Expression of four protein kinase C isoforms in rat fibroblasts. Distinct subcellular distribution and regulation by calcium and phorbol esters.

Protein kinase C (PKC), the major receptor for tumor-promoting phorbol esters, consists of a family of at least eight distinct lipid-regulated enzymes. How the various PKC isozymes are regulated in vivo and how they couple to particular cellular responses is largely unknown. We have examined the expression and regulation of PKC isoforms in R6 rat embryo fibroblasts. Northern and Western blot analyses indicate that these cells express four PKC isoforms, cPKC alpha, nPKC epsilon, nPKC delta, and nPKC zeta; of which nPKC epsilon and nPKC delta are the most abundant. In agreement with the simultaneous presence of cPKC and nPKC isozymes, both Ca(2+)-dependent and -independent PKC activities were detected in extracts of these cells. cPKC alpha and nPKC zeta were predominantly localized in the cytosol when subcellular fractionation was carried out in the presence of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid. When cell lysis was carried out in the presence of Ca2+, greater than 50% of cPKC alpha redistributed to the particulate fraction, whereas nPKC zeta remained in the cytosol. In contrast to cPKC alpha and nPKC zeta, 60-80% of nPKC epsilon and nPKC delta were located in a Ca(2+)-insensitive, membrane-bound form. Treatment of R6 cells with 12-O-tetradecanoyl phorbol 13-acetate (TPA), resulted in the translocation of all four PKC isozymes to the membrane fraction, and the subsequent down-regulation of cPKC alpha, nPKC zeta, and nPKC delta, nPKC epsilon, however, was only partially down-regulated in response to long-term TPA exposure. Overproduction of exogenous cPKC beta I in R6 cells conferred partial resistance of nPKC delta to TPA-induced down-regulation and potentiated the resistance of nPKC epsilon to down-regulation. These results demonstrate that the multiple isoforms of PKC which coexist within a single cell type are differentially regulated by extra- and intracellular stimuli and may thereby influence growth control and transformation via distinct mechanisms.     

Expression of protein kinase C isoenzymes in thymocyte subpopulations and their differential regulation

The expression of the different protein kinase C (PKC) isozymes in mouse thymocytes was studied to determine if there is a correlation between isozyme expression and thymocyte phenotype. Expression of PKC isozymes in thymocyte subsets (distinguished by the CD4 or CD8 Ag) was determined by message amplification phenotyping. The expression of mRNA for PKC-alpha, -beta, -epsilon, and -zeta, but not -gamma or -delta isozymes, was detected in all of the unstimulated thymocyte subpopulations analyzed. Thus no differences in the pattern of PKC isozyme expression were found that could be correlated with thymocyte phenotype. However, it was noted that the levels of PKC mRNA expression were affected by different stimuli in unfractionated thymocytes. Whereas mRNA levels of PKC-alpha and -beta were down-regulated by PMA and ionomycin treatment, no significant changes were seen in the levels of PKC-epsilon mRNA with these agents. PKC-epsilon mRNA decreased in thymocytes exposed to Con A similar to what has been reported for PKC-epsilon protein. PKC-zeta mRNA was also down-regulated by PMA or ionomycin, and the combination of both compounds caused a more rapid and drastic effect. Finally, PKC-delta mRNA expression was induced transiently in thymocytes only after exposure to PMA or Con A, and this induction was inhibited by ionomycin treatment. These results indicate that message levels of specific isoforms of PKC are uniquely regulated and suggest an additional level of control of PKC activity in activated lymphocytes.     

IL-1beta enhances beta2-adrenergic receptor expression in human airway epithelial cells by activating PKC

Protein kinase C (PKC)-activated signal transduction pathways regulate cell growth and differentiation in many cell types. We have observed that interleukin (IL)-1beta upregulates beta2-adrenergic receptor (beta2-AR) density and beta2-AR mRNA in human airway epithelial cells (e.g., BEAS-2B). We therefore tested the hypothesis that PKC-activated pathways mediate IL-1beta-induced beta-AR upregulation. The role of PKC was assessed from the effects of 1) the PKC activator phorbol 12-myristate 13-acetate (PMA) on beta-AR density, 2) selective PKC inhibitors (calphostin C and Ro-31-8220) on beta-AR density, and 3) IL-1beta treatment on the cellular distribution of PKC isozymes. Recombinant human IL-1beta (0.2 nM for 18 h) increased beta-AR density to 213% of control values (P < 0.001). PMA (1 microM for 18 h) increased beta-AR density to 225% of control values (P < 0.005), whereas Ro-31-8220 and calphostin C inhibited the IL-1beta-induced upregulation of beta-AR in dose-dependent fashion. PKC isozymes detected by Western blotting included alpha, betaII, epsilon, mu, zeta, and lambda/iota. IL-1beta increased PKC-mu immunoreactivity in the membrane fraction and had no effect on the distribution of the other PKC isozymes identified. These data indicate that IL-1beta-induced beta-AR upregulation is mimicked by PKC activators and blocked by PKC inhibitors and appears to involve selective activation of the PKC-mu isozyme. We conclude that signal transduction pathways activated by PKC-mu upregulate beta2-AR expression in human airway epithelial cells.     

Nonequivalent effects of PKC activation by PMA on murine CD4 and CD8 cell-surface expression

The membrane glycoproteins CD4 (L3T4) and CD8 (Lyt2) are expressed on distinct populations of mature murine T lymphocytes, and are thought to be receptors for monomorphic determinants expressed on MHC class II and class I molecules, respectively. Although they differ in their ligand specificity, it has been presumed that CD4 and CD8 perform equivalent functions in the T cells that bear them. Since activation of protein kinase C (PKC) is known to cause rapid down-regulation of various receptors, including the T cell receptor complex (TcR complex), we treated cells with phorbol 12-myristate 13-acetate (PMA), a PKC activator, to determine whether cell-surface expression of CD4 and CD8 would be similarly affected by this intracellular mediator. Brief or relatively prolonged treatment with PMA induced mature murine T cells to reduce their surface expression of the TcR complex and of CD4, but not of CD8. Similarly, PMA rapidly induced transfected L cells to down-regulate surface CD4 expression, but had no effect on surface CD8 expression. Most significantly, PMA treatment induced CD4+CD8+ immature thymocytes to rapidly reduce their surface CD4 expression, but, again, it had no immediate effect on the surface expression of CD8. These results indicate that CD4 and TcR complex cell-surface expression are both sensitive to PKC activation by brief treatment with PMA, whereas CD8 expression is not, and suggest that CD4 and CD8 surface expression levels are regulated by distinct intracellular mechanisms.     

Constitutive presence of a catalytic fragment of protein kinase C epsilon in a small cell lung carcinoma cell line.

Protein kinase C (PKC) has been implicated in a variety of cellular responses such as proliferation, differentiation, and secretion. We assessed the role of PKC in the mitogenic effects of gastrin-releasing peptide (in a small cell lung cancer (SCLC) cell line. Using antisera that specifically recognize the PKC isoforms alpha, beta, gamma, delta, and epsilon, we determined that PKC epsilon is the major isoform in the SCLC cell line NCI-N417, followed by PKC alpha and delta. In addition to the 90-kDa PKC epsilon, our anti-PKC epsilon antiserum specifically detected a 40-kDa immunoreactive protein. Treatment of the cells with either 20 nM phorbol myristate acetate or 50 nM GRP enhanced significantly the level of the 40-kDa protein in a time-dependent (1-8 h), cycloheximide-sensitive fashion. Subcellular fractionation revealed that 90% of PKC epsilon was in particulate form, while the 40-kDa immunoreactive protein was cytosolic. To test the hypothesis that the 40-kDa soluble protein represented a catalytically independent PKC epsilon fragment, cytosolic extracts were assayed for kinase activity. 45-50% of the activity was apparent in the absence of the PKC activators phosphatidylserine and diacylglycerol. This effector-independent kinase activity was further purified by affinity chromatography using a synthetic peptide corresponding to the pseudosubstrate region of PKC epsilon (ERMRPRKRQGAVRRRV) coupled to Sepharose. The partially purified protein, recognized by the anti-PKC epsilon antiserum, exhibited histone kinase activity with kinetics similar to those of the tryptically generated catalytic fragment of brain PKC epsilon. This activity was inhibited by staurosporine (IC50 = 1 x 10(-8) M) and by the pseudosubstrate inhibitor peptide (IC50 = 7.7 x 10(-8) M). The SCLC kinase and the brain PKC epsilon catalytic fragment were similar as indicated by the relative sizes of the PKC epsilon immunoreactive peptides generated with protease V8 from Staphylococcus aureus (Mr approximately 37,000, 34,000, 28,000, 26,000, and 25,000). Taken together, we conclude that a variant SCLC cell line expresses a constitutively active catalytic fragment of PKC epsilon. Regulation by 12-O-tetradecanoyl-13-acetate or GRP via de novo protein synthesis suggests a novel mechanism of control of PKC diversity with implications for small cell lung cancer and possibly other malignancies.     

Identification and functional characterization of protein kinase C isozymes in platelets and HEL cells.

To determine if selective activation of individual isozymes of protein kinase C (PKC) might explain the apparently divergent effects of PKC stimulation on platelets, we purified and characterized the isozymes from both platelets and human erythroleukemia (HEL) cells, a cell line that has many features of megakaryocytes. Two peaks of platelet PKC activity were resolved by hydroxylapatite chromatography; immunoblot analysis revealed that these two peaks represented the alpha and beta isozymes of PKC. In contrast, HEL cells produced only a single peak that contained the beta isozyme. None of the other PKC isozymes were detected in these fractions. The cytosol of platelets and HEL cells, however, were both found to contain the PKC-delta isozyme. Northern hybridization analyses and mRNA amplification by the polymerase chain reaction demonstrated the presence of mRNA encoding the alpha, beta, and delta PKC isozymes in platelets, but only the beta and delta isozymes in HEL cells. Phorbol myristate acetate (PMA), thrombin, or an endoperoxide analog induced the phosphorylation of the 47-kDa substrate of PKC (pleckstrin) found in platelets and HEL cells; preincubation of either HEL cells or platelets with PMA reduced the intracellular Ca2+ rise induced by thrombin. Thus, although both HEL cells and platelets contain PKC-beta and the recently described PKC-delta isozymes, the widely distributed alpha isozyme of PKC is absent in HEL cells; however, isozymes other than PKC-alpha are sufficient for some PMA-mediated functions that are similar to those seen in stimulated platelets.     

Selective translocation of protein kinase C-delta in PC12 cells during nerve growth factor-induced neuritogenesis.

The specific intracellular signals initiated by nerve growth factor (NGF) that lead to neurite formation in PC12 rat pheochromocytoma cells are as of yet unclear. Protein kinase C-delta (PKC delta) is translocated from the soluble to the particulate subcellular fraction during NGF-induced-neuritogenesis; however, this does not occur after treatment with the epidermal growth factor, which is mitogenic but does not induce neurite formation. PC12 cells also contain both Ca(2+)-sensitive and Ca(2+)-independent PKC enzymatic activities, and express mRNA and immunoreactive proteins corresponding to the PKC isoforms alpha, beta, delta, epsilon, and zeta. There are transient decreases in the levels of immunoreactive PKCs alpha, beta, and epsilon after 1-3 days of NGF treatment, and after 7 days there is a 2.5-fold increase in the level of PKC alpha, and a 1.8-fold increase in total cellular PKC activity. NGF-induced PC12 cell neuritogenesis is enhanced by 12-O-tetradecanoyl phorbol-13-acetate (TPA) in a TPA dose- and time-dependent manner, and this differentiation coincides with abrogation of the down-regulation of PKC delta and other PKC isoforms, when the cells are treated with TPA. Thus a selective activation of PKC delta may play a role in neuritogenic signals in PC12 cells.     

Regulation of 5-hydroxytryptamine-induced signal transduction in canine cultured aorta smooth muscle cells by phorbol ester.

Regulation of the increase in inositol phosphates (IPs) production and intracellular Ca2+ concentration ([Ca2+]i) by protein kinase C (PKC) was investigated in cultured canine aorta smooth muscle cells (ASMCs). Stimulation of ASMCs by 5-hydroxytryptamine (5-HT) led to IPs formation and caused an initial transient [Ca2+]i peak followed by a sustained elevation of [Ca2+]i in a concentration-dependent manner. Pretreatment of ASMCs with phorbol 12-myristate 13-acetate (PMA) for 30 min almost abolished the 5-HT-induced IPs formation and Ca2+ mobilization. This inhibition was reduced after long-term incubating the cells with PMA. Prior treatment of ASMCs with staurosporine or GF109203X, PKC inhibitors, inhibited the ability of PMA to attenuate 5-HT-induced responses, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC. In parallel with the effect of PMA on the 5-HT-induced IP formation and Ca2+ mobilization, the translocation and down-regulation of PKC isozymes were determined by Western blotting with antibodies against different PKC isozymes. The results revealed that treatment of ASMCs with PMA for various times, translocation of PKC-alpha, betaI, betaII, delta, epsilon, theta, and zeta isozymes from the cytosol to the membrane was seen after 5-min, 30-min, 2-h, and 4-h treatment. However, 24-h treatment caused a partial down-regulation of these PKC isozymes. In conclusion, these results demonstrate that translocation of PKC-alpha, betaI, betaII, delta, epsilon, theta, and zeta induced by PMA caused an attenuation of 5-HT-induced IPs accumulation and Ca2+ mobilization in ASMCs.     

IL-10 production induced by HIV-1 Tat stimulation of human monocytes is dependent on the activation of PKC beta(II) and delta isozymes

The effect of HIV-1 Tat protein on the production of IL-10, an immunosuppressive cytokine, was examined in human primary monocytes obtained from healthy HIV-1-negative blood donors. As expected and in agreement with our previous data, a dose-dependent induction of IL-10 was observed. In addition, we showed that this induction is mediated by the PKC pathway: in the presence of Ro 31-8220, an inhibitor of all PKC isozymes, or after 48 h of PMA treatment, Tat protein becomes unable to stimulate IL-10 production. Among the 11 PKC isozymes, eight (PKC alpha, beta(I), beta(II), delta, epsilon, eta, zeta, mu) are expressed in monocytes. In this study, by analyzing the translocation to the membrane after Tat stimulation, we showed that PKC alpha, beta(I), beta(II), delta and epsilon isozymes are activated by Tat. Moreover, by combining different approaches including selective PKC inhibitors (G?6983, G?6976, hispidin and rottlerin), we showed that PKC beta(II) and delta isozymes are essential for the activation of IL-10 production in human monocytes following stimulation by HIV-1 Tat protein.     

Activation of protein kinase C isozymes is associated with post-mitotic events in intestinal epithelial cells in situ

The mechanisms underlying control of cell growth and differentiation in epithelial tissues are poorly understood. Protein kinase C (PKC) isozymes, members of a large family of serine/threonine kinases of fundamental importance in signal transduction, have been increasingly implicated in the regulation of cell growth, differentiation, and function. Using the rat intestinal epithelium as a model system, we have examined PKC-specific activity as well as individual PKC isozyme expression and distribution (i.e., activation status) in epithelial cells in situ. Increased PKC activity was detected in differentiating and functional cells relative to immature proliferating crypt cells. Immunofluorescence and Western blot analysis using a panel of isozyme- specific antibodies revealed that PKC alpha, beta II, delta, epsilon, and zeta are expressed in rat intestinal epithelial cells and exhibit distinct subcellular distribution patterns along the crypt-villus unit. The combined morphological and biochemical approach used permitted analysis of the activation status of specific PKC isozymes at the individual cell level. These studies showed that marked changes in membrane association and level of expression for PKC alpha, beta II, delta, and zeta occur as cells cease division in the mid-crypt region and begin differentiation. Additional changes in PKC activation status are observed with acquisition of mature function on the villus. These studies clearly demonstrate naturally occurring alterations in PKC isozyme activation status at the individual cell level within the context of a developing tissue. Direct activation of PKC in an immature intestinal crypt cell line was shown to result in growth inhibition and coincident translocation of PKC alpha from the cytosolic to the particulate subcellular fraction, paralleling observations made in situ and providing further support for a role of intestinal PKC isozymes in post-mitotic events. PKC isozymes were also found to be tightly associated with cytoskeletal elements, suggesting participation in control of the structural organization of the enterocyte. Taken together, the results presented strongly suggest an involvement of PKC isoforms in cellular processes related to growth cessation, differentiation, and function of intestinal epithelial cells in situ.     

Selective stimulation of thymocyte precursors mediated by specific cytokines. Different CD3+ subsets are generated by IL-1 versus IL-2.

The sequence of activation signals that stimulate proliferation, differentiation, and selection of mature T cell subsets from immature, dull-CD5+/CD4-, CD8- double negative (bCD5), (dCD5/DN) thymocytes are still unclear. However, it is likely that cytokines play integral roles in these events. Here we report that IL-1, in the presence of Con A, supports the proliferation and differentiation of highly purified dCD5/DN precursors into bright-CD5+ DN, CD2- lymphocytes with an apparently mature phenotype. These cells express CD3 and preferentially express the products of two TCR gene families, V beta 8 and V beta 6, whose expression is dependent on the allelic expression of the Mls-1 locus. Experiments, using DN thymocytes mixed with purified dCD5 subset of DN cells from a congenic strain of mice (i.e., expressing two different alleles of CD5) have shown that the cells that are stimulated by IL-1 and comitogen are derived from the immature dCD5 subset and not from the mature bCD5 cells contained within the DN subset. In contrast, IL-2 with the co-mitogen stimulates three- to fourfold higher levels of proliferation, from the same purified immature precursor population, and nearly a twofold increase in cell yield. However, the cells that were generated from precursor thymic cells stimulated with IL-2 represent a completely different T cell subset compared to IL-1-generated cells; these IL-2-stimulated cells express comparable levels of CD3, but also express substantial levels of CD2 and the TCR-gamma/delta, and a subset expresses CD8. These data suggest that these two TCR-alpha/beta and TCR-gamma/delta subsets of mature thymocytes use different cytokines and therefore possibly different stromal interactions to initiate differentiation.     

Microgravity modifies protein kinase C isoform translocation in the human monocytic cell line U937 and human peripheral blood T-cells

Individual protein kinase C (PKC) isoforms fulfill distinct roles in the regulation of the commitment to differentiation, cell cycle arrest, and apoptosis in both monocytes and T-cells. The human monocyte like cell line U937 and T-cells were exposed to microgravity, during spaceflight and the translocation (a critical step in PKC signaling) of individual isoforms to cell particulate fraction examined. PKC activating phorbol esters induced a rapid translocation of several PKC isoforms to the particulate fraction of U937 monocytes under terrestrial gravity (1 g) conditions in the laboratory. In microgravity, the translocation of PKC beta II, delta, and epsilon in response to phorbol esters was reduced in microgravity compared to 1 g, but was enhanced in weak hypergravity (1.4 g). All isoforms showed a net increase in particulate PKC following phorbol ester stimulation, except PKC delta which showed a net decrease in microgravity. In T-cells, phorbol ester induced translocation of PKC delta was reduced in microgravity, compared to 1 g, while PKC beta II translocation was not significantly different at the two g-levels. These data show that microgravity differentially alters the translocation of individual PKC isoforms in monocytes and T-cells, thus providing a partial explanation for the modifications previously observed in the activation of these cell types under microgravity.     

Protein kinase C isoforms from Giardia duodenalis: identification and functional characterization of a β-like molecule during encystment

Protein kinase C (PKC) is a family of serine/threonine kinases that regulate many different cellular processes such as cell growth and differentiation in eukaryotic cells. Using specific polyclonal antibodies raised against mammalian PKC isoforms, it was demonstrated here for the first time that Giardia duodenalis expresses several PKC isoforms (beta, delta, epsilon, theta and zeta). All PKC isoforms detected showed changes in their expression pattern during encystment induction. In addition, selective PKC inhibitors blocked the encystment in a dose-dependent manner, suggesting that PKC isozymes may play important roles during this differentiation process. We have characterized here the only conventional-type PKC member found so far in Giardia, which showed an increased expression and changes in its intracellular localization pattern during cyst formation. The purified protein obtained by chromatography on DEAE-cellulose followed by size-exclusion chromatography, displayed in vitro kinase activity using histone HI-IIIS as substrate, which was dependent on cofactors required by conventional PKCs, i.e., phospholipids and calcium. An open reading frame in the Giardia Genome Database that encodes a homolog of PKCβ catalytic domain was identified and cloned. The expressed recombinant protein was also recognized by a mammalian anti-PKCβ antibody and was referred as giardial PKCβ on the basis of all these experimental evidence.     

An epsilonPKC-selective inhibitor attenuates back phosphorylation of a low molecular weight protein in cardiac myocytes

We have studied epsilon PKC-mediated phosphorylation events in neonatal cardiac myocytes using back phosphorylation. 3 nM 4-beta 12-myristate-13-acetate (PMA)-intact cell treatment preferentially activates epsilon PKC in these cells (Circ. Res. 76 (1995) 654) and caused decreased 32P incorporation (back phosphorylation) into an approximately 18-kDa protein. This response required physiological levels of free Mg(2+) and short (3-5 min) incubation periods in back phosphorylation assays. Introduction of a selective epsilon PKC translocation inhibitor (epsilon V1) into these cells attenuated the 3 nM PMA-induced back phosphorylation response while translocation inhibitors to the classical PKC or deltaPKC isozymes were without effect. Pretreatment of our cells with endothelin-1 (ET1) had similar effects to 3 nM PMA albeit the magnitude of the ET1 back phosphorylation response was about one-half that of 3 nM PMA. Our results suggest that epsilon PKC phosphorylates an approximately 18-kDa protein found in the particulate cell fraction of neonatal cardiac myocytes. Epsilon PKC modulates diverse cardiac responses including contraction, ion channel functions, hypertrophy, and ischemic preconditioning. Characterization of epsilon PKC-selective phosphotransferase events may reveal novel regulatory mechanisms for this enzyme in neonatal cardiac myocytes.     

Activation of distinct protein kinase C isozymes by phorbol esters: correlation with induction of interleukin 1 beta gene expression

Treatment of human promyelocytic leukemia cells U937 with phorbol 12-myristate 13-acetate (TPA) induces them to differentiate into monocytic cells [Harris, P., & Ralph, P. (1985) J. Leukocyte Biol. 37, 407-422]. Here we investigated the effects of TPA on interleukin 1 gene expression and the possible role of protein kinase C (PKC) in this process. Addition of TPA to serum-starved U937 cells induced the expression of the interleukin 1 beta (IL-1 beta) gene. This effect was apparent as early as 2 h and peaked at 24 h in the presence of 5 X 10(-8) M TPA. Higher concentrations of TPA, which partially or totally depleted protein kinase C levels in the cells (10(-9)-2 X 10(-5) M), had an inhibitory effect on IL-1 beta mRNA expression. Cell-permeable 1,2-dioctanoyl-sn-glycerol (diC8), a diacylglycerol that activates PKC in intact cells and cell-free systems, did not mimic the effect of TPA on the IL-1 beta mRNA induction. To determine the protein kinase C isozymes present in the control and TPA- (5 X 10(-8) M) treated U937 cells, we prepared antipeptide antibodies that specifically recognize the alpha, beta, and gamma isoforms of protein kinase C in rat brain cytosol and U937 cell extracts. In "control" U937 cells, 30% of PKC alpha was particulate, and PKC beta was cytosolic, while there was no detectable PKC gamma.(ABSTRACT TRUNCATED AT 250 WORDS)     

PKC isozyme selective regulation of cloned human cardiac delayed slow rectifier K current

Delayed rectifying K(+) channel, I(Ks), plays a vital role in normal and arrhythmogenic heart. I(Ks) is modulated by PKC but the identity of which PKC isozymes is involved in this modulation is not known. To dissect the role of individual PKC isozymes in the regulation of I(Ks), human cardiac I(Ks) channel (minK+KvLQT1) was expressed in Xenopus oocytes. Peptide PKC isozyme-specific activator and inhibitors, in addition to the general PKC activator, PMA, were used. Whole-cell I(Ks) was recorded using two-electrode voltage clamp technique. PMA and epsilon PKC specific activator peptide, but not the inactive analog, 4alphaPDD, significantly increased I(Ks). Peptide specific inhibitors for beta(II)PKC, and a general PKC inhibitor, calphostin C antagonized PMA-induced activation of I(Ks). However, control peptide, pentalysine, and specific inhibitor peptide for alphaPKC, beta(I)PKC, deltaPKC, or etaPKC did not alter PMA effect on I(Ks). The present study demonstrates that beta(II)PKC, epsilon PKC but not beta(I)PKC, alphaPKC, deltaPKC, and etaPKC, are involved in PMA-induced activation of the cloned human I(Ks) expressed in Xenopus oocyte. Furthermore, this is the first report to dissect the fine functional role of beta(II)PKC and beta(I)PKC in the regulation of I(Ks). Identification of the particular isozyme(s) that mediates the regulation of I(Ks) channels is of importance for the understanding of the mechanism of ion channel regulation and the development of new therapeutic agents.     

Increased degradation of protein kinase C without diminution of mRNA level after treatment of WEHI-231 B lymphoma cells with phorbol esters

Immunoblot analysis of WEHI-231 B lymphoma cell homogenates revealed that both type II, a major component, and type III, a minor component, protein kinase C (PKC) were present. Northern blot analysis of PKC mRNA showed a higher level of beta II and beta I mRNA (encoding type II PKC) than of alpha mRNA (encoding type III PKC). Short term (3 min) treatment with phorbol 12-myristate 13-acetate (PMA) caused a rapid loss of PKC in cytosol and a concomitant increase in the particulate fraction. After prolonged (24 hr) exposure, the level of both PKC isozymes were decreased. However, the corresponding mRNA levels remained intact. PMA did not inhibit the anti-IgM-mediated increase in [Ca2+]i in PKC-depleted cells.     

Thymic nurse cells exclusively bind and internalize CD4+CD8+ thymocytes.

Thymic nurse cells (TNC) contain 20-200 thymocytes within specialized vacuoles in their cytoplasm. The purpose of the uptake of thymocytes by TNCs is unknown. TNCs also have the capacity to present self-antigens, which implies that they may serve a function in the process of thymic education. We have recently reported the development of thymic nurse cell lines that have the ability to bind and internalize T cells. Here, we use one of these TNC lines to identify the thymocyte subpopulation(s) involved in this internalization process. TNCs exposed to freshly isolated thymocytes bind and internalize CD4 and CD8 expressing thymocytes (CD4+CD8+ or double positives) exclusively. More specifically, a subset of the double-positive thymocyte population displayed binding capacity. These double-positive cells express cell surface alpha beta type T cell antigen receptor (TCR), as well as CD3 epsilon. Binding was not inhibited in the presence of antibodies against CD3, CD4, CD8, Class I antigens, or Class II antigens. These results describe two significant events in T cell development. First, TNCs exclusively bind and internalize a subset of alpha beta TCR expressing double-positive T cells. Also, binding is facilitated through a mechanism other than TCR recognition of major histocompatibility complex antigens. This suggests that thymocyte internalization may be independent of the process used by TNCs to present self-antigen.