBu) significantly inhibited ACh-induced accumulation of [3H]inositol mono-, bis-, and trisphosphates. However, interpretation of this result as proof of PKC involvement was complicated by the failure of 1,2-dioctanoylglycerol, 1,2-didecanoylglycerol, or 1-oleoyl-2-acetylglycerol pretreatments to mimic the phorbol ester effect. Further evidence against PKC involvement was obtained using the PKC inhibitor sphingosine; PDBu inhibition of inositol phosphate formation was not reversed by sphingosine pretreatments at concentrations which blocked ACh-stimulated PKC activation of inositol trisphosphate phosphatase activity. These results suggest that there may be phorbol effects not mediated by PKC.     

Evidence against the regulation of caldesmon inhibitory activity by p42/p44erk mitogen-activated protein kinase in vitro and demonstration of another caldesmon kinase in intact gizzard smooth muscle.

The effect of direct phosphorylation by recombinant p44erk1 mitogen-activated protein kinase on the inhibitory activity of caldesmon and its C-terminal fragment H1 was studied in vitro. Neither inhibition of actin-tropomyosin activated ATPase of heavy meromyosin by caldesmon or H1, nor inhibition of the actin-tropomyosin motility over heavy meromyosin by H1 was significantly affected by the phosphorylation while only a moderate effect on the actin-activated component of heavy meromyosin ATPase inhibition was observed. Phosphopeptide mapping of caldesmon immunoprecipitated from [32P]PO4-labelled intact gizzard strips revealed that it is predominantly phosphorylated at mitogen-activated protein kinase sites in unstimulated tissue and that it is stimulated for 1 h with phorbol 12,13-dibutyrate. We find that phorbol 12,13-dibutyrate also induces a transitory phosphorylation of caldesmon peaking at 15 min after addition and this phosphorylation is not attributed to mitogen-activated protein kinase, protein kinase C, Ca2+/calmodulin-dependent kinase II or casein kinase II. We suggest that a yet unidentified kinase, rather than mitogen-activated protein kinase, may be involved in regulation of the caldesmon function in vivo.