Protein kinase C isoforms in pituitary cells displaying differential sensitivity to phorbol ester

Investigations with protein kinase C (PKC) isoform-specific antisera, revealed distinct profiles of PKC isoform content amongst pituitary tissues. Western analysis revealed the and isoforms of PKC are present in rat anterior and posterior pituitary tissue as well as in the GH₃ somatomammotrophic cell line. AtT-20/D16-V corticotrophic and T3-1 gonadotrophic murine cell lines contained no PKC-. The or isoforms were undetected in any pituitary tissue. PKC activity measurements revealed Ca²⁺-independent PKCs in T3-1 and GH₃ cells which were more sensitive to activation by phorbol-dibutyrate (PDBu) than the corresponding PKC activity found in COS cells. However, Ca²⁺-dependent PKC activities were of similar sensitivity to PDBu in GH₃, T3-1 and COS cells, indicating that functional differences observed in PDBu-sensitivity in these cells may be due to differential activation of Ca²⁺-independent PKC isoforms. Moreover, substrate-specificity of these PKCs were also compared indicating that the amount of Ca²⁺-dependency of the observed PKC activity from the same pituitary tissue is dependent upon the substrate utilized by the PKC isotypes present. These findings explain differential sensitivities of PKC-mediated actions that have previously been observed in a range of pituitary cells. (Mol Cell Biochem 000-000, 1999)     

Protein kinase C negatively modulated by phorbol ester

Pretreatment of protein kinase C with 12-O-tetradecanoylphorbol-13-acetate (TPA) and phospholipid resulted in complete inhibition of ATP/phosphotransferase activity, irreversibly. The inactivation by TPA required the phospholipid, and TPA alone did not cause inactivation. Ca2+ and diacylglycerol mimicked TPA. This action of TPA was not general for all protein kinases as it did not accelerate the inactivation of the catalytic subunit of cAMP-dependent protein kinase by phospholipid. The addition of MgATP to the reaction mixture completely protected protein kinase C from being inactivated by TPA, in the presence of phospholipid. The nucleotide-binding site of the enzyme was probably influenced by the binding of TPA and phospholipid.     

Protein kinase C contains two phorbol ester binding domains

A series of deletion and truncation mutants of protein kinase C (PKC) were expressed in the baculovirus-insect cell expression system in order to elucidate the ability of various domains of the enzyme to bind phorbol dibutyrate (PDBu). A PKC truncation mutant consisting of only the catalytic domain of the enzyme did not bind [3H]PDBu, whereas a PKC truncation mutant consisting of the regulatory domain (containing the tandem cysteine-rich putative zinc finger regions) bound [3H]PDBu. Deletion of the second conserved region (C2) of PKC did not abolish [3H]PDBu binding, whereas a deletion of the first conserved region (C1) of PKC, containing the two cysteine-rich sequences, completely abolished [3H]PDBu binding. Additional truncation and deletion mutants helped to localize the region necessary for [3H]PDBu binding; all PKC mutants that contained either one of the cysteine-rich zinc finger-like regions possessed phorbol ester binding activity. Scatchard analyses of these mutants indicated that each bound [3H]PDBu with equivalent affinity (21-41 nM); approximately 10-20-fold less than the native enzyme. In addition, a peptide of 146 amino acid residues from the first cysteine-rich region, as well as a peptide of only 86 amino acids residues from the second cysteine-rich region, both bound [3H]PDBu with high affinity (31 +/- 4 and 59 +/- 13 nM, respectively). These data establish that PKC contains two phorbol ester binding domains which may function in its regulation.     

Analysis of phorbol ester receptors in phorbol ester unresponsive 3T3 cell variants

The phorbol ester tumor promoters induce multiple cellular responses in cell culture, including mitogenesis. We have analyzed 3 variants of mouse 3T3 cells mitogenically unresponsive to the phorbol esters for phorbol ester receptors. All resembled control 3T3 cells in their specific [³H]phorbol 12,13-dibutyrate binding. The variants thus appear to be altered at steps distal to receptor occupancy in the mitogenic response to the phorbol esters.     

Bimodal effect of phorbol ester on B cell activation. Implication for the role of protein kinase C.

The role of protein kinase C PKC in B cell activation is controversial. These studies were undertaken to determine whether protein kinase C has a stimulatory or inhibitory role in B cell activation. We found that treatment of B cells for a short period of time (30 min) with the PKC activator phorbol 12,13-dibutyrate (PDBU) primed the cells for enhanced proliferative responses to anti-immunoglobulin (anti-Ig) antibody whereas treatment for a longer period of time (3 h or more) resulted in suppression of proliferation. The enhanced proliferative response to treatment of B cells with PDBU for short periods of time was associated with inhibition of anti-Ig-stimulated increases in phosphatidyl 4,5-bisphosphate (PIP2) hydrolysis and inhibition of increases in [Ca2+]i, indicating that activation of PKC per se might be sufficient for enhancing B cell activation. The time-dependent effect of phorbol esters on the inhibition of B cell proliferation was found to be closely correlated with the kinetics of disappearance of PKC as measured by Western blot and by enzymatic activity but not with inhibition of [Ca2+]i and PIP2. These data demonstrate a bimodal time-dependent effect of PDBU on B cell activation and suggest that (a) the inhibitory effect of phorbol ester on anti-Ig-induced proliferation may be due to the disappearance of PKC rather than to the inhibition of PIP2 and Ca2+; and (b) the early activation of PKC is a stimulatory rather than an inhibitory signal in the induction of B lymphocyte proliferation by anti-Ig.     

Protein kinase C activity and phorbol ester binding to rat myogenic cells during growth and differentiation

Phorbol esters have been reported to induce opposite responses in fetal myoblasts and in satellite cells isolated from adult skeletal muscles. We examined the possibility that different levels of protein kinase C (PKC) activity and different phorbol ester binding characteristics account for these responses. For this purpose, the subcellular distributions of PKC were compared in primary cultures of myogenic cells from fetal and adult rat muscles and in the L6 cell line. Cells were used at the proliferative stage or after differentiation into myotubes. Binding of phorbol dibutyrate (PDBu) was assayed. In all three cell types, the levels of PKC specific activity were comparable at the proliferating and the differentiated stages, and partial translocation of PKC activity from the membrane to the cytosolic compartment was observed after differentiation into myotubes. PDBu binding, which had a Kd of 6 to 13 nM in proliferative cells, rose to between 30 and 52 nM in myotubes. Simultaneously, a small increase was observed in the total number of PDBu binding sites. These results suggest that the role of PKC might change with the stage of differentiation. They also imply that the difference described by others between the sensitivity to phorbol esters of fetal myoblasts and satellite cells is not connected with the phorbol ester receptor (i.e., PKC), but might be caused by events subsequent to PKC activation.     

Protein kinase C as a possible receptor protein of tumor-promoting phorbol esters

A tumor-promoting phorbol ester, [3H]phorbol-12,13-dibutyrate, may bind to a homogeneous preparation of Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C) in the simultaneous presence of Ca2+ and phospholipid. This tumor promoter does not bind simply to phospholipid nor to the enzyme per se irrespective of the presence and absence of Ca2+. All four components mentioned above appear to be bound together, and the quaternary complex thus produced is enzymatically fully active for protein phosphorylation. Phosphatidylserine is most effective. Various other phorbol derivatives which are active in tumor promotion compete with [3H]phorbol-12,13-dibutyrate for the binding, and an apparent dissociation binding constant of the tumor promoter is 8 nM. This value is identical with the activation constant for protein kinase C and remarkably similar to the dissociation binding constant that is described for intact cell surface receptors. The binding of the phorbol ester is prevented specifically by the addition of diacylglycerol, which serves as activator of protein kinase C under physiological conditions. Scatchard analysis suggests that one molecule of the tumor promoter may bind to every molecule of protein kinase C in the presence of Ca2+ and excess phospholipid. It is suggestive that protein kinase C is a phorbol ester-receptive protein, and the results presented seem to provide clues for clarifying the mechanism of tumor promotion.     

Protein kinase C activity following exposure to magnetic field and phorbol ester

We examined the separate and combined effects of 60 Hz sinusoidal magnetic fields (MFs) and a phorbol ester on protein kinase C (PKC) activity in HL60 cells. No enhancement in PKC activity was observed when a cell culture was exposed to a 1.1 mT (rms) MF alone or to a combination of MF and 2 μM phorbol 12-myristate 13-acetate (PMA) for 1 h. In a second set of experiments, cells were preexposed to a less than optimal concentration of PMA (50 nM) for 45 min, followed by a 15 min exposure to both PMA and MF. The data showed a greater decrease in cytosolic PKC activity and a larger increase in membrane activity than was induced by either 1 h PMA treatment alone or PMA and sham MF exposure. One logical conclusion from these data is that MFs may be acting in a synergistic manner on a pathway that has already been activated. Therefore, we suggest that MFs, rather than producing biological effects by a new pathway or mechanism of interaction, exert their effect(s) by interacting with already functioning reactions or pathways. If correct, the question of an MF's mechanism of interaction refocuses on how weak fields might enhance or depress a molecular reaction in progress, rather than on finding a new transduction pathway. Bioelectromagnetics 19:469–476, 1998. © 1998 Wiley-Liss, Inc.     

New insights into the regulation of protein kinase C and novel phorbol ester receptors.

Protein kinase C (PKC), a family of related serine-threonine kinases, is a key player in the cellular responses mediated by the second messenger diacylglycerol (DAG) and the phorbol ester tumor promoters. The traditional view of PKCs as DAG/phospholipid-regulated proteins has expanded in the last few years by three seminal discoveries. First, PKC activity and maturation is controlled by autophosphorylation and transphosphorylation mechanisms, which includes phosphorylation of PKC isozymes by phosphoinositide-dependent protein kinases (PDKs) and tyrosine kinases. Second, PKC activity and localization are regulated by direct interaction with different types of interacting proteins. Protein-protein interactions are now recognized as important mechanisms that target individual PKCs to different intracellular compartments and confer selectivity by associating individual isozymes with specific substrates. Last, the discovery of novel phorbol ester receptors lacking kinase activity allows us to speculate that some of the biological responses elicited by phorbol esters or by activation of receptors coupled to elevation in DAG levels could be mediated by PKC-independent pathways.     

Subcellular distribution of protein kinase C/phorbol ester receptors in differentiating mouse keratinocytes

The activation of protein kinase C (PKC) by diacylglycerol or tumor promoters plays a pivotal role in signal transduction and subsequent activation of cellular processes. Since the activity of this enzyme is dependent on its immediate lipid domain, its relative distribution within the cell may be an important regulatory mechanism. We report here a relative decrease in PKC/phorbol ester receptor associated with the particulate fraction of mouse keratinocytes induced to differentiate by two separate systems. First, proliferating keratinocytes maintained in low Ca2+ (0.09 mM) serum-free medium were induced to differentiate rapidly by the addition of Ca2+ (1.8 mM). A 1.4-fold decrease in the percent of total phorbol receptor binding activity present in the particulate fraction and concomitant increase in binding in the cytosol fraction was evident 20 min after the Ca2+ addition. Second, in keratinocytes that differentiate over a 6 day cultivation period in serum-containing medium with Ca2+ concentration of 1.8 mM, a significant decrease in the percent of the phorbol receptor binding activity present in the particulate fraction was observed as the culture begins to differentiate on days 3 and 4. Maximal phorbol ester binding in the particulate fraction corresponded to the proliferative phase of the culture (day 2), while lower levels of PKC/phorbol ester binding to particulate fractions were noted during the early differentiative phase (days 3 and 4). Addition of the synthetic diacylglycerols 1-oleoyl-2-acetylglycerol or L-alpha-1,2 dioctanyl glycerol at 30 micrograms/ml to proliferating keratinocyte cultures induced a modest increase in two markers of terminal differentiation: cornified envelope formation and transglutaminase levels. These findings, taken together, support the hypothesis that PKC activation plays a role in the initial signalling events for keratinocyte differentiation.     

Protein kinase C activation in a 3T3 cell variant morphologically unresponsive to tumor-promoting phorbol esters

To investigate the mechanism of the morphological changes induced in cells by tumor-promoting phorbol esters, we isolated a 3T3 cell variant which was morphologically unresponsive to phorbol esters and analyzed the activation of protein kinase C induced by the phorbol esters in it. The variant resembled the parent cells in its activation and appeared to have been altered at some step distal to the early events of protein kinase C activation.     

Selective post-transcriptional down-regulation of protein kinase C isoenzymes in leukemic T cells chronically treated with phorbol ester

Binding to, and activation of, protein kinase C (PKC) by phorbol ester (PE) tumor promoters may underlie their tumor-promoting activity. To study the effects of long-term PE treatment on regulation of cellular PKC, we adapted the human leukemic T cell line, Jurkat (JK), to continuous growth in the presence of PE. Such cells (JKPE) displayed loss of CD2 and CD3 cell surface molecules, known to play an important role in agonist-stimulated T cell activation, unresponsiveness to stimuli that induce interleukin 2 (IL2) receptor expression or IL2 production, change in the expression of several cell cycle-regulated genes, and a 6-fold reduction in cellular PKC enzymatic activity. This reduction was accompanied by the disappearance of a major approximately 82-kDa immunoreactive protein in JKPE cytosol when cell extracts were immunoblotted with a polyclonal anti-PKC peptide antibody cross-reactive with the PKC isoenzymes, alpha, beta, and gamma. Analysis with additional anti-peptide antibodies specific for alpha, beta, or gamma PKC indicated that all three types of PKC are expressed in JK cells; however, JKPE cells lost a major approximately 82 kDa immunoreactive cytosolic protein detectable with anti-PKC alpha antibody. In contrast, levels of expression and subcellular distribution of immunoreactive PKC beta and PKC gamma, as well as levels of mRNA specific for the three PKC isoenzymes, were not significantly affected by chronic PE treatment. These results indicate that PE-mediated reduction of PKC in JKPE cells is selective and occurs at the protein, not mRNA, level, and support the notion that distinct isoenzymes encoded by the PKC multigene family may be independently regulated. Moreover, the correlation between phenotypic and functional changes on one hand, and the selective reduction of PKC alpha on the other, raises the possibility that expression of CD2 and/or CD3 and functional activation in JKPE cells are preferentially regulated by PKC alpha.     

Evidence for protein kinase C--independent pathways mediating phorbol ester induced plasmacytoid differentiation of B chronic lymphocytic leukemia cells.

The effects of phorbol esters on many cell types are known to be mediated through activation of the protein kinase C (PKC) signal transduction pathway. By using the specific inhibitor of this enzyme 1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine dihydrochloride (H7) we have assessed the role of PKC activation in phorbol ester (phorbol 12-myristate 13-acetate, PMA)-induced plasmacytoid differentiation of B chronic lymphocytic leukemia cells (B-CLL) as a model of terminal differentiation of human B lymphocytes. H7 affected a dose-dependent inhibition of PMA-induced thymidine and uridine uptake with ID50 values of 41 microM and 32 microM, respectively. A comparable ID50 value (34 microM) was obtained for H7 inhibition of B-CLL PKC activity in a cell-free system. PMA-induced changes in cell morphology, expression of CD20, CD37 and FMC7 surface antigens together with increased secretion of immunoglobulin were variably abrogated by H7 suggesting that PKC activation is more important in B cell activation/DNA synthesis than in the differentiative response. Consistent with this, expression of a sizable proportion of PMA-inducible genes was not significantly affected by H7. These data are consistent with the existence of a PMA-activated, PKC-independent signal transduction pathway which may be important, though by itself apparently insufficient, for eliciting full terminal differentiation in B lymphocytes.     

Protein kinase C and phorbol ester receptor expression related to growth and differentiation of nullipotent and pluripotent embryonal carcinoma cells

We have characterized effects of phorbol, 12-myristate 13 acetate (PMA) on growth and differentiation in a nullipotent embryonal carcinoma (EC) cell line, F9, in a pluripotent EC line, P19, and in the differentiated derivatives of these cells, In P19EC and F9EC PMA addition resulted in inhibition of growth, while in the differentiated derivates PMA was mitogenic. PMA did not induce differentiation in EC cells but potentiated the retinoic acid (RA) induced differentiation in P19EC, although, not in F9EC. Rapid morphological changes by PMA were seen in P19EC and two differentiated derivatives which represent different stages of differentiation. In F9 no rapid morphological changes were induced by PMA. Using [3H]phorbol dibutyrate as a ligand we showed that during differentiation into endoderm-like cells the number of phorbol ester receptors increases, while in epithelial-like derivatives no increase is found. In differentiated cells with an increased number of phorbol ester receptors, the cytoplasmic Ca2+- and phospholipid-dependent protein kinase (the putative receptor for phorbol esters) activity was also increased. Only in those derivatives where the number of phorbol ester receptors is increased, is the binding of epidermal growth factor (EGF) inhibited by PMA. These results suggest a relationship between levels of expression of phorbol ester receptors, cytoplasmic protein kinase C and biological effects, namely rapid morphological changes, altered growth, potentiation of RA induced differentiation, and inhibition of EGF binding.     

Protein kinase C phosphorylates glutamyl-tRNA synthetase in rabbit reticulocytes stimulated by tumor promoting phorbol esters

A high Mr synthetase core complex isolated from higher eukaryotes contains aminoacyl-tRNA synthetases specific for arginine, aspartic acid, glutamic acid, glutamine, isoleucine, leucine, lysine, methionine, and proline. Previously, five of the synthetases were shown to be phosphorylated in reticulocytes, and the glutaminyl- and aspartyl-tRNA synthetases were shown to be selectively phosphorylated in response to 8-bromo cAMP (Pendergast, A. M., Venema, R. C., and Traugh, J. A. (1987) J. Biol. Chem. 262, 5939-5942). Exposure of reticulocytes to phorbol 12-myristate 13-acetate stimulates the selective phosphorylation of one synthetase in the complex, glutamyl-tRNA synthetase. Only the glutamyl-tRNA synthetase is modified to a significant extent when the purified complex is phosphorylated in vitro by protein kinase C; up to 0.7 mol of phosphate is incorporated per mol of synthetase. Two-dimensional phosphopeptide mapping shows a single tryptic phosphopeptide, which is identical for the enzyme modified in vitro by protein kinase C or in phorbol 12-myristate 13-acetate-stimulated cells. Phosphorylation in vivo is reproducibly accompanied by a 38 +/- 10% reduction in aminoacylation activity of partially purified glutamyl-tRNA synthetase assayed in vitro. Phosphorylation in vitro has no detectable effect on aminoacylation. This difference may be due to the absence of a required effector molecule which alters activity by interaction with the phosphorylated synthetase. Glutamyl-tRNA synthetase is one of a growing number of translational components, including initiation factors, which are coordinately modified by protein kinase C in response to phorbol 12-myristate 13-acetate.     

Protein kinase C blockers and neutrophil receptors for leukotriene B4

Three protein kinase C blockers (staurosporin, Cl, and sphinganine) acted temperature- and time-dependently on human neutrophils to lower the affinity and number of high affinity plasmalemma receptors available to leukotriene B4. The drugs did not alter the ligand's binding to isolated plasma membranes or reduce intact cell binding of platelet-activating factor. Thus, protein kinase C may regulate the expression of certain receptors in resting cells and blockers of this enzyme, by interfering with receptor expression, have secondary effects that complicate their use as pharmacological probes.     

Protein kinase C-associated kinase (PKK) mediates Bcl10-independent NF-kappa B activation induced by phorbol ester

Protein kinase C-associated kinase (PKK) is a recently described kinase of unknown function that was identified on the basis of its specific interaction with PKC beta. PKK contains N-terminal kinase and C-terminal ankyrin repeats domains linked to an intermediate region. Here we report that the kinase domain of PKK is highly homologous to that of two mediators of nuclear factor-kappa B (NF-kappa B) activation, RICK and RIP, but these related kinases have different C-terminal domains for binding to upstream factors. We find that expression of PKK, like RICK and RIP, induces NF-kappa B activation. Mutational analysis revealed that the kinase domain of PKK is essential for NF-kappa B activation, whereas replacement of serine residues in the putative activation loop did not affect the ability of PKK to activate NF-kappa B. A catalytic inactive PKK mutant inhibited NF-kappa B activation induced by phorbol ester and Ca(2+)-ionophore, but it did not block that mediated by tumor necrosis factor alpha, interleukin-1 beta, or Nod1. Inhibition of NF-kappa B activation by dominant negative PKK was reverted by co-expression of PKC beta I, suggesting a functional association between PKK and PKC beta I. PKK-mediated NF-kappa B activation required IKK alpha and IKK beta but not IKK gamma, the regulatory subunit of the IKK complex. Moreover, NF-kappa B activation induced by PKK was not inhibited by dominant negative Bimp1 and proceeded in the absence of Bcl10, two components of a recently described PKC signaling pathway. These results suggest that PKK is a member of the RICK/RIP family of kinases, which is involved in a PKC-activated NF-kappa B signaling pathway that is independent of Bcl10 and IKK gamma.     

Diacylglycerol kinase gamma is one of the specific receptors of tumor-promoting phorbol esters.

Diacylglycerol kinase (DGK) and protein kinase C (PKC) are two different enzyme families that interact with diacylglycerol. Both enzymes contain cysteine-rich C1 domains with a zinc finger-like structure. Most of the C1 domains of PKCs show strong phorbol-12,13-dibutyrate (PDBu) binding with nanomolar dissociation constants (K(d)'s). However, there has been no experimental evidence that phorbol esters bind to the C1 domains of DGKs. We focused on DGK gamma because its C1A domain has a high degree of sequence homology to those of PKCs, and because DGK gamma translocates from the cytoplasm to the plasma membrane following 12-O-tetradecanoylphorbol-13-acetate treatment similar to PKCs. Two C1 domains of DGK gamma (DGK gamma-C1A and DGK gamma-C1B) were synthesized and tested for their PDBu binding along with whole DGK gamma (Flag-DGK gamma) expressed in COS-7 cells. DGK gamma-C1A and Flag-DGK gamma showed strong PDBu binding affinity, while DGK gamma-C1B was completely inactive. Scatchard analysis of DGK gamma-C1A and Flag-DGK gamma gave K(d)'s of 3.1 and 4.4 nM, respectively, indicating that the major PDBu binding site of DGK gamma is C1A. This is the first evidence that DGK gamma is a specific receptor of tumor-promoting phorbol esters.     

Protein kinase C in the nervous system of insects: effects of phorbol esters on cholinergic synapses

Specific high affinity binding sites for phorbol esters have been demonstrated in the nervous tissue of locust. The binding was reduced by the kinase C-inhibitor polymyxin B. Phorbol esters in micromolar concentrations activated protein kinase C in subcellular preparations from locust ganglia. The same concentrations of phorbol esters significantly enhanced the high affinity transport of choline and the release of acetylcholine by locust synaptosomes.