Epsilon Activity Research Articles

Preconditioning with spreading depression activates specifically protein kinase Cdelta.

Preconditioning with brief ischemia or spreading depression (SD) confers tolerance in cortical neurons to subsequent episode of ischemia. In myocardium a similar preconditioning is achieved by mechanisms, which are mediated by protein kinase C (PKC) alpha, delta, epsilon or zeta isoform. We induced SD by cortical application of KCl in the rat and analyzed cortical tissues after recovery of 30 min, 4 h and 12 h. While no changes at protein levels or activity of PKCalpha, epsilon or zeta were detected, a considerable increase in membrane translocation of PKCdelta was seen at 30 min and 12 h. A significant increase at mRNA level, protein amount and autophosphorylation at 12 h confirmed the late activation of PKCdelta, which may be involved in neuronal protection by preconditioning.

Involvement of the essential yeast DNA polymerases in induced gene conversion.

In the yeast Saccharomyces cerevisiae three different DNA polymerases alpha, delta and epsilon are involved in DNA replication. DNA polymerase alpha is responsible for initiation of DNA synthesis and polymerases delta and epsilon are required for elongation of DNA strand during replication. DNA polymerases delta and epsilon are also involved in DNA repair. In this work we studied the role of these three DNA polymerases in the process of recombinational synthesis. Using thermo-sensitive heteroallelic mutants in genes encoding DNA polymerases we studied their role in the process of induced gene conversion. Mutant strains were treated with mutagens, incubated under permissive or restrictive conditions and the numbers of convertants obtained were compared. A very high difference in the number of convertants between restrictive and permissive conditions was observed for polymerases alpha and delta, which suggests that these two polymerases play an important role in DNA synthesis during mitotic gene conversion. Marginal dependence of gene conversion on the activity of polymerase epsilon indicates that this DNA polymerase may be involved in this process but rather as an auxiliary enzyme.

A neutralizing antibody against human DNA polymerase epsilon inhibits cellular but not SV40 DNA replication.

The contribution of human DNA polymerase epsilon to nuclear DNA replication was studied. Antibody K18 that specifically inhibits DNA polymerase activity of human DNA polymerase epsilon in vitro significantly inhibits DNA synthesis both when microinjected into nuclei of exponentially growing human fibroblasts and in isolated HeLa cell nuclei. The capability of this neutralizing antibody to inhibit DNA synthesis in cells is comparable to that of monoclonal antibody SJK-132-20 against DNA polymerase alpha. Contrary to the antibody against DNA polymerase alpha, antibody K18 against DNA polymerase epsilon did not inhibit SV40 DNA replication in vitro. These results indicate that DNA polymerase epsilon plays a role in replicative DNA synthesis in proliferating human cells like DNA polymerase alpha, and that this role for DNA polymerase epsilon cannot be modeled by SV40 DNA replication.

Administration of dexamethasone up-regulates protein kinase C activity and the expression of gamma and epsilon protein kinase C isozymes in the rat brain.

Altered hypothalamic-pituitary-adrenal (HPA) function (increased plasma cortisol level) has been shown to be associated with mood and behavior. Protein kinase C (PKC), an important component of the phosphatidyl-inositol signal transduction system, plays a major role in mediating various physiological functions. The present study investigates the effects of acute (single) and repeated (10-day) administrations of 0.5 or 1.0 mg/kg doses of dexamethasone (DEX), a synthetic glucocorticoid, on Bmax and KD of [3H]phorbol 12,13-dibutyrate ([3H]PDBu) binding, PKC activity, and protein expression of PKC isozymes alpha, beta, gamma, delta, and epsilon in the membrane and the cytosolic fractions of rat cortex and hippocampus. It was observed that repeated administration of 1.0 mg/kg DEX for 10 days caused a significant increase in Bmax of [3H]PDBu binding to PKC, in PKC activity, and in expressed protein levels of the gamma and epsilon isozymes in both the cytosolic and the membrane fractions of the cortex and the hippocampus, whereas a lower dose of DEX (0.5 mg/kg for 10 days) caused these changes only in the hippocampus. On the other hand, a single administration of DEX (0.5 or 1.0 mg/kg) had no significant effect on PKC in the cortex or in the hippocampus. These results suggest that alterations in HPA function from repeated administration of glucocorticoids may modulate PKC-mediated functions.

DNA polymerase epsilon encoded by cdc20+ is required for chromosomal DNA replication in the fission yeast Schizosaccharomyces pombe.

DNA polymerase II (PolII), the homologue of mammalian DNA polymerase epsilon, is essential for chromosomal DNA replication in the budding yeast Saccharomyces cerevisiae and also participates in S-phase checkpoint control. An important issue is whether chromosomal DNA replication in other eukaryotes, including the fission yeast Schizosaccharomyces pombe--in which the characteristics of replication origins are poorly defined--also requires DNA polymerase epsilon. It has been shown that DNA polymerase epsilon is not required for the in vitro replication of SV40 DNA by human cell extracts. We have cloned and sequenced S. pombe pol2+, which is identical to the cell-cycle gene cdc20+, encoding the catalytic polypeptide of DNA polymerase epsilon (Pol epsilon). The predicted amino acid sequence of Pol epsilon is highly homologous to that of S. cerevisiae PolII and human Pol epsilon. Consistent with this, the Pol epsilon polypeptide was recognized by polyclonal antibodies against S. cerevisiae PolII holoenzyme (PolII*). The terminal morphology of cells containing the disrupted pol2 gene was similar to that of DNA replication mutant cells and cdc20 mutant cells. Furthermore, the Pol epsilon activity from temperature-sensitive S. pombe cdc20 mutant cells was temperature-sensitive, and chromosomal DNA replication in the mutant cells was inhibited at the restrictive temperatures. These data strongly suggest that Pol epsilon is required for normal chromosomal DNA replication in S. pombe, as is PolII in S. cerevisiae. Thus, eukaryotic chromosomal DNA is replicated differently from that of viral SV40 DNA.

Autoinhibition of Casein Kinase I ε (CKIε) Is Relieved by Protein Phosphatases and Limited Proteolysis

Casein kinase I epsilon (CKI epsilon) is a member of the CKI gene family, members of which are involved in the control of SV40 DNA replication, DNA repair, and cell metabolism. The mechanisms that regulate CKI epsilon activity and substrate specificity are not well understood. We report that CKI epsilon, which contains a highly phosphorylated 123-amino acid carboxyl-terminal extension not present in CKI alpha, is substantially less active than CKI alpha in phosphorylating a number of substrates including SV40 large T antigen and is unable to inhibit the initiation of SV40 DNA replication. Two mechanisms for the activation of CKI epsilon have been identified. First, limited tryptic digestion of CKI epsilon produces a protease-resistant amino-terminal 39-kDa core kinase with several-fold enhanced activity. Second, phosphatase treatment of CKI epsilon activates CKI epsilon 5-20-fold toward T antigen. Similar treatment of a truncated form of CKI epsilon produced only a 2-fold activation. Notably, this activation was transient; reautophosphorylation led to a rapid down-regulation of the kinase within 5 min. Phosphatase treatment also activated CKI epsilon toward the novel substrates I kappa B alpha and Ets-1. These mechanisms may serve to regulate CKI epsilon and related forms of CKI in the cell, perhaps in response to DNA damage.

The regulatory functions of the gamma and epsilon subunits from chloroplast CF1 are transferred to the core complex, alpha3beta3, from thermophilic bacterial F1.

The expression plasmids for the subunit gamma (gamma(c)) and the subunit epsilon (epsilon(c)) of chloroplast coupling factor (CF1) from spinach were constructed, and the desired proteins were expressed in Escherichia coli. Both expressed subunits were obtained as inclusion bodies. When recombinant gamma(c) was mixed with recombinant alpha and beta subunits of F1 from thermophilic Bacillus PS3 (TF1), a chimeric subunit complex (alpha3beta3gamma(c)) was reconstituted and it showed significant ATP hydrolysis activity. The ATP hydrolysis activity of this complex was enhanced in the presence of dithiothreitol and suppressed by the addition of CuCl2, which induces formation of a disulfide bond between two cysteine residues in gamma(c). Hence, this complex has similar modulation characteristics as CF1. The effects of recombinant epsilon(c) and epsilon subunit from TF1 (epsilon(t)) on alpha3beta3gamma(c) were also investigated. Epsilon(c) strongly inhibited the ATP hydrolysis activity of chimeric alpha3beta3gamma(c) complex but epsilon(t) did not. The inhibition was abolished and the ATP hydrolysis activity was recovered when methanol was added to the assay medium. The addition of epsilon(c) or epsilon(t) to the alpha3beta3gamma(t) complex, which is the authentic subunit complex from TF1, resulted in weak stimulation of the ATP hydrolysis activity. These results suggest that (a) the specific regulatory function of gamma(c) can be transferred to the bacterial subunit complex; (b) the interaction between the gamma(c) subunit and epsilon(c) strongly affects the enzyme activity, which was catalyzed at the catalytic sites that reside on the alpha3beta3 core.

Regulation of DNA metabolic enzymes upon induction of preB cell development and V(D)J recombination: up-regulation of DNA polymerase delta.

Withdrawal of interleukin-7 from cultured murine preB lymphocytes induces cell differentiation including V(D)J immunoglobulin gene rearrangements and cell cycle arrest. Advanced steps of the V(D)J recombination reaction involve processing of coding ends by several largely unidentified DNA metabolic enzymes. We have analyzed expression and activity of DNA polymerases alpha, beta, delta and epsilon, proliferating cell nuclear antigen (PCNA), topoisomerases I and II, terminal deoxynucleotidyl transferase (TdT) and DNA ligases I, III and IV upon induction of preB cell differentiation. Despite the immediate arrest of cell proliferation, DNA polymerase delta protein levels remained unchanged for approximately 2 days and its activity was up-regulated several-fold, while PCNA was continuously present. Activity of DNA polymerases alpha,beta and epsilon decreased. Expression and activity of DNA ligase I were drastically reduced, while those of DNA ligases III and IV remained virtually constant. No changes in DNA topoisomerases I or II expression and activity occurred and TdT expression was moderately increased early after induction. Our results render DNA polymerase delta a likely candidate acting in DNA synthesis related to V(D)J recombination in lymphocytes.

Inhibition of the 3′→ 5′ Exonuclease of Human DNA Polymerase ε by Fludarabine-terminated DNA

Incorporation of the anticancer drug fludarabine (9-beta-D-arabinofuranosyl-2-fluoroadenine 5'-monophosphate; F-ara-AMP) into the 3'-end of DNA during replication causes termination of DNA strand elongation and is strongly correlated with loss of clonogenicity. Because the proofreading mechanisms that remove 3'-F-ara-AMP from DNA represent a possible means of resistance to the drug, the present study investigated the excision of incorporated F-ara-AMP from DNA by the 3' --> 5'-exonuclease activity of DNA polymerase epsilon from human leukemia CEM cells. Using the drug-containing and normal deoxynucleotide oligomers (21-base) annealed to M13mp18(+) DNA as the excision substrates, we demonstrated that DNA polymerase epsilon was unable to effectively remove F-ara-AMP from the 3'-end of the oligomer. However, 3'-terminal dAMP and subsequently other deoxynucleotides were readily excised from DNA in a distributive fashion. Kinetic evaluation demonstrated that although DNA polymerase epsilon has a higher affinity for F-ara-AMP-terminated DNA (Km = 7.1 pM) than for dAMP-terminated DNA of otherwise identical sequence (Km = 265 pM), excision of F-ara-AMP proceeded at a substantially slower rate (Vmax = 0.053 pmol/min/mg) than for 3'-terminal dAMP (Vmax = 1.96 pmol/min/mg). When the 3'-5' phosphodiester bond between F-ara-AMP at the 3'-terminus and the adjacent normal deoxynucleotide was cleaved by DNA polymerase epsilon, the reaction products appeared to remain associated with the enzyme but without the formation of a covalent bond. No further excision of the remaining oligomers was observed after the addition of fresh DNA polymerase epsilon to the reaction. Furthermore, the addition of DNA polymerase alpha and deoxynucleoside triphosphates to the excision reaction failed to extend the oligomers. After DNA polymerase epsilon had been incubated with 3'-F-ara-AMP-21-mer for 10 min, the enzyme was no longer able to excise 3'-terminal dAMP from a freshly added normal 21-mer annealed to M13mp18(+) template. We conclude that the 3' --> 5' exonuclease of human DNA polymerase epsilon can remove 3'-terminal F-ara-AMP from DNA with difficulty and that this excision results in a mechanism-mediated formation of "dead end complex."

Further Characterization of HeLa DNA Polymerase ∊

DNA polymerase epsilon (pol epsilon) from HeLa cells was purified to near homogeneity, utilizing Mono S fast protein liquid chromatography for complete separation from pol alpha. The purified pol epsilon preparation showed two polypeptides of > 200 and 55 kDa and a small amount of active 122-kDa proteolysis product on denaturing polyacrylamide gels. Pol epsilon (as well as pols alpha and delta) is optimally active in 100-150 mM potassium glutamate and 15 mM MgCl2. Replication factors RF-A and RF-C, proliferating cell nuclear antigen, and Escherichia coli single-stranded DNA binding protein showed no significant effect on this preparation's pol epsilon activity, processivity, or substrate specificity. The size of the pol epsilon transcript for the catalytic subunit (> 200 kDa) was investigated in both normal human fibroblasts and HeLa cells. A 7.7-kilobase transcript was detected which was 5-16-fold more prevalent in proliferating than in quiescent HeLa cells. No significant difference in the level of pol epsilon transcript in HeLa cells or fibroblasts was seen after ultraviolet irradiation. Mouse polyclonal antiserum was produced to a 144-amino acid fragment of pol epsilon fused to staphylococcal protein A. This non-neutralizing polyclonal antiserum specifically recognized the catalytic subunit of pol epsilon by immunoblotting, but not that of pol alpha, beta, or delta. In addition, mouse polyclonal antiserum raised against column-purified pol epsilon was able to recognize and to neutralize pol epsilon, and a mouse monoclonal antibody was raised which was able to recognize specifically the catalytic subunit of pol epsilon.

DNA polymerase epsilon interacts with proliferating cell nuclear antigen in primer recognition and elongation.

Kinetic analysis of DNA polymerase epsilon in its interaction with the homopolymeric template-primer poly(dA)/oligo(dT) and a singly-primed synthetic oligonucleotide of defined sequence indicated that primer utilization is inhibited by single-stranded DNA. Long single-stranded DNA regions appear to sequester DNA polymerase epsilon via nonproductive binding, thus reducing its catalytic efficiency. Proliferating cell nuclear antigen can reduce this nonproductive effect by increasing the rate of primer binding by DNA polymerase epsilon. Once the complex between DNA polymerase epsilon and the primer is formed, proliferating cell nuclear antigen can increase the rate of nucleotide incorporation. The results suggested a dual role of proliferating cell nuclear antigen in stimulating the activity of DNA polymerase epsilon, namely, first to facilitate primer binding and second to stimulate the synthetic activity itself. A model for the interaction between these two proteins in DNA synthesis is discussed.

Fludarabine inhibits DNA replication: a rationale for its use in the treatment of acute leukemias.

Fludarabine is a prodrug that must enter cells and be phosphorylated to the nucleoside triphosphate, F-ara-ATP, to elicit biological activity. F-ara-ATP serves as an inhibitory alternative substrate in several key processes involved in DNA synthesis. The enzymes required in DNA synthesis and affected by F-ara-ATP are ribonucleotide reductase, DNA primase, DNA polymerases, 3'-5' exonuclease activity of DNA polymerases delta and epsilon, and DNA ligase I. The action of fludarabine on DNA replication provides a compelling rationale to use this agent for leukemias where target cells are actively synthesizing DNA, for example acute myelogenous leukemia. Additionally, the role of F-ara-ATP to potentiate the activity of deoxycytidine kinase makes it an appropriate candidate to use in combination with other nucleoside analogs which require deoxycytidine kinase for their activation. The present article reviews the effect of fludarabine on enzymes involved in DNA synthesis and the role of fludarabine in combination with arabinosylcytosine for the treatment of diseases other than indolent leukemias.

Isolation and characterization of DNA polymerase epsilon from the silk glands of Bombyx mori

The silk gland of Bombyx mori, an endomitotically replicative tissue shows high levels of DNA polymerases alpha, delta, and epsilon activities. The ratio of polymerase alpha to that of delta plus epsilon is maintained at 1.1 to 1.3 in both the posterior and middle silk glands for the entire duration of late larval development. The three activities copurify in the initial stages of fractionation through phosphocellulose and DE52 but polymerase alpha gets resolved from the others on hydroxylapatite column. Separation between polymerase delta and epsilon is achieved by chromatography on QAE-Sephadex. DNA polymerase epsilon is a heterodimer comprising of 215- and 42-kDa subunits. The activity is maximum at pH 6.5 and the Km values for dNTPs vary between 3-9 microM. The enzyme possesses an intrinsically associated exonuclease activity which functions in the mismatch repair during DNA synthesis. Both polymerase and 3'-->5' exonuclease activities are associated with the 215-kDa subunit. By itself, DNA polymerase epsilon is processive and the catalytic activity is not enhanced by externally added bPCNA (Bombyx-proliferating cell nuclear antigen, an auxiliary protein for DNA polymerase delta). The enzyme resembles polymerase delta in having the exonuclease activity and in its response to aphidicolin or substrate analogs, but could be distinguished from the latter by its lack of response to the bPCNA and sensitivity to dimethyl sulfoxide. The two enzymes show partial immunological cross-reactivity with each other but no immunological relatedness to polymerase alpha. The absence of the repair enzyme DNA polymerase beta and the presence of substantial levels of polymerase epsilon in the silk glands suggest a possible role for the latter in DNA repair in that tissue.

Effects of the anticancer drug cis-diamminedichloroplatinum(II) on the activities of calf thymus DNA polymerase epsilon.

DNA polymerase (pol) epsilon is essential for DNA replication and is thought to be a component of DNA repair systems in eukaryotic cells. The activities of pol epsilon have been examined using a series of synthetic oligonucleotides designed with cis-diamminedichloroplatinum(II) (cis-DDP)-modified specific guanine residues. Pol epsilon was incapable of synthesis over cis-DDP-modified single guanine or adjacent guanine residues present in the template strand. Both single and double guanines modified by cis-DDP present at the 3'-OH end of a primer strand completely inhibited the synthetic activity of pol epsilon and, in addition, sequestered pol epsilon at the platinated 3'-OH termini. The sequestering of pol epsilon on cis-DDP modified DNA may interfere with the function of this enzyme in DNA repair in vivo. The intrinsic 3' to 5' proofreading exonuclease activity of pol epsilon was also examined. Pol epsilon was capable of degrading a single-strand template with internal cis-DDP-modified guanines up to, but not through, the platinated nucleotides. A single platinated guanosine was sufficient to block the 3' to 5' exonuclease activity of pol epsilon. These results suggest that cis-DDP-DNA adducts inhibit DNA synthesis mediated by DNA polymerase epsilon and that platinated sites can arrest the nuclease of pol epsilon, a function exhibited during DNA repair.

The recognition of a recombinant human Fc epsilon fragment by the subclasses of IgG autoanti-IgE: disproportional subclasses distribution of complexed autoantibody.

Circulating IgG autoanti-IgE is detectable in a large proportion of individuals with allergic asthma where it is suggested to be potentially involved in the removal of IgE-allergen complexes. Since such a putative role will largely be determined by the subclass profile of complexed (i.e. IgE-bound) IgG anti-IgE, a study was undertaken to determine the subclass distribution of complexed IgG anti-IgE antibody in the sera of asthmatic patients. The study exploits the heat-labile property of IgE by heating (30 min at 56 degrees C) serum to liberate bound anti-IgE, pre- and post-heated sera are then assayed for IgG subclass anti-recombinant human Fc epsilon (rFc epsilon) activities by ELISA and any heat-induced increase in antibody activity is taken as a measure of complexed anti-IgE. This has revealed a disproportionately high amount of IgG4 in complexed (but not free) IgG anti-IgE. The propensity of IgG4 to form complexes with IgE has important biological consequences, particularly with regard to the activation of C1q and Fc gamma R by other subclasses.

Purification and characterization of protein kinase C .epsilon. from rabbit brain

Protein kinase C epsilon was chromatographically purified from rabbit brain to electrophoretic homogeneity. We identified the enzyme as the epsilon species of novel-type protein kinase C (nPKC epsilon), originally discovered and defined by cDNA cloning [Ohno, S., et al. (1988) Cell 53, 731-741], on the basis of the following observations: (i) the enzyme reacts specifically with an antipeptidic antiserum to nPKC epsilon but not with antisera to any of the other molecular species of PKC thus far known; (ii) it exhibits enzymatic behavior essentially identical to that of a recombinant nPKC epsilon purified from transfected COS cells [Konno, Y., et al. (1989) J. Biochem. 106, 673-678] and distinct from that of conventional PKC (alpha, beta I/II, and gamma) in its dependence on magnesium concentration and cofactors such as phospholipids, calcium, and phorbol ester; and (iii) it has an apparent molecular weight of 95.7K +/- 0.4K on SDS-PAGE, significantly greater than the other conventional and novel PKCs thus far identified. Notably, calcium exhibits a complex effect, both positive and negative, on the kinase activity of epsilon depending on the kind of substrate and the coexisting phospholipid, calling for a modification of the current notion that epsilon is a kinase unresponsive to calcium. The amount of epsilon species in the brain was estimated to be comparable to that of each conventional species, indicating that epsilon stands as one of the major PKC family members in brain. Furthermore, the enzyme shows a broader substrate spectrum than conventional PKC when examined with endogenous substrates, implying that it may cover a wider or different range of physiological functions.(ABSTRACT TRUNCATED AT 250 WORDS)

Synthesis of DNA by DNA polymerase epsilon in vitro.

The isolation of DNA polymerase (Pol) epsilon from extracts of HeLa cells is described. The final fractions contained two major subunits of 210 and 50 kDa which cosedimented with Pol epsilon activity, similar to those described previously (Syvaoja, J., and Linn, S. (1989) J. Biol. Chem. 264, 2489-2497). The properties of the human Pol epsilon and the yeast Pol epsilon were compared. Both enzymes elongated singly primed single-stranded circular DNA templates. Yeast Pol epsilon required the presence of a DNA binding protein (SSB) whereas human Pol epsilon required the addition of SSB, Activator 1 and proliferating cell nuclear antigen (PCNA) for maximal activity. Both enzymes were totally unable to elongate primed DNA templates in the presence of salt; however, activity could be restored by the addition of Activator 1 and PCNA. Like Pol delta, Pol epsilon formed complexes with SSB-coated primed DNA templates in the presence of Activator 1 and PCNA which could be isolated by filtration through Bio-Gel A-5m columns. Unlike Pol delta, Pol epsilon bound to SSB-coated primed DNA in the absence of the auxiliary factors. In the presence of salt, Pol epsilon complexes were less stable than they were in the absence of salt. In the in vitro simian virus 40 (SV40) T antigen-dependent synthesis of DNA containing the SV40 origin of replication, yeast Pol epsilon but not human Pol epsilon could substitute for yeast or human Pol delta in the generation of long DNA products. However, human Pol epsilon did increase slightly the length of DNA chains formed by the DNA polymerase alpha-primase complex in SV40 DNA synthesis. The bearing of this observation on the requirement for a PCNA-dependent DNA polymerase in the synthesis and maturation of Okazaki fragments is discussed. However, no unique role for human Pol epsilon in the in vitro SV40 DNA replication system was detected.

Identification and tryptic cleavage of the catalytic core of HeLa and calf thymus DNA polymerase epsilon.

DNA polymerase epsilon, formerly known as a proliferating cell nuclear antigen-independent form of DNA polymerase delta, has been shown elsewhere to be catalytically and structurally distinct from DNA polymerase delta. The catalytic activity of HeLa DNA polymerase epsilon, an enzyme consisting of greater than 200- and 55-kDa polypeptides, was assigned to the larger polypeptide by polymerase trap reaction. This catalytic polypeptide was cleaved by incubation with trypsin into two polypeptide fragments with molecular masses of 122 and 136 kDa, the former of which was relatively resistant to further proteolysis and possessed the polymerase activity. The cleavage increased the polymerase and exonuclease activities of the enzyme some 2-3-fold. DNA polymerase epsilon was also purified in a smaller 140-kDa form from calf thymus. The digestion of this form of the enzyme by trypsin also generated a 122-kDa polypeptide. These results suggest that the catalytic core of DNA polymerase epsilon is a 258-kDa polypeptide that is composed of two segments linked with a protease-sensitive area. One of the segments harbors both DNA polymerase and 3'----5' exonuclease activities. In spite of the different polypeptide structures, the catalytic properties of the HeLa enzyme, its trypsin-digested form, and the calf thymus enzyme remained essentially the same.

Expression and properties of two distinct classes of the phorbol ester receptor family, four conventional protein kinase C types, and a novel protein kinase C.

Five rabbit cDNAs, encoding four conventional protein kinase Cs (PKCs), alpha, beta I, beta II, and gamma, and a novel PKC-related protein (nPKC epsilon) were transfected into COS cells. Antisera raised against a bacterially synthesized fragment of PKC alpha or nPKC epsilon and against a chemically synthesized peptide of PKC beta I or beta II, specifically identified the corresponding species in the transfected cells. All four PKCs and nPKC epsilon expressed by transfection served as phorbol ester receptors. Phorbol 12,13-dibutyrate (PDBu)-binding activities of all PKCs and nPKC epsilon required phospholipid but not magnesium. The phosphatidylserine requirement for the activity of nPKC epsilon is independent of Ca2+ and similar to that for PKC alpha observed at 0.03 mM Ca2+. Calcium dependence of the binding activity was observed only for the four conventional PKCs. Scatchard plot analysis clearly showed that the dissociation constants of PDBu for all four PKCs were nearly the same (approximately 25 nM) in the presence of Ca2+, and that the value for nPKC epsilon was slightly higher (84 nM) and independent of Ca2+. The latter value is comparable to those observed in several cell types under conditions of Ca2+ chelation. Translocation of conventional PKC alpha to the membranes was induced with phorbol ester in a Ca2+-dependent manner, whereas the PDBu-stimulated translocation of nPKC epsilon did not require Ca2+. These results, together with previous studies on the enzymological characteristics of nPKC epsilon (Ohno, S., Akita, Y., Konno, Y., Imajoh, S., and Suzuki, K. (1988) Cell 53, 731-741), suggest that nPKC epsilon plays an important role in a transmembrane signaling pathway distinct from that involving conventional PKCs.

Enzymatic Properties of a Novel Phorbol Ester Receptor/Protein Kinase, nPKC1

A protein kinase C-related cDNA encodes a novel phorbol ester receptor/protein kinase, nPKC epsilon, clearly distinct from the four "conventional" PKCs [Ohno, S., Akita, Y., Konno, Y., Imajoh, S., & Suzuki, K. (1988) Cell 53, 731-741]. We purified nPKC epsilon from COS cells transfected with nPKC cDNA and compared its enzymatic properties with a conventional PKC, PKC alpha. nPKC epsilon was eluted from a hydroxyapatite column at a position coincident with type II PKC and thus was separated from type III PKC (PKC alpha), the only PKC expressed in COS cells. The protein kinase activity of nPKC epsilon is activated by phospholipids and diacylglycerols (or phorbol esters) in a manner similar to conventional PKCs. However, the cofactor dependencies and substrate specificities were clearly different from PKC alpha. A phospholipid, cardiolipin, enhances the kinase activity three- to fourfold compared with phosphatidylserine. The optimum Mg2+ concentration (3 mM) is clearly different from those of conventional PKCs (10-20 mM). The activation of nPKC epsilon by these cofactors is totally independent of Ca2+. Similar to conventional PKCs, nPKC epsilon autophosphorylates serine and threonine residues, indicating the specificity of the kinase to these amino acid residues. However, it shows a clearly different substrate specificity against exogenous substrates in that myelin basic proteins rather than histone are good substrates. These properties of nPKC epsilon permit clear discrimination of nPKC epsilon from conventional PKCs.