Protein Kinase C Gene Research Articles

Localization of the two protein kinase Cβ-mRNA subtypes in cat visual system

Protein kinase C (PKC) consists of a family of different subtypes encoded by different PKC genes. We investigated the distribution of PKCβ1 and PKCβ2 in the visual system of the adult cat by in situ hybridization using oligonucleotide probes complementary to the PKCβ1 and PKCβ2 mRNAs, two splicing variants of the same gene transcript. In the primary visual cortex PKCβ1 and PKCβ2 were both present. The laminar distribution patterns found for the two PKC subtypes were identical. A remarkable finding was the difference between the laminar distribution of the PKCβs in areas 17 and 18 when compared with area 19. In all three areas the highest expression levels were found in layer VI, moderately high levels were found in layers II, III and V, while layer I was devoid of signal. In area 17 and 18 layer IV stood out by its low PKCβ signal. In sharp contrast, layer IV of area 19 was indiscernible from the superficial layers because of an evenly high signal. In the dLGN of the adult cat PKCβ1 and PKCβ2 mRNAs were distributed rather homogeneously over the different layers, but the expression levels for PKCβ1 were clearly higher than those for PKCβ2.

Characterization of PKC2, a gene encoding a second protein kinase C isotype of Saccharomyces cerevisiae

Background: Protein kinase C (PKC) has attracted considerable attention over the past decade, primarily because of its presumed role in cellular growth control and tumourigenesis. Mammalian cells express at least 10 different isozymes of PKC; it is this complexity that has made elucidating the precise functions of PKC: so difficult. The identification of PKC homologues in organisms such as Drosophila, Xenopus, Dictyostelium, Aplysia and Caenorhabditis indicates that the enzyme is evolutionarily conserved, and this has stimulated our search for counterparts in the yeast Saccharomyces cerevisiae, in which powerful genetic analyses can be used. To date, only one PKC homologue, PKC1, has been identified in yeast and no biochemical activity has been definitively ascribed to the encoded protein. This, and the inability to identify other PKC homologues in yeast by DNA hybridization, has led to doubts about the existence of PKC isozymes in yeast. We have taken the approach of screening yeast expression libraries with anti-PKC antibodies in an attempt to identify further homologues.Results: We have identified a novel PKC isozyme, Pkc2p, encoded by the gene PKC2. We report here the sequence of PKC2 and a comparison showing its similarity to other PKCs. Phylogenetic analysis suggests that all known PKC genes, including PKC2, originated from a common ancestor. Disruption of the PKC2 protein-coding region, deleting the entire catalytic domain of the encoded enzyme, is not lethal to yeast growing on rich media. However, the pkc2 mutant, unlike wild-type strains, fails to grow on minimal media containing limited concentrations of amino acids. This implicates Pkc2p in the response of yeast cells to amino-acid starvation.Conclusion: We have shown that yeast cells do express more than one PKC isozyme, by identifying and characterizing a novel PKC gene PKC2, the product of which may be involved in the cellular response to amino-acid starvation.

PKC gamma gene expression is delayed in postnatal central nervous system of mi/mi mice.

In the central nervous system (CNS), the expression of protein kinase C (PKC) genes is strictly controlled by the developmental stage. We have examined the expression of PKC genes (cPKC alpha, beta, gamma, and nPKC delta, epsilon) in the process of the postnatal development in normal (+/+) C57BL/6 and microphthalmic (mi/mi) C57BL/6 mouse brains by Northern blotting and in situ hybridization. By Northern blotting, the expression level of cPKC gamma mRNA in mi/mi mice was significantly lower than that in +/+ littermates at d 9, 13, and 17. By in situ hybridization analysis, cPKC gamma mRNA-positive cells were detected in hippocampal and Purkinje cells in +/+ and mi/mi mice, but the magnitude of the signals in mi/mi mice was lower than that of +/+ mice, and the number of positive cells was smaller, whereas other isozymes (cPKC alpha, beta, and nPKC delta, epsilon) showed no significant difference between normal and mi/mi mice. The neuronal morphometric analysis by anti-P400 antibody revealed the same number and expression level of P400 protein in cerebellar Purkinje cells compared with +/+ mice. These results indicate that the deficiency of mi gene product causes the delayed expression of the cPKC gamma gene.

Phorbol ester-induced myeloid differentiation is mediated by protein kinase C-alpha and -delta and not by protein kinase C-beta II, -epsilon, -zeta, and -eta.

It is generally accepted that the multiple, similar protein kinase C (PKC) isozymes are responsible for different specialized physiological processes, but evidence that directly assigns specific functions to specific isozymes is scarce. To test whether specific PKC isozymes are involved in myeloid differentiation, we have studied the effect of overexpression of PKC-alpha, -beta II, -delta, -epsilon, -zeta and -eta in 32D, a mouse myeloid progenitor cell line that does not differentiate in response to 12-O-tetradecanoylphorbol-13-acetate (TPA). No significant morphological or phenotypic changes could be observed in unstimulated cells that overexpress any of these isozymes. However, the cell lines that overexpressed PKC-alpha or -delta had acquired the ability to become mature macrophages 2-6 h after TPA stimulation. The overexpression of PKC-beta II, -epsilon, -zeta, or -eta, in contrast, did not permit TPA-induced differentiation. These results indicate that only these two members of the PKC gene family can participate in TPA-induced myeloid differentiation.

Activation and substrate specificity of the human protein kinase C alpha and zeta isoenzymes.

Protein kinase C (PKC), a class of serine/threonine kinases activated by Ca2+ and/or phospholipids, is involved in a variety of cellular processes such as proliferation, differentiation and secretion. Nine members of the PKC gene family are known; these are differentially expressed in eukaryotic cells and can be divided into two sub-groups: the Ca(2+)-dependent (classical) PKC isoenzymes alpha, beta I, beta II and gamma, and the Ca(2+)-independent neoPKC isoenzymes delta, epsilon, zeta, eta and theta. A detailed biochemical characterisation of these PKC isoenzymes is one prerequisite for the elucidation of their distinct roles within cellular signal transduction. In this study, we report the cloning of a human PKC-zeta cDNA, its expression in recombinant baculovirus-infected insect cells and the partial purification of the PKC-zeta isoenzyme. In comparison to highly purified human PKC alpha, a representative of the classical PKC subgroup, purified PKC zeta was characterised with respect to activator requirement, substrate specificity, proteolytic activation and sensitivity towards PKC inhibitors. In contrast to PKC alpha, PKC zeta exhibits a constitutive kinase activity which is independent of Ca2+, phosphatidylserine and diacylglycerol. Arachidonic acid alone or a combination of gamma-linolenic acid and phosphatidylserine slightly enhance PKC zeta activity. In the presence of the classical PKC activators phosphatidylserine/diacylglycerol, PKC alpha phosphorylates a PKC-alpha pseudosubstrate-derived peptide, an epidermal-growth-factor-receptor-derived peptide, histone III-S and myelin basic protein to an equal extent, whilst PKC zeta phosphorylates only the PKC-alpha-derived peptide. However, arachidonic acid greatly diminishes PKC-alpha activity towards the epidermal-growth-factor-receptor-derived peptide, histone III-S and myelin basic protein, but enhances PKC-zeta activity towards the PKC-alpha-derived peptide. These results indicate a possible modulation of substrate specificity of these two PKC isoenzymes by (the binding of) different activators (to their regulatory domains). In the case of PKC zeta, this finding is strengthened by the fact that the epidermal growth factor receptor-derived peptide, which is not a substrate for the holoenzyme, is significantly phosphorylated by a protein fragment generated by limited proteolysis and comprising only the kinase domain. Furthermore, PKC zeta, in contrast to PKC alpha, is insensitive to PKC inhibitors known to interfere either with the regulatory or the catalytic domain and cannot be activated by phorbol ester treatment of NIH 3T3 cells or insect cells, overexpressing the respective PKC isoenzyme. The potential implications of these findings on the mechanism(s) of activation and the substrate specificity of PKC zeta are discussed.

Molecular cloning and characterization of PKC theta, a novel member of the protein kinase C (PKC) gene family expressed predominantly in hematopoietic cells.

Members of the protein kinase C (PKC) family of serine/threonine kinases play a key role in regulating the differentiation and growth of diverse cell types and, to date, the cloning of seven mammalian PKC genes encoding eight distinct isoforms has been reported. Here we describe the molecular cloning and deduced primary structure of a cDNA encoding a novel PKC isoform, termed PKC theta, which was isolated in the course of attempts to identify PKC genes that are expressed selectively in hematopoietic cells. Degenerate oligonucleotide primers corresponding to conserved sequence motifs, which distinguish the PKC family from other protein kinases, were employed in polymerase chain reactions (PCR) to amplify partial core sequences of putative PKC genes from a human peripheral blood lymphocyte-derived cDNA library. DNA sequencing of selected clones revealed several PKC-related sequences, including one that, on the basis of sequence comparison with known PKC isoforms, represented a novel PKC isoform. The complete cDNA sequence was determined by anchored PCR cloning and sequencing the entire coding sequence, using cDNA derived from a human leukemic T cell line (Jurkat). Included within this approximately 2.7-kilobase pair cDNA is an open reading frame of 2,118 nucleotides encoding a putative 82-kDa protein. The deduced primary structure contains consensus sequences characteristic of protein kinase catalytic domains and, based on its amino acid sequence and domain structure, is a member of the PKC family. PKC theta displays the highest homology to PKC delta, lacks the Ca(2+)-binding C2 domain and, thus, belongs to the subfamily of Ca(2+)-independent PKC enzymes which also includes the delta, epsilon, zeta, and eta isoforms. RNase protection assays and semiquantitative PCR analysis indicated that, although PKC theta transcripts are expressed ubiquitously, the highest levels are found in hematopoietic tissues and cell lines, including T cells and thymocytes. In contrast, the expression levels in the brain and testes are considerably lower, and no transcripts were detected in several human carcinoma cell lines. A rabbit antiserum raised against a unique (V3 domain) bacterially expressed PKC theta fragment immunoprecipitated specifically an 82-kDa protein from Jurkat cell lysates. Thus, PKC theta represents an additional member of the PKC family, and its predominant expression in hematopoietic cells suggests that it may play a role in signal transduction and growth regulatory pathways unique to these cells.

Mitogen-induced human T cell proliferation is associated with increased expression of selected PKC genes

The induction of T cell proliferation and differentiation into mature effector cells is dependent on two principal exogenous signals that are provided by the antigen or mitogen and IL2. The enzyme protein kinase C (PKC) has a major role in the antigen-receptor signalling pathway in T cells, but appears not to be involved in signalling via the IL2-receptor (IL2-R). Since both pathways trigger a series of sequentially coordinated transcriptional events in which numerous genes are activated, we tested whether a T cell mitogen acting via the TCR/CD3 complex, and IL2, affect the expression of the conventional, Ca 2+-dependent, PKC genes (α, β and γ) in T cells. Stimulation of human peripheral blood lymphocytes or an enriched population of human T cells with phytohemagglutinin resulted in augmented mRNA levels of PKCα and PKCβ, but not PKCγ-gene. The response peaked at 24–48 hr when a 3–5-fold increase was observed. Stimulation of IL2-Rα-expressing T cells with human recombinant IL2 induced cell proliferation and transcription of the IL2-Rα gene (> 100-fold), but did not change mRNA levels of PKCa or PKCβ genes. The results suggest that stimulation of human T cells with mitogens acting via the TCR/CD3 complex, that involve activation of PKC, is accompanied also by a late activation of selected PKC genes. By contrast, agonists such as IL2, that operate via a different signalling pathway, do not modify the expression of any of the known conventional PKC genes.

Regulation of protein kinase C gene expression by retinoic acid in B16 mouse melanoma cells

We have previously shown that the retinoic acid (RA)-induced growth arrest and differentiation of B16 mouse melanoma cells is accompanied by a large increase in the amount and activity of protein kinase C (PKC). Since PKC is a multigene family, we investigated which isoforms were expressed in control and RA-treated B16 melanoma cells, and characterized the manner by which RA regulates PKC gene expression. We found that RA treatment of B16 cells resulted in an increase in PKCα mRNA beginning at 4–8 h and reached a maximum of 10- to 12-fold over control levels by 48 h. There was also a small amount of PKCγ mRNA, present only in 48-h RA-treated cells, but no PKCβ mRNA was detected. The effect of RA on PKCα mRNA induction was not direct since the induction was abolished when cycloheximide was included in the incubation medium. Nuclear run-on experiments showed that the RA-induced increase in PKCα steady-state mRNA was not entirely due to an increase in transcriptional activity, as the increase in PKCα transcription was only 2- to 3-fold over control, which is not enough to account for the 10- to 15-fold increase in steady state levels. There was also no change in PKCα mRNA stability in RA-treated B16 cells compared to untreated cells. The 10.9-kb PKCα message in both control and RA-treated cells was less stable than the 3.8-kb PKCα message. Therefore, we propose that the major level of control of PKCα mRNA levels by RA is post-transcriptional, either RNA processing or transport out of the nucleus.

Isolation and characterization of PKC-L, a new member of the protein kinase C-related gene family specifically expressed in lung, skin, and heart.:

We have isolated and characterized a new human cDNA, coding for a protein kinase, related to the protein kinase C (PKC) gene family. Although this protein kinase shares some homologous sequences and structural features with the four members of the PKC family initially isolated (alpha, beta I, beta II, and gamma), it shows more homology with the recently described PKC-related subfamily, encoded by the cDNAs delta, epsilon, and zeta. The transcript for this gene product, termed PKC-L, is most abundant in lung tissue, less expressed in heart and skin tissue, and exhibited very low expression in brain tissue. Thus, its tissue distribution is different from that described for other mammalian members of the PKC gene family, their expression being enriched in brain tissues. PKC-L is also expressed in several human cell lines, including the human epidermoid carcinoma line A431. The ability of phorbol esters to bind to and stimulate the kinase activity of PKC-L was revealed by introducing the cDNA into COS cells.

Transcriptional activation of the cardiac myosin light chain 2 and atrial natriuretic factor genes by protein kinase C in neonatal rat ventricular myocytes.

A cultured myocardial cell model was used to examine the role of protein kinase C-dependent pathways in the transcriptional activation of two cardiac muscle genes [myosin light chain 2 (MLC-2) and atrial natriuretic factor (ANF)] during alpha-adrenergic receptor-mediated hypertrophy. Phorbol ester (phorbol 12-myristate 13-acetate) and the alpha-adrenergic agonist phenylephrine both activate protein kinase C (PKC) and induce 4- to 5-fold increases in the expression of MLC-2 and ANF promoter/luciferase reporter genes with little effect on Rous sarcoma virus/luciferase or minimal prolactin promoter/luciferase genes. To further assess the role of PKC in cardiac gene regulation, PKC expression vectors encoding constitutively activated PKC-alpha or PKC-beta, or a catalytically inactive PKC, were transiently cotransfected with the cardiac promoter/luciferase constructs. Cotransfection of either activated PKC-alpha or PKC-beta cDNA induces the expression of MLC-2 and ANF promoter/luciferase genes and of a reporter gene responsive to the transcription factor AP-1. The Rous sarcoma virus/luciferase and minimal prolactin promoter/luciferase genes are not concomitantly induced by cotransfectin with the PKC genes, indicating specificity of the transcriptional effect. The finding that activated PKC increases cardiac gene transcription suggests that activation of this enzyme may be a proximal signal for coregulation of two cardiac genes, MLC-2 and ANF, during the course of myocardial cell hypertrophy.

Deficient protein kinase C activity in turnip, a Drosophila learning mutant

The Drosophila mutant turnip was initially isolated based on poor learning performance (Quinn, W.G., Sziber, P.P., and Booker, R. (1979) Nature 277, 212-214). Here we show that turnip is dramatically reduced in protein kinase C (PKC) activity. In addition, turnip flies are deficient in phosphorylation of a 76-kDa head membrane protein (hereafter pp76) which is a major substrate for protein kinase C in homogenates of wild-type flies. Reduced PKC activity, defective pp76 phosphorylation, and most of turnip's learning deficiency co-map genetically to a region on the X-chromosome, 18A5-18D1-2, spanned by the deletion Df(1)JA27. Apparently turnip+ is not a structural gene for PKC because Drosophila PKC genes map elsewhere in the genome. Our results suggest that turnip gene product is required for activation of PKC and that PKC plays a role in associative learning in Drosophila.

Protein kinase C subspecies distinguish major cell types in rat hippocampus: an immunocytochemical and in situ hybridization histochemical study.

This study compares the distribution of protein kinase C (PKC) alpha and beta with the distribution of PKC epsilon in the hippocampal formation of rats by immunocytochemistry and in situ hybridization histochemistry. Alpha and PKC beta are members of the group A PKC genes that were first described; PKC epsilon is a member of the group B PKC genes that were more recently identified by molecular cloning. A combination of all three gene products and their mRNAs overlapped in their distributions in dentate granule cells and pyramidal and nonpyramidal neurons. However, each subspecies predominated in one of the major cell types. PKC alpha-immunoreactivity and mRNA were most intense in CA2-3 pyramidal cells and dendrites, whereas PKC beta-immunoreactivity and mRNA were most intense in CA1 pyramidal cells and dendrites. PKC epsilon-immunoreactivity and mRNA were concentrated in dentate granule cells and CA3 pyramidal cells. Furthermore, PKC epsilon-immunoreactivity was detectable in mossy fibers. Each subspecies labeled different kinds of interneurons that were particularly numerous in, but not restricted to, the hilus. These data support the contention that different subtypes of hippocampal neurons are distinguished by the expression of different combinations of PKC subspecies under resting conditions.

Isolation and Characterization of PKC-L, A New Member of the Protein Kinase C-Related Gene Family Specifically Expressed in Lung, Skin, and Heart

We have isolated and characterized a new human cDNA, coding for a protein kinase, related to the protein kinase C (PKC) gene family. Although this protein kinase shares some homologous sequences and structural features with the four members of the PKC family initially isolated (alpha, beta I, beta II, and gamma), it shows more homology with the recently described PKC-related subfamily, encoded by the cDNAs delta, epsilon, and zeta. The transcript for this gene product, termed PKC-L, is most abundant in lung tissue, less expressed in heart and skin tissue, and exhibited very low expression in brain tissue. Thus, its tissue distribution is different from that described for other mammalian members of the PKC gene family, their expression being enriched in brain tissues. PKC-L is also expressed in several human cell lines, including the human epidermoid carcinoma line A431. The ability of phorbol esters to bind to and stimulate the kinase activity of PKC-L was revealed by introducing the cDNA into COS cells.

Isolation and characterization of PKC-L, a new member of the protein kinase C-related gene family specifically expressed in lung, skin, and heart.

We have isolated and characterized a new human cDNA, coding for a protein kinase, related to the protein kinase C (PKC) gene family. Although this protein kinase shares some homologous sequences and structural features with the four members of the PKC family initially isolated (alpha, beta I, beta II, and gamma), it shows more homology with the recently described PKC-related subfamily, encoded by the cDNAs delta, epsilon, and zeta. The transcript for this gene product, termed PKC-L, is most abundant in lung tissue, less expressed in heart and skin tissue, and exhibited very low expression in brain tissue. Thus, its tissue distribution is different from that described for other mammalian members of the PKC gene family, their expression being enriched in brain tissues. PKC-L is also expressed in several human cell lines, including the human epidermoid carcinoma line A431. The ability of phorbol esters to bind to and stimulate the kinase activity of PKC-L was revealed by introducing the cDNA into COS cells.

Expression of Protein Kinase C Isozyme in Epidermal Langerhans Cells of the Mouse

Protein kinase C (PKC) is encoded by a complex of a gene family, and its multiple isozymes are expressed in various mammalian tissues. We examined whether PKC is expressed in epidermal Langerhans cells (LC) of the mouse by using monoclonal antibodies specific to PKC I, PKC II, and PKC III isozymes (respective products of PKC genes gamma, beta, and alpha). Immunohistochemical and immunoblotting studies revealed that LC of adult C57BL/6 mice express PKC II, while PKC I and PKC III were not detected. In keratinocytes, none of the isozymes were detected. These results suggest that PKC II is a novel marker of LC and that it possibly plays a regulatory role in epidermal LC of the mouse in vivo.

Differential responsiveness to phorbol esters correlates with differential expression of protein kinase C in KG-1 and KG-1a human myeloid leukemia cells

The KG-1 myeloid leukemia cell line differentiates into macrophages in response to 12- O-tetradecanoylphorbol 13-acetate (TPA), while its spontaneous variant, the KG-1a cell line, is resistant to the differentiative effects of TPA. To determine the mechanism underlying these differential responses to TPA, experiments were performed to determine the relative numbers of TPA binding sites, protein kinase C (PKC) enzyme activity levels, PKC translocation responses and PKC gene expression in these cell lines. KG-1a cells exhibited 40% fewer high affinity binding sites for TPA than KG-1 cells. Although PKC translocation from cytosol to the membrane fraction was observed in both cell types, total PKC activity, measured in vitro using type III-S histone as substrate, was reduced by 70% in KG-1a cells. These biochemical differences between the parental line and the phorbol ester non-responsive variant were correlated with the depressed level of PKC-β RNA abundance in KG-1a cells. Both lines expressed PKC-α RNA at comparable levels. Chronic exposure to TPA resulted in down-regulation of PKC enzyme activity in both cell lines, and a selective decrease in PKC-β RNA transcripts in both cell types. In contrast, chronic TPA treatment had no effect on the levels of PKC-α RNA in KG-1 and KG-1a cells. Our results indicate a correlation between the level of PKC-β expression and the responsiveness of myeloid lineage precursor cells to the differentiative effects of TPA.

Isolation and characterization of two new Drosophila protein kinase C genes, including one specifically expressed in photoreceptor cells

We have isolated and characterized two new protein kinase C (PKC) genes from D. melanogaster. One, dPKC98F, maps to chromosome region 98F and displays over 60% amino acid sequence identity with members of a recently described “PKC-related” subfamily in mammals. The other, dPKC53E(ey), maps to region 53E 4–7 on the second chromosome and lies within 50 kb of a PKC gene previously characterized (dPKC). While dPKC98F transcripts are expressed throughout development, expression of the two genes mapping at cytogenetic location 53E is primarily in adults. dPKC98F and the previously reported 53E gene are transcribed predominantly in brain tissue. In contrast, dPKC53E(ey) is transcribed only in photoreceptor cells. We will discuss the significance of this tissue-specific localization with regard to phototransduction.

Expression of protein kinase C genes during ontogenic development of the central nervous system.

We have analyzed the RNA expression of three protein kinase C (PKC) genes (alpha, beta, and gamma) in human and murine central nervous systems during embryonic-fetal, perinatal, and adult life. Analysis of human brain poly(A)+ RNA indicates that expression of PKC alpha and beta genes can be detected as early as 6 weeks postconception, undergoes a gradual increase until 9 weeks postconception, and reaches its highest level in the adult stage, and that the PKC gamma gene, although not expressed during embryonic and early fetal development, is abundantly expressed in the adult period. Similar developmental patterns were observed in human spinal cord and medulla oblongata. A detailed analysis of PKC gene expression during mammalian ontogeny was performed on poly(A)+ RNA from the brain cells of murine embryos at different stages of development and the brain cells of neonatal and adult mice. The ontogenetic patterns were similar to those observed for human brain. Furthermore, we observed that the expression of PKC gamma is induced in the peri- and postnatal phases. These results suggest that expression of PKC alpha, beta, and gamma genes possibly mediates the development of central neuronal functions, and expression of PKC gamma in particular may be involved in the development of peri- and postnatal functions.

Expression of Protein Kinase C Genes during Ontogenic Development of the Central Nervous System

We have analyzed the RNA expression of three protein kinase C (PKC) genes (alpha, beta, and gamma) in human and murine central nervous systems during embryonic-fetal, perinatal, and adult life. Analysis of human brain poly(A)+ RNA indicates that expression of PKC alpha and beta genes can be detected as early as 6 weeks postconception, undergoes a gradual increase until 9 weeks postconception, and reaches its highest level in the adult stage, and that the PKC gamma gene, although not expressed during embryonic and early fetal development, is abundantly expressed in the adult period. Similar developmental patterns were observed in human spinal cord and medulla oblongata. A detailed analysis of PKC gene expression during mammalian ontogeny was performed on poly(A)+ RNA from the brain cells of murine embryos at different stages of development and the brain cells of neonatal and adult mice. The ontogenetic patterns were similar to those observed for human brain. Furthermore, we observed that the expression of PKC gamma is induced in the peri- and postnatal phases. These results suggest that expression of PKC alpha, beta, and gamma genes possibly mediates the development of central neuronal functions, and expression of PKC gamma in particular may be involved in the development of peri- and postnatal functions.

A protein kinase C cDNA without the regulatory domain is active after transfection in vivo in the absence of phorbol ester.

We constructed mutant protein kinase C (PKC) cDNAs which expressed PKC activity in vivo in the absence of phorbol ester activation. A hybrid PKC gene, PKAC, was constructed by substituting the coding region for the N-terminal 253 amino acids of PKC alpha with the N-terminal 17 amino acids of the cyclic AMP-dependent protein kinase catalytic subunit (PKA). A truncated PKC gene, delta PKC beta, lacking the coding region for amino acid positions 6 to 159 of PKC beta was also constructed. These mutant kinase genes expressed under the control of the SR alpha promoter activated the c-fos gene enhancer in Jurkat cells and initiated maturation of Xenopus laevis oocytes. Phorbol ester binding activity was absent in both constructs but was preserved in another hybrid gene, PKCA, which was composed of the coding region for 1 to 253 amino acids of PKC alpha at the N-terminal side and the coding region for 18 to 350 amino acids of PKA at the C-terminal side. These results indicate that elimination of the regulatory domain of PKC produces constitutively active PKC that can bypass activation by the phorbol ester. delta PKC beta, in synergy with a calcium ionophore, was capable of activating the interleukin 2 promoter, indicating that cooperation of PKC-dependent and calcium-dependent pathways is necessary for activation of the interleukin 2 gene.