The heterogeneity of protein kinase C in various rat tissues

Expression of multiple subspecies of protein kinase C (PKC) was studied in various rat tissues. Three types of the enzyme designated type I, II, and III were analyzed, which have the structures of gamma-, beta- (beta I- and beta II-), and alpha-sequence, respectively. Type I enzyme was found only in the central nervous tissue, whereas type III enzyme appeared to be commonly present in various tissues such as liver, spleen, lung, testis, heart, and kidney. Type II enzyme was also found in these tissues. However, immunoblot and biochemical analysis indicated that type II enzyme of lung and heart was distinct from that of other tissues. The tissue-specific expression of PKC suggests that each subspecies of this enzyme has a defined function in processing and modulating tissue responses to external stimuli.     

The heterogeneity and differential expression of multiple species of the protein kinase C family

Although once considered as a single entity, enzymological and molecular cloning analysis has revealed that protein kinase C exists as a family of multiple subspecies with subtle individual characteristics. The members of this family have closely related structures, but their mode of activation, and kinetic and catalytic properties appear to differ slightly from one another. Biochemical and immunocytochemical studies indicate their differential regional expression and distinct cellular localization. It is attractive to surmise that each member of this family has a defined function in processing and modulating the physiological and pathological response of different cell types to a variety of external stimuli.     

Differential expression of multiple protein kinase C subspecies in rat central nervous tissue

Protein kinase C from a number of areas of rat central nervous tissue was resolved into three distinct fractions upon hydroxyapatite column chromatography. One of the enzyme fractions, designated type II, could be further distinguished into two subspecies with polyclonal antisera, which were raised against synthetic peptides specific for the predicted amino acid sequences of two alternative cDNA clones encoding this enzyme type. Using a combination of these biochemical and immunological techniques, the relative activity of the multiple subspecies of protein kinase C was assessed for each brain area. A distinct regional pattern of expression was found, which per se may be an important factor in determining the response of different neuronal cell types to extracellular stimuli.     

Diacylglycerol kinase in the central nervous system--molecular heterogeneity and gene expression.

Diacylglycerol (DAG) is one of the important second messengers, which serves as an activator of protein kinase C (PKC). DAG kinase (DGK) phosphorylates DAG to generate phosphatidic acid, thus DGK is considered to be a regulator of PKC activity through attenuation of DAG. Recent studies have revealed molecular structures of several DGK isozymes from mammalian species, and showed that most of the isozymes are expressed in the brain in various amounts. We have cloned four DGK isozyme cDNAs from rat brain library (DGK alpha, -beta, -gamma, and -zeta) (previously also designated DGK-I, -II, -III, and -IV, respectively) and examined their mRNA expressions in rat brain by in situ hybridization histochemistry. Interestingly, it is revealed that the mRNA for each isozyme is expressed in a distinct pattern in the brain; DGK alpha is expressed in oligodendrocytes, glial cells that form myelin; DGK beta in neurons of the caudate-putamen; DGK gamma predominantly in the cerebellar Purkinje cells; and DGK zeta in the cerebellar and cerebral cortices. Molecular diversity and distinct expression patterns of DGK isozymes suggest a physiological importance for the enzyme in brain function. Furthermore, functional implications of these DGK isozymes are briefly discussed.     

Cloning and differential expression of new calcium, calmodulin-dependent protein kinase II isoforms in Xenopus laevis oocytes and several adult tissues

The different oligomers composing the high molecular weight calcium/calmodulin-dependent protein kinase II (CaMKII) holoenzyme, previously shown to be transiently activated during Xenopus oocyte maturation, migrate on SDS-PAGE as proteins of 83, 72, 62, 56, and 52 kDa and have all been cloned. The holoenzyme consists of the CaMKII isoforms gammaB, gammaC, and delta12, already described in other species, while gammaJ, gammaK, gammaL, gammaM, and gammaN are now described for the first time. The gamma-isoforms are splice variants of the gamma-gene, containing in their variable region different combinations of known exons and one, two or three novel exons. Semi-quantitative RT-PCR revealed that all isoforms identified in prophase oocytes are also expressed in adult tissues with a tissue-specific expression pattern. At least thirty different CaMKII isoforms could be identified in different Xenopus adult tissues, most of which are described here for the first time.     

Evidence for clonal heterogeneity of the expression of six protein kinase C isoforms in murine B and T lymphocytes.

Protein kinase C (PKC), which plays a pivotal role in lymphocyte activation, represents a homologous family of at least nine proteins. Seven genes that encode PKC proteins have been identified. Since the regulatory properties and substrate specificities of the isoforms are not identical in vitro, it is possible that each isoform plays a unique role in cell activation. Toward an understanding of the role of PKC isoforms in lymphocyte activation we have studied the expression of mRNA encoding six of the isoforms (alpha, beta, gamma, delta, epsilon, and zeta) in T cell clones and B cell lines. PKC isoform phenotyping was done by MAPPing using isoform-specific primers and slot-blot analyses of mRNA were performed using specific probes. T cell clones and B cell lines were determined to express levels of the delta, epsilon, and zeta isoforms of PKC that were detectable by MAPPing. Plasmacytomas did not express PKC-beta message detectable by MAPPing. Slot blot analyses and Western blot analyses with peptide-specific antibody confirmed that B cell plasmacytomas did not express PKC-beta mRNA or protein. T cell clones and B cell lines were similar in that none expressed PKC-gamma. In cells that expressed PKC isoforms that were detectable by the MAPPing protocol, there was heterogeneity in the relative abundance of isoform mRNA (PKC-delta and -beta) and protein (PKC-beta and -epsilon). Such diversity of isoform expression could be responsible for the differential responsiveness of lymphocyte clones to activating stimuli.     

Cloning and expression of multiple protein kinase C cDNAs

Three different protein kinase C related cDNA clones were isolated from a rat brain cDNA library and designated PKC-I, PKC-II, and PKC-III. These each encode very similar, but distinct, polypeptides that contain a region homologous with other protein kinases. COS cells transfected with either PKC-I or PKC-II specifically bind at least 5-fold more 3H-PDBu (phorbol ester) than control cells. An increase in Ca2+, phosphatidylserine, and diacylglycerol/phorbol-ester-dependent protein kinase activity is also observed in COS cells transfected with either PKC-I or PKC-II. The physiological implications of the discovery of three protein-kinase-C-related cDNAs are discussed.     

Dose-dependent differential expression of protein kinase C isozymes in mouse lymphocytes after gamma irradiation in vivo and ex vivo

Protein kinase C (PKC, now known as Prkc) plays an important role in the response of cells to radiation, but little is known about the specific response of each isozyme in the radiation-induced response of cells in whole animals. However, most studies are based on single cells. There is a paucity of data on signaling after whole-body irradiation. In this study, a comparison has been made between the expression of Prkc isozymes after in vivo and ex vivo irradiation. There was a significant difference in the dose response of the isozymes. In animals in which lymphocytes were irradiated ex vivo, the expression of the Prkca isozyme was found to be maximum at 3 Gy, while in vivo irradiation did not increase the expression beyond that of 1 Gy. Prkcd was marginally activated after 0.1 Gy ex vivo irradiation, whereas there was significant activation of expression after in vivo irradiation with 3 Gy. The response of Prkcz was found to be similar to that of Prkcd. Prkc is a crucial enzyme that is being used to manipulate the response of tumors to radiotherapy. Conventional radiotherapy is delivered at low doses, and hence only those isozymes that are activated at these doses should be taken into consideration. Moreover, the differences between the response of a single cell and that of the whole animal must be considered.     

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.     

Reciprocal regulation of protein kinase C isoforms results in differential cellular responsiveness

Immunological homeostasis is often maintained by counteractive functions of two different cell types or two different receptors signaling through different intermediates in the same cell. One of these signaling intermediates is protein kinase C (PKC). Ten differentially regulated PKC isoforms are integral to receptor-triggered responses in different cells. So far, eight PKC isoforms are reported to be expressed in macrophages. Whether a single receptor differentially uses PKC isoforms to regulate counteractive effector functions has never been addressed. As CD40 is the only receptor characterized to trigger counteractive functions, we examined the relative role of PKC isoforms in the CD40-induced macrophage functions. We report that in BALB/c mouse macrophages, higher doses of CD40 stimulation induce optimum phosphorylation and translocation of PKCα, βI, βII, and ε whereas lower doses of CD40 stimulation activates PKCδ, ζ, and λ. Infection of macrophages with the protozoan parasite Leishmania major impairs PKCα, βI, βII, and ε isoforms but enhances PKCδ, ζ, and λ isoforms, suggesting a reciprocity among these PKC isoforms. Indeed, PKCα, βI, βII, and ε isoforms mediate CD40-induced p38MAPK phosphorylation, IL-12 expression, and Leishmania killing; PKCδ and ζ/λ mediate ERK1/2 phosphorylation, IL-10 production, and parasite growth. Treatment of the susceptible BALB/c mice with the lentivirally expressed PKCδ- or ζ-specific short hairpin RNA significantly reduces the infection and reinstates host-protective IFN-γ-dominated T cell response, defining the differential roles for PKC isoforms in immune homeostasis and novel PKC-targeted immunotherapeutic and parasite-derived immune evasion strategies.     

Localization of subspecies of protein kinase C in the mammalian central nervous system

Activation of protein kinase C (PKC) is regulated by dual second messengers; diacylglycerol (DG) produced by receptor mediated hydrolysis of phosphatidylinositol and Ca²⁺ which is released by inositol 1,4,5-triphosphate (IP3) from intracellular stores in the endoplasmic reticulum. In the mammalian central nervous system, available evidence suggests that PKC plays a prominent role in the processing of neuronal signals and in the short-term or long-term modulation of synaptic transmission. This enzyme is a member of a family consisting of at least eight subspecies, , βI, βII, γ, δ, , ζ and η. The homologous structure of each subspecies makes difficult resolution of the enzymological properties of the enzyme. The distinct functional roles of PKC subspecies in mammalian tissues have been elucidated by defining the localization of each subspecies. We identified -, βI-, βII- and γ-PKC subspecies in the rat brain by in situ hybridization and by light and electron microscopic immunohistochemistry, using antibodies specific for each subspecies. Most immunoreactions of the , βI, βII and γ subspecies were evident in neurons and there were few, if any, in glial cells. In this article, we summarize known cellular and subcellular localizations of PKC subspecies in mammalian CNS and some aspects of current studies in neuronal functions regulated by this enzyme are discussed.     

Heterogeneity of protein kinase C expression and regulation in T lymphocytes

The purpose of the present study was to examine protein kinase C (PKC) isotype expression in T lymphoblasts derived from peripheral blood and the T leukaemic cell Jurkat. Using antisera reactive with PKC alpha, beta 1, and beta 2 and gamma, it was observed that T cells expressed two PKC isotypes, PKC alpha and beta 1. No PKC gamma was detected in T lymphocytes. In lymphoblasts, high levels of PKC beta compared to PKC alpha were found whereas Jurkat cells expressed high levels of alpha compared to PKC beta. Differences in the calcium sensitivity of phorbol ester-induced phosphorylation were observed in Jurkat and T lymphoblasts which correlated with the relative levels of PKC alpha and beta isotypes expressed by the cells.     

Novel protein kinase C isoforms and mitogen-activated kinase kinase mediate phorbol ester-induced osteopontin expression

BACKGROUND AND AIMS: The expression of osteopontin (OPN), a protein postulated to play a role in tumorigenesis, is induced by the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA) in vivo and in the in vitro initiation-promotion skin carcinogenesis model (JB6 cells). Although TPA-induced OPN expression in JB6 cells has been suggested to involve protein kinase C (PKC), the PKC isoforms and the downstream pathway mediating OPN expression have not been extensively studied. METHODS: Using the JB6 cell model, we determined the involvement of PKC isoforms, mitogen-activated protein kinase kinase (MAPK kinase/MEK) and MAPK in TPA-induced OPN expression using inhibitors specific to PKC isoforms and MEK and performing Northern blot analyses. Western blot analyses of cells treated with specific inhibitors were also performed to determine whether PKC isoforms or MEK were involved in activation of MAPK. KEY RESULTS: TPA increased the steady-state level of OPN mRNA as early as 2-4h and this expression persisted for at least 4 days. TPA induction of OPN expression in JB6 cells is mediated through PKC epsilon and PKC delta, which also mediated the phosphorylation of MAPK. Additionally, inhibition of MEK activity, which activates MAPK, attenuated TPA-induced OPN expression. These findings suggest that activation of MAPK is important in mediating OPN expression. CONCLUSION: TPA-induced steady-state OPN mRNA expression in mouse JB6 cells involves the activation of MAPK mediated through PKC epsilon and/or PKC delta.     

Tissue distribution and developmental expression of protein kinase C isozymes

Protein kinase C is a ubiquitous enzyme found in a variety of mammalian tissues and is especially highly enriched in brain and lymphoid organs. Based on biochemical and immunological analyses, we have identified three types of protein kinase C isozyme (designated types I-III) from rat brain. Monospecific antibodies against each of the protein kinase C isozymes were prepared for the determination of tissue distribution, subcellular localization, and developmental changes of these enzymes. The various protein kinase C isozymes were found to be distinctively distributed in different tissues: the type I enzyme in brain; the type II enzyme in brain, pituitary and pineal glands, spleen, thymus, retina, lung, and intestine; and the type III enzyme in brain, pineal gland, retina, and spleen. The rat brain enzymes were differentially distributed in different subcellular fractions. The type I enzyme appeared to be most lipophilic and was recovered mostly in the particulate fractions (80-90%) regardless of the EGTA- or Ca2+-containing buffer used in the homogenization. Significant amounts (30-40%) of the type II and III enzymes were recovered in the cytosolic fraction with EGTA-containing buffer. The expressions of different protein kinase C isozymes appear to be differently controlled during development. In rat brain, both type II and III enzymes were found to increase progressively from 3 days before birth up to 2-3 weeks of age and remained constant thereafter. However, the expression of the type I enzyme displayed a different developmental pattern; it was very low within 1 week, and an abrupt increase was observed between 2 and 3 weeks of age. In thymus, the type II enzyme was found to be maximal shortly after birth; whereas the same kinase in spleen was very low within 2 weeks of age, and a significant increase was observed between 2 and 3 weeks. These results demonstrate that protein kinase C isozymes are distinctively distributed in different tissues and subcellular locales and that their expressions are controlled differently during development.     

Phorbol ester induces differential membrane-association of protein kinase C subspecies in human platelets

Human platelets contained proteins which cross-reacted with antisera specific for brain protein kinase C-alpha and -beta. When platelets were incubated with 12-O-tetradecanoylphorbol-13-acetate there was a rapid accumulation of protein kinase C-alpha in the particulate fraction associated with a loss of this subspecies from the soluble fraction. No particulate accumulation or soluble loss of protein kinase C-beta could be detected when platelets were incubated with the phorbol ester.     

Cellular distribution and developmental expression of AMP-activated protein kinase isoforms in mouse central nervous system

The mammalian AMP-activated protein kinase is a heterotrimeric serine/threonine protein kinase with multiple isoforms for each subunit (alpha, beta, and gamma) and is activated under conditions of metabolic stress. It is widely expressed in many tissues, including the brain, although its expression pattern throughout the CNS is unknown. We show that brain mRNA levels for the alpha2 and beta2 subunits were increased between embryonic days 10 and 14, whereas expression of alpha1, beta1, and gamma1 subunits was consistent at all ages examined. Immunostaining revealed a mainly neuronal distribution of all isoforms. The alpha2 catalytic subunit was highly expressed in neurons and activated astrocytes, whereas the alpha1 catalytic subunit showed low expression in neuropil. The gamma1 noncatalytic subunit was highly expressed by neurons, but not by astrocytes. Expression of the beta1 and beta2 noncatalytic subunits varied, but some neurons, such as granule cells of olfactory bulb, did not express detectable levels of either beta isoform. Preferential nuclear localization of the alpha2, beta1, and gamma1 subunits suggests new functions of the AMP-activated protein kinase, and the different expression patterns and cellular localization between the two catalytic subunits alpha1 and alpha2 point to different physiological roles.     

The expression and stage-specific localization of protein kinase C isotypes during mouse preimplantation development

Signaling events mediate many processes that act during embryogenesis to initiate the program of early development. Within the cell many of these changes are mediated through the activation or inactivation of kinases and phosphatases. Protein kinase C (PKC) is one kinase that has been shown to be involved in at least two developmental transitions during early development, fertilization and embryonic compaction. PKC is a family of kinases whose various isotypes have differing requirements for activation of the kinase that include the availability of calcium, diacylglycerol, and negatively charged phospholipids. The presence of more than one isotype in an egg or blastomere of the embryo would provide the possibility that different isotypes mediate distinct signaling pathways in the cells. To address this possibility the different isotypes of PKC were examined at the mRNA and protein levels during preimplantation development in the mouse. Our results demonstrate that seven isotypes of PKC are present during preimplantation development in mouse, some are of maternal origin and others appear after fertilization. Two isotypes have a stage-dependent nuclear localization. In addition, within each blastomere PKC isotypes occupy different subcellular locations in a stage-dependent fashion.