Calcium-independent Protein Kinase C Research Articles

Phosphorylation of the Platelet p47 Phosphoprotein Is Mediated by the Lipid Products of Phosphoinositide 3-Kinase

Platelet stimulation by thrombin or the thrombin receptor activating peptide (TRAP) results in the activation of phosphoinositide 3-kinase and the production of the novel polyphosphoinositides phosphatidylinositol 3,4-bisphosphate (PtdIns-3,4-P2) and phosphatidylinositol 3,4,5-trisphosphate (PtdIns-3,4,5-P3). We have shown previously that these lipids activate calcium-independent protein kinase C (PKC) isoforms in vitro (Toker, A., Meyer, M., Reddy, K. K., Falck, J. R., Aneja, R., Aneja, S., Parra, A., Burns, D. J., Ballas, L. M. and Cantley, L. C. (1994) J. Biol. Chem. 269, 32358-32367). Activation of platelet PKC in response to TRAP is detected by the phosphorylation of the major PKC substrate in platelets, the p47 phosphoprotein, also known as pleckstrin. Here we provide evidence for two phases of pleckstrin phosphorylation in response to TRAP. A rapid phase of pleckstrin phosphorylation (< 1 min) precedes the peak of PtdIns-3,4-P2 production and is unaffected by concentrations of wortmannin (10-100 nM) that block production of this lipid. However prolonged phosphorylation of pleckstrin (> 2 min) is inhibited by wortmannin concentrations that block PtdIns-3,4-P2 production. Phorbol ester-mediated pleckstrin phosphorylation was not affected by wortmannin and wortmannin had no effect on purified platelet PKC activity. Phosphorylation of pleckstrin could be induced using permeabilized platelets supplied with exogenous gamma-32P[ATP] and synthetic dipalmitoyl PtdIns-3,4,5-P3 and dipalmitoyl PtdIns-3,4-P2 micelles, but not with dipalmitoyl phosphatidylinositol 3-phosphate or phosphatidylinositol 4,5-bisphosphate. These results suggest two modes of stimulating pleckstrin phosphorylation: a rapid activation of PKC (via diacylglycerol and calcium) followed by a slower activation of calcium-independent PKCs via PtdIns-3,4-P2.

Inhibitory Effect of Annexin V on Protein Kinase C Activity in Mesangial Cell Lysates

Annexin V belongs to a large family of calcium-binding and phospholipid-binding proteins and may act as an endogenous regulator of the protein kinase C (PKC) activity. This study examines the effect of annexin V on the in vitro PKC activity in cultured mesangial cells using histone H1, the peptide [Ser25]PKC-(19-31), or endogenous proteins as substrates. The SDS/PAGE pattern of 32P-labeled mesangial proteins showed that the calcium-independent PKC [(n+a)PKC] phosphorylated several proteins from 70 kDa to 40 kDa and 22 kDa to 15 kDa. Three additional proteins from 34 kDa to 29 kDa, including annexin I and its proteolytic forms, were detected after activation of calcium-dependent PKC (cPKC). Increasing concentrations of annexin V did not alter the phosphorylation of (n+a)PKC substrates. By contrast, specific phosphorylation of proteins and annexin I by cPKC, was reduced in a dose-dependent manner. Addition of high concentration of calcium and phosphatidylserine did not reverse the inhibitory effect of annexin V. Annexin V also inhibited the phosphorylation of histone H1 or peptide [Ser25]PKC-(19-31) by cPKC. Moreover, removal of annexin V from cytosols increased the annexin I phosphorylation by these isoforms. From these results, we propose that annexin V may regulate the signal-transduction pathway involving the activation of cPKC, as they act in vitro as an inhibitor of these kinases.

Increased expression of a high molecular weight (130 KD) protein kinase C isoform in a differentiation‐defective ras‐transfected keratinocyte line

The role of ras on protein kinase C (PKC) signaling was examined in two keratinocyte cell lines. Increasing the level of extracellular calcium from 0.15 mM to 1.0 mM induces some features of differentiation in the spontaneously immortalized HaCaT line, but fails to do so in a c-H-ras-transfected subline (ras-HaCaT). Raising extracellular calcium also induced a transient increase in membrane-associated PKC activity 5 min after calcium addition, in HaCaT, but not in the ras-HaCaT cells. Partial purification of PKC from the membrane/particulate fraction revealed the major isoform expressed in HaCaT to be an 80 KD species recognized by the anti-PKC alpha antibody. In ras-HaCaT, the major expressed isoform is a 130 KD species recognized by the PKC beta antibody. The kinase activity of the partially purified high molecular weight PKC is phospholipid dependent but calcium independent. Further evaluation of PKC in the HaCaT and ras-HaCaT membrane/particulate cell fraction by immunoblotting using affinity-purified antibodies against PKC alpha, beta, delta, epsilon and zeta revealed a 130 KD band reacting with the PKC delta antibody. Increased expression of this high molecular weight protein was observed in ras-HaCaT. Immunoprecipitation of PKC in ras-HaCaT using the PKC delta antibody also revealed a 130 KD species. Analysis of the PKC delta immunoprecipitate demonstrated a phospholipid, but not calcium-dependent kinase which autophosphorylated. These results suggest that the 130 KD protein may be a novel (calcium-independent) PKC (nPKC) isoform and increased expression in the ras-transfected HaCaT may be a consequence of oncogenic ras expression. This 130 KD species may also play a role in the ras-associated inhibition of differentiation in HaCaT.

CD14-dependent activation of protein kinase C and mitogen-activated protein kinases (p42 and p44) in human monocytes treated with bacterial lipopolysaccharide.

To investigate mechanisms of mononuclear phagocyte cell signaling, the effects of bacterial LPS on protein kinase activities in normal human peripheral blood monocytes were examined. Incubation of intact monocytes with LPS brought about time- and concentration-dependent increases in myelin basic protein (MBP) phosphotransferase activity in high speed supernatants of cell lysates. Anion-exchange chromatography on Mono Q demonstrated that LPS treatment resulted in two principal peaks of stimulated MBP kinase activity. Evidence was obtained to indicate that the first eluted peak of MBP kinase activity is accounted for by p42 and p44 mitogen-activated protein (MAP) kinases. Thus, 1) MBP kinase activity within peak 1 was quantitatively precipitated by anti-MAP kinase Abs, 2) the enzyme effectively phosphorylated a specific peptide substrate, 3) peak 1 contained proteins of subunit size M(r) 42,000 and M(r) 44,000 that reacted specifically with anti-MAP kinase Abs, and that 4) were recognized by anti-phosphotyrosine Abs only after stimulation of cells with LPS. Studies of the second peak of LPS-stimulated MBP kinase activity indicate that it is an isoform of protein kinase C (PKC) because: 1) enzyme activity was quantitatively immunoprecipitated by anti-PKC Abs, 2) the activity of the enzyme was potently and selectively inhibited by a specific peptide modeled on the autoinhibitory domain of PKC, and 3) the presence of a protein of subunit size M(r) 80,000 recognized by anti-PKC Abs. Because the second peak of MBP kinase activity (like the first) was active in the absence of added calcium and in the presence of 2 mM EGTA, it appears to be a type II, calcium-independent isoform of PKC. Abs to CD14 completely abrogated LPS-induced activation of both Mono Q peaks of MBP phosphotransferase activity. These results indicate that LPS coordinately activates both an apparently calcium-independent PKC and MAP kinase in mononuclear phagocytes and these responses appear to be initiated by signaling through the cell surface receptor, CD14.

Protein Kinase C Isoforms in Human and Rat Colonic Mucosa

The protein kinase C (PKC) family of enzymes plays a key role in the regulation of cellular events, including cell proliferation and differentiation. Work from our laboratory has shown that the effects of dietary fat and fiber on colonic cell proliferation were positively correlated with membrane/cytosol PKC activity ratios (Chapkin et al., 1993, J. Nutr. 123, 649-655). The presence and subcellular distribution of specific PKC isoforms in rat and human colon were therefore determined in cytosolic and membrane extracts. Tissue extracts were probed with antibodies to individual PKC isoforms. PKC α, β, δ, ϵ, and ζ were detected in both rat and human colonic mucosa, while PKC η was detected in human colonic mucosa only. PKC α, β, and ζ were predominantly localized in the cytosolic fraction, whereas the majority of PKC δ, ϵ, and η were found in the membrane-associated fraction. Presence of mRNA for individual PKC isoforms was determined by reverse transcriptase PCR (RT-PCR). Using rat colonic mucose, mRNA for PKC α, β, δ, ϵ, η and ζ were detected by RT-PCR with identity confirmed by sequencing. The relative steady-state levels of PKC isoforms in human colon adenocarcinoma as compared with normal colonic mucosa were determined, with adenocarcinomas having higher amounts of cytosolic PKC β, δ, ϵ, η and ζ. PKC isoforms were also detected in viable, exfoliated colonic cells isolated from human feces, demonstrating that this noninvasive method can be utilized to examine PKC expression in colonic cells. These results demonstrate that colonic mucose expresses both calcium-dependent (classical) and calcium-independent (novel and atypical) PKC isoforms with distinct subcellular distributions for each. The dynamics of these PKC isoforms may have implications in the development of colon carcinogenesis.

12-deoxyphorbol-13-O-phenylacetate 20 acetate [an agonist of protein kinase C?1 (PKC?1)] induces DNA synthesis, interleukin-2 (IL-2) production, IL-2 receptor ?-chain (CD25) and ?-chain (CD122) expression, and translocation of PKC? isozyme in human peripheral blood lymphocytes: Evidence for a role of PKC?1 in human T cell activation

To determine a role of protein kinase C (PKC) isozymes in lymphocyte activation, human peripheral blood mononuclear cells were activated with 12-deoxyphorbol-13-O-phenylacetate (dPP; an agonist of both calcium-dependent and calcium-independent PKC isozymes), thymeleatoxin (TX; an activator of calcium-dependent PKC alpha, beta, and gamma), and 12-deoxyphorbol-13-O-phenylacetate 20 acetate (dPPA; an activator of PKC beta 1 isozyme) and examined for DNA synthesis, lymphocyte proliferation, interleukin-2 (IL-2) production, expression of IL-2 receptor alpha and beta chains on CD3+, CD4+, and CD8+ T lymphocytes and CD20+ B lymphocytes, and translocation of PKC beta isozyme from cytosol to membrane fraction. The results show that dPPA activates lymphocytes by inducing the above changes in a manner analogous to that of dPP, TX, and phorbol myristate acetate. These data suggest that PKC beta 1 is involved in the activation of human peripheral blood T and B lymphocytes.

Human immunodeficiency virus-1 recombinant gp120 induces changes in protein kinase C isozymes — A preliminary report

Human immunodeficiency virus 1 (HIV-1) and its purified proteins activate target cell functions. Because protein kinase C (PKC) plays a crucial role in signal transduction and there is a molecular heterogeneity of PKC, we compared the effect of recombinant HIV-1 gp 120 and phorbol ester (PMA) on PKC isozymes in monocytic U937 cells, with isozyme-specific antibodies using flow cytometry. All PKC isozymes except PKC-γ were present in U937 cells. Both PMA and HIV-1 gp120 increased levels of calcium-dependent and -independent PKC isozymes. The most striking change was observed in PKC-ξ isozymes levels. This study for the first time demonstrate the HIV-1 gp120 affects calcium-independent PKC isozymes in U937 cells.

Resolution on major protein kinase substrates in neutrophil cytosol in response to DAG/PS and arachidonic acid stimulation and the selective action of various protein kinase inhibitors

Stimulation of human neutrophils induces phosphorylation of several cellular proteins. Human neutrophils possess calcium-dependent protein kinase C (PKC) α and β isoforms and calcium-independent n isoforms. Little is known, however, of the physiological substrates of each isoform. In this study, we characterized the substrates of calcium-dependent and -independent PKC isoforms and the substrate of PKC activated by arachidonic acid. Furthermore, we found that the PKC inhibitor H-7 failed to inhibit phosphorylation of endogenous substrates of calcium-independent PKC activity. These results may help to understand the role of PKC in neutrophil activation and shed light on the different responses elicited by H-7 in intact cells.

Thyrotropin-releasing hormone downregulates pyroglutamyl peptidase II activity in adenohypophyseal cells.

Pyroglutamyl peptidase II (PPII) is a thyrotropin-releasing hormone (TRH) hydrolyzing ectoenzyme with a narrow specificity. In the adenohypophysis, it is present on lactotropes. This study was undertaken in order to determine whether TRH itself regulates PPII activity in the adenohypophysis. After 5 days in culture, dispersed cells from female pituitaries expressed detectable levels of PPII activity when 10(-8) M 3,3',5'-triiodo-L-thyronine was present throughout the culture. 10(-6) M TRH decreased PPII activity with a maximal effect (down to 46% of initial values) at 16 h and an ED50 of 10(-9) M. [3Me-His2]TRH, a potent agonist of the TRH receptor was effective at lower concentrations (ED50: 1.6 x 10(-10) M). Phorbol-12-myristate-13-acetate (PMA; 10(-6) M), a protein kinase C (PKC) activator, diminished PPII activity to 61% or initial values with an ED50 of 2.2 x 10(-8) M. Maximal effects of PMA and TRH were not additive. Neither PMA nor TRH effects were reversed by inhibitors of protein kinases (1-(5-isoquinolinesulfonyl)-2-methyl-piperazine or sphingosine or staurosporine); TRH-induced downregulation of the enzyme was not modified by PMA pretreatment. TRH had no effect on two other ectopeptidases, endopeptidase 24.11 and dipeptidyl aminopeptidase IV. These data demonstrate that TRH specifically downregulates PPII activity in adenohypophyseal cells through TRH receptor activation and suggest that the activation of a presumably calcium-independent PKC mimics the TRH effect. TRH regulation of PPII activity may contribute to adjust lactotrope responsiveness to TRH.

Differential Expression of Protein Kinase C Isoenzymes in Normal and Psoriatic Adult Human Skin: Reduced Expression of Protein Kinase C-βII in Psoriasis

Psoriatic lesions contain elevated levels of 1,2-diacylglycerol, the physiologic activator of protein kinase C (PKC), suggesting that PKC activation may be aberrant in psoriasis. We therefore have investigated the expression and properties of PKC isozymes in normal and psoriatic skin and in human skin cells. Chromatographic and immunoblot analyses revealed the presence of the calcium-dependent PKC isozymes PKC-alpha and -beta, but not -gamma, in normal human epidermis. PKC-beta was more prominent, constituting two thirds of the total calcium-dependent PKC activity. In psoriatic lesions, expression of both PKC-alpha and -beta was decreased, with preferential reduction (80%) of PKC-beta. Northern analysis and semi-quantitative polymerase chain reaction (PCR) indicated no change in the mRNA levels of PKC-alpha and -beta between normal and psoriatic epidermis. In normal epidermis, PKC-alpha was expressed mainly in the lower epidermis, whereas PKC-beta was localized to the upper cell layers, with very intense staining of CD1a+ Langerhans cells. In psoriasis, PKC-alpha staining was present in the lower epidermis, whereas PKC-beta staining was essentially absent, with the exception of some positive inflammatory cells. In addition to PKC-alpha and beta, immunoblot and Northern/PCR analysis revealed expression of four calcium-independent PKC isozymes, delta, epsilon, zeta, and eta, in both normal and psoriatic skin. There were no significant differences in mRNA levels among any of these PKC isozymes, between normal and psoriatic skin. Soluble PKC-zeta protein was modestly increased (twofold) in psoriatic, compared to normal, skin, whereas the levels of PKC-delta, epsilon, and eta were unchanged. Analysis of PKC isozyme expression in the three major cell types of human epidermis revealed that Langerhans cells and keratinocytes were the major sources of PKC-beta and PKC-zeta, respectively. These data demonstrate the diversity of PKC isozyme expression in human skin, and suggest that alterations of PKC-beta and -zeta may participate in the aberrant regulation of growth and differentiation observed in psoriasis.

Endogenous protein kinase-C activation in osteoblast-like cells modulates responsiveness to estrogen and estrogen receptor levels.

The osteoblast-like osteosarcoma cell line ROS 17/2.8, which expresses very low levels of estrogen receptor (ER), was stably transfected with the mouse ER in order to more easily evaluate the physiological role of estrogens in bone cell homeostasis. These transfected ROS.SMER 14 cells are highly responsive to estrogenic stimulation at subconfluence, but become refractory to estrogenic stimulation when postconfluency is reached. The purpose of these studies was to determine the mechanisms underlying this loss of responsiveness in these ER stably transfected cells at postconfluence. When proliferative capacity was evaluated by bromodeoxyuridine immunocytochemistry, approximately 70% of the subconfluent cells were actively dividing, whereas none of the postconfluent cells underwent division. Subconfluent cells were found to contain 2500-3000 ER-binding sites/cell, whereas the ER in postconfluent cells was low and often undetectable. Steady state ER mRNA levels were not significantly modified by postconfluency. ER protein levels were also unaffected by confluency status. Since protein kinase-C (PKC) has been reported to influence cell proliferation and steroid hormone receptor binding, PKC activity was measured in sub- and postconfluent cells. Calcium-dependent PKC activity was approximately about 2-fold higher in postconfluent compared to subconfluent cells, whereas no differences were discerned in calcium-independent PKC activity. In an effort to examine the role of PKC in greater detail, postconfluent cells were treated with PKC inhibitors (H-7 or staurosporine) or with the tumor promoter TPA (12-O-tetradecanoylphorbol-13-acetate) to down-regulate PKC activity, and changes in ER were evaluated. Inhibition or down-regulation of the PKC activity in postconfluent cells enhanced ER-binding capacity in a dose-dependent manner and estrogen responsiveness of an exogenous reporter gene and of the endogenous alkaline phosphatase, representing an endogenous estrogen-stimulated gene. These data indicate that there is an interaction between the PKC and ER signaling systems in bone cells and that this interaction may be influenced by the proliferative and/or differentiative state of the cells, resulting in modulation of hormone responsiveness.

Selective regulation of protein kinase C isoenzymes by oleic acid in human platelets.

Cis-unsaturated fatty acids activate soluble protein kinase C (PKC) in vitro and in intact platelets. The following studies were conducted to determine the effects of oleate on individual isoenzymes of PKC in human platelets. Human platelets were found to contain predominantly PKC alpha, beta I, beta II, and delta with minor immunoreactivity for PKC epsilon, zeta, and eta. In intact platelets, sodium oleate caused a time-dependent redistribution of PKC alpha, beta II, and delta from cytosol to membrane fractions with little effects on PKC beta I. On the other hand, PMA and thrombin induced translocation of all four isoenzymes of PKC. In vitro, oleate partially activated (50% of Vmax) purified calcium-dependent PKC (alpha, beta I, and beta II) with an EC50 of 50 microM whereas it fully activated (100% of Vmax) purified calcium-independent PKC (predominantly delta) with an EC50 of 5 microM. The selective effects of oleate on PKC isoenzymes were investigated in platelet cytosol which contains endogenous PKC and its physiologic substrates. Under these conditions, oleate potently activated calcium-independent PKC causing the phosphorylation of the 40-kDa substrate. Activation of calcium-dependent isoforms occurred only at higher concentrations of oleate. Thus, oleate activates multiple isoenzymes of PKC with predominant effects on calcium-independent PKC.

Tissue and cellular distribution of the extended family of protein kinase C isoenzymes

Polyclonal isoenzyme-specific antisera were developed against four calcium-independent protein kinase C (PKC) isoenzymes (delta, epsilon, epsilon', and zeta) as well as the calcium-dependent isoforms (alpha, beta I, beta II, and gamma). These antisera showed high specificities, high titers, and high binding affinities (3-370 nM) for the peptide antigens to which they were raised. Each antiserum detected a species of the predicted molecular weight by Western blot that could be blocked with the immunizing peptide. PKC was sequentially purified from rat brain, and the calcium-dependent forms were finally resolved by hydroxyapatite chromatography. Peak I reacted exclusively with antisera to PKC gamma, peak II with PKC beta I and -beta II, and peak III with PKC alpha. These same fractions, however, were devoid of immunoreactivity for the calcium-independent isoenzymes. The PKC isoenzymes demonstrated a distinctive tissue distribution when evaluated by Western blot and immunocytochemistry. PCK delta was present in brain, heart, spleen, lung, liver, ovary, pancreas, and adrenal tissues. PKC epsilon was present in brain, kidney, and pancreas, whereas PKC epsilon' was present predominantly in brain. PKC zeta was present in most tissues, particularly the lung, brain, and liver. Both PKC delta and PKC zeta showed some heterogeneity of size among the different tissues. PKC alpha was present in all organs and tissues examined. PKC beta I and -beta II were present in greatest amount in brain and spleen. Although the brain contained the most PKC gamma immunoreactivity, some immunostaining was also seen in adrenal tissue. These studies provide the first evidence of selective organ and tissue distributions of the calcium-independent PKC isoenzymes.

Heterogeneity of protein kinase C in cultured rat mesangial cells

Two forms of protein kinase C (PKC) activity in cytosol of cultured rat mesangial cells have been characterized in vitro by using histone H1 or endogenous proteins as substrates. Histones H1-phosphorylation was significantly increased only when calcium, phosphatidylserine (PS) and 1,2-diacylglycerol (DAG) or phorbol myristate acetate (PMA) were present together in the incubation medium. EGTA, a calcium chelator, completely inhibited this activity. Upon hydroxyapatite chromatography (HPLC), the PKC activity was eluted as a main peak at 150 mM potassium phosphate with a shoulder at 180 mM. Both peaks corresponded to the type III PKC from rat brain and were identified as PKC α isoform by immunoblot analysis. In contrast with what was observed using histone H1, the increased phosphorylation of endogenous proteins in the presence of a mixture of Ca 2+/PS, plus either DAG or PMA, was only partly reduced by EGTA. Moreover, the level of the PKC activity detected in the presence of EGTA was comparable to the level of kinase activity, measured in the presence of PS alone or associated with DAG or PMA. This suggests that mesangial cells contain PKC activity which does not absolutely require calcium. Polyacrylamide gel electrophoresis revealed that patterns of phosphorylated mesangial cell proteins are different depending on whether calcium was added or not. In the presence of calcium, PKC strongly phosphorylated the proteins of 53,000 molecular weight, a doublet of 37,000–39,000, the 24,000 and the triplet of 17,000–20,000–22,000 molecular weight. The additional of EGTA to the assays suppressed completely the labelling of most proteins; only the 20,000 molecular weight protein remained strongly labelled, while the 39,000 molecular weight band was only faintly visible. The same patterns of phosphorylations were obtained after omission of calcium in the assays containing only PS and DAG (or PMA). So, the main substrates of calcium-dependent PKC are proteins of 53,000, 39,000, 37,000, 22,000, 24,000 and 17,000 molecular weight while the protein of 20,000 moeecular weight appears to be the main substrate of calcium-independent PKC. The existence in mesangial cells of at least two forms of PKC, which phosphorylate specific endogenous proteins, emphasizes the complexity of the phospholipid-dependent regulatory cascade and raises the possibility that actions of different regulators may be transduced through distinct PKC isozymes.

Selective changes in protein kinase C isoenzymes in rat liver nuclei during liver regeneration

Using isoenzyme-specific antisera, protein kinase C (PKC) α and PKC δ were detected in total liver homogenate and in isolated nuclei. PKC β I, β II, ε, ε′, and ζ were not detected. During liver regeneration, nuclear PKCα levels decreased while PKCδ levels increased. These studies demonstrate, for the first time, the presence of a calcium-independent PKC isoenzyme in liver nuclei and suggest that PKCα and PKCδ may have different roles in liver regeneration and cell proliferation.