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U Kikkawa

Publications and source records attributed to U Kikkawa.

At least 73 records · Page 4Linked to original sources
PubMed1995

Differential down-regulation of protein kinase C subspecies in normal human melanocytes: possible involvement of the zeta subspecies in growth regulation.

Normal human melanocytes are often grown in vitro in the continuous presence of 12-O-tetradecanoylphorbol-13-acetate (TPA) for growth in vitro. The expression of protein kinase C (PKC) subspecies, which are the major cellular receptors for phorbol esters, was examined in melanocytes after long-term treatment with TPA to investigate the role of PKC subspecies in TPA-dependent cell growth. The PKC enzyme activity detected in quiescent melanocytes was almost completely depleted in cells after incubation with 85 nM TPA for 48 h. Immunoblot analysis indicated that, among the PKC subspecies alpha, beta, delta, epsilon, and zeta expressed in quiescent cells, alpha-, beta-, delta-, and epsilon-PKC were significantly down-regulated, whereas zeta-PKC remained at detectable levels in TPA-treated cells. TPA did not significantly affect the expression or subcellular distribution of zeta-PKC in melanocytes. Immunoprecipitation assay revealed that the enzyme activity of zeta-PKC was increased in both the cytosol and particulate cell fractions, but the increase was much greater in the latter. The activation of zeta-PKC lasted for 24 to 48 h after the addition of TPA; thereafter, zeta-PKC activity returned to basal levels. DNA synthesis was shown to change concomitantly with the activation of zeta-PKC in TPA-treated cells. These results indicate that TPA induces not only the down-regulation of alpha-, beta-, delta-, and epsilon-PKC, but also long-term activation of zeta-PKC in melanocytes, and that activation of zeta-PKC parallels the growth of normal human melanocytes.

Cell Division↗
PubMed1994

The pleckstrin homology domain of RAC protein kinase associates with the regulatory domain of protein kinase C zeta.

Binding proteins to the pleckstrin homology domain of RAC protein kinase were screened by using glutathione S-transferase fusion protein system. Proteins in CHO cell extract of approximate molecular mass of 76 kD and 200 kD bound specifically to the pleckstrin homology domain of RAC protein kinase in vitro. The 76 kD protein was identified as protein kinase C zeta by immunoblot analysis. Studies of the association between the pleckstrin homology domain-truncated mutants and protein kinase C zeta indicated that the amino-terminal portion of the pleckstrin homology domain is essential for the binding and the whole structure of the domain is important for the efficient binding to protein kinase C zeta. The pleckstrin homology domain of RAC protein kinase was shown to recognize the regulatory domain of protein kinase C zeta. The protein-protein interaction between RAC protein kinase and protein kinase C through the pleckstrin homology domain might be important for the regulation of these protein kinases.

Animals↗
PubMed1994

Molecular cloning of rat RAC protein kinase alpha and beta and their association with protein kinase C zeta.

cDNAs of RAC protein kinases (RAC-PK) alpha and beta were cloned from a rat testis cDNA library. The predicted open reading frames encode 480 and 481 amino acids of RAC-PK alpha and beta, respectively, and the rat RAC-PK alpha and beta have sequences conserved among different mammalian species such as the pleckstrin homology domain at their amino-terminal region and the protein-serine/threonine kinase catalytic domain at their carboxyl-terminal region. RNA blot analysis showed wide distribution of two RAC-PK in rat tissues. Immunoprecipitation analysis revealed that RAC-PK alpha and beta associate with protein kinase C zeta through the pleckstrin homology domain in vitro, suggesting the interaction between RAC-PK and protein kinase C.

Amino Acid Sequence↗
PubMed1994

Purification and characterization of protein kinase C from a higher plant, Brassica campestris L.

Protein kinase C (PKC) was partially purified from Brassica campestris L., by successive chromatographies on DEAE-cellulose membrane, hydroxyapatite and phenyl-5PW columns. The purified preparation showed typical characteristics of the conventional type of mammalian PKC that responds to Ca2+, phosphatidylserine, and diacylglycerol or the tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate. The plant PKC activity was apparently associated with a 75-kDa polypeptide that was recognized by an antibody against the catalytic domain of rat PKC. Substrate specificity of the plant PKC was similar to that of the rat PKC. A synthetic peptide corresponding to residues 4-14 of myelin basic protein, which is a selective substrate for the mammalian PKC, was phosphorylated efficiently by the plant PKC. These results indicate the existence of a PKC equivalent in higher plant cells.

Brassica↗
PubMed1992

Cell division arrest induced by phorbol ester in CHO cells overexpressing protein kinase C-delta subspecies.

Several lines of CHO cells stably overexpressing protein kinase C (PKC) subspecies to various extents were established by the DNA-mediated transfer. Upon treatment with phorbol 12-myristate 13-acetate, the growth of the cells expressing the PKC-delta subspecies was markedly inhibited, whereas cell lines expressing PKC-alpha, PKC-beta II, and PKC-zeta subspecies were not significantly affected. Flow cytometric analysis indicated that all cell lines overexpressing PKC-delta subspecies accumulated in G2/M phase in response to phorbol 12-myristate 13-acetate. In these arrested cells, dikaryons were predominant, implying that phorbol ester-induced inhibition of cell division is specific to telophase. These results suggest PKC-delta subspecies may play a role in the normal cell cycle progression.

Animals↗
PubMed1992

Casein kinase II is a negative regulator of c-Jun DNA binding and AP-1 activity.

c-Jun, a major component of the inducible transcription factor AP-1, is a phosphoprotein. In nonstimulated fibroblasts and epithelial cells, c-Jun is phosphorylated on a cluster of two to three sites abutting its DNA-binding domain. Phosphorylation of these sites inhibits DNA binding, and their dephosphorylation correlates with increased AP-1 activity. We show that two of these sites, Thr-231 and Ser-249, are phosphorylated by casein kinase II (CKII). Substitution of the third site, Ser-243, by Phe interferes with phosphorylation of the inhibitory sites in vivo and by purified CKII in vitro. Microinjection into living cells of synthetic peptides that are specific competitive substrates or inhibitors of CKII results in induction of AP-1 activity and c-Jun expression. Microinjection of CKII suppresses induction of AP-1 by either phorbol ester or an inhibitory peptide. These results suggest that one of the roles of CKII, a major nuclear protein kinase with no known functions, is to attenuate AP-1 activity through phosphorylation of c-Jun.

Amino Acid Sequence↗
PubMed1992

Isolation and characterization of delta-subspecies of protein kinase C from rat brain.

The delta-subspecies of protein kinase C (delta PKC) was purified to near homogeneity from the Triton X-100 extract of the rat brain particulate fraction by successive chromatographies on S-Sepharose fast flow, phenyl 5PW, heparin 5PW, hydroxyapatite, and Mono Q columns. The purified enzyme was a doublet with molecular masses of 78 and 76 kDa on SDS/PAGE. The doublet proteins were separated partially by Mono Q column chromatography; both were recognized by the antibodies raised against synthetic oligopeptides, parts of the deduced amino acid sequence of the rat delta PKC. Protein phosphatase 2A treatment suggested that the 78-kDa protein was a phosphorylated form of the 76-kDa protein. To confirm the structural and genetic identity of the doublet proteins, delta PKC was expressed in COS 7 cells by transfecting its cDNA-constructed plasmid and was purified for comparison. This recombinant enzyme was also a doublet. The enzymes isolated from the brain and COS 7 cells showed identical reactivities with delta PKC-specific antibodies, chromatographic behaviors, and V8 protease peptide mappings. In addition, these two enzyme preparations were indistinguishable from each other in their responses to phosphatidylserine, diacylglycerol, phorbol esters, free fatty acids, Ca2+, and enzyme inhibitors. Comparison was also made between the enzymologic properties of delta PKC and alpha PKC, which were distinctly different from each other.

Animals↗
PubMed1992

Isolation and characterization of the epsilon subspecies of protein kinase C from rat brain.

The epsilon subspecies of protein kinase C (epsilon PKC) was purified to near homogeneity from the soluble fraction of rat brain by successive chromatographies on DEAE-cellulose, threonine-Sepharose, phenyl-5PW, Mono Q, heparin-5PW, and hydroxyapatite columns. The enzyme from COS-7 cells that were transfected with an epsilon PKC cDNA expression plasmid showed the same elution profile. The purified enzyme from the brain was a double (96 and 93 kDa) on SDS/PAGE. Both the doublet proteins were recognized by antibodies raised against several oligopeptides that were parts of the deduced amino acid sequence of the rat brain epsilon PKC. When treated with potato acid phosphatase, both doublet proteins disappeared with the concomitant appearance of a single protein at 90 kDa, suggesting that epsilon PKC exists in the tissue as phosphorylated forms. The physiological significance of this phosphorylation is unknown. The enzymes from the rat brain and COS-7 cells were indistinguishable from each other in their kinetic and catalytic properties. Unlike alpha-, beta I-, beta II-, and gamma PKC, epsilon PKC was independent of Ca2+ but absolutely required phosphatidylserine and diacylglycerol for its activation; a tumor-promoting phorbol ester could replace diacylglycerol. epsilon PKC showed enzymological properties similar to those of delta PKC, except that epsilon PKC but not delta PKC was greatly activated by free arachidonic acid. Immunoblot analysis revealed that, in marked contrast to delta PKC, epsilon PKC is expressed predominantly in the brain tissue and only in trace amounts in heart, lung, spleen, thymus, and testis.

Alkaloids↗
PubMed1992

Catalytic properties of yeast protein kinase C: difference between the yeast and mammalian enzymes.

With bovine myelin basic protein as a model common substrate, protein kinases C (PKC) purified from yeast (Saccharomyces cerevisiae) and mammalian tissue (rat brain) were shown to exhibit clearly different catalytic properties. The major sites of phosphorylation in bovine myelin basic protein by the yeast PKC were identified: Thr-19, Thr-34, and Thr-65. These sites are distinctly different from those for the mammalian PKC: Ser-8, Ser-46, Ser-55, Ser-110, Ser-132, Ser-151, and Ser-161, which were previously identified [Kishimoto, A., Nishiyama, K., Nakanishi, H., Uratsuji, Y., Nomura, H., Takeyama, Y., & Nishizuka, Y. (1985) J. Biol. Chem. 160, 12492-12499]. The results suggest that the yeast and mammalian enzymes may play distinct roles in cellular regulation. No evidence is available, however, that a yeast-type PKC exists in mammalian tissues. An oligopeptide containing the sequence around Thr-19 of bovine myelin basic protein, Lys-Tyr-Leu-Ala-Ser-Ala-Ser-Thr(19)-Met-Asp-His-Ala, can be used as a substrate for selective assaying of the yeast PKC.

Amino Acid Sequence↗
PubMed1992

Molecular cloning of casein kinase II alpha subunit from Dictyostelium discoideum and its expression in the life cycle.

A Dictyostelium discoideum cDNA encoding an alpha-type subunit of casein kinase II was isolated, and its cDNA was used to study developmental expression of casein kinase II during the Dictyostelium life cycle. The 1.3-kb cDNA insert contained an open reading frame of 337 amino acids (M(r) 39,900). The deduced amino acid sequence has high homology with those of casein kinase II alpha subunits from other species. Genomic Southern blot analysis suggested that there is a single gene encoding casein kinase II alpha subunit in D. discoideum. Northern (RNA) blot analysis showed that the casein kinase II alpha-subunit gene is expressed constitutively as a 1.9-kb mRNA throughout vegetative growth and multicellular development. Casein kinase purified from normal vegetative cells contained a major protein band of approximately 36 kDa, which was recognized by antisera raised against rat testis casein kinase II. Comparison of the in vitro transcription/translation product of the alpha-subunit cDNA clone and the purified 36-kDa protein by partial proteolysis indicated that the isolated cDNA clone encodes the Dictyostelium casein kinase II alpha subunit. No protein corresponding to a beta subunit was detected in purified casein kinase. Immunoblot analysis using anti-rat casein kinase II sera showed that the alpha subunit of casein kinase II is expressed constitutively like its mRNA during the life cycle of D. discoideum. Casein kinase II activity measured by using a specific peptide substrate paralleled the level of alpha subunit detected by immunoblotting during the life cycle, with a maximum variation of approximately 2-fold. We were unable to obtain disruptants of the casein kinase II alpha gene, suggesting that there is a single casein kinase II alpha gene, which is essential for vegetative growth of D. discoideum.

Amino Acid Sequence↗
PubMed1992

The family of protein kinase C in transmembrane signalling for cellular regulation.

Signal-induced hydrolysis of inositol phospholipid produces two second messengers, diacylglycerol and inositol trisphosphate. Diacylglycerol activates protein kinase C, whereas inositol trisphosphate mobilizes Ca2+ from its internal store. Analogously, signal-induced hydrolysis of choline phospholipid generates two second messengers, unsaturated free fatty acid and lysophosphatidylcholine. The free fatty acid synergizes with diacylglycerol to activate protein kinase C and causes full activation of the enzyme even at the basal level of Ca2+. On the other hand, lysophosphatidylcholine dramatically enhances cellular responses such as cell proliferation and differentiation under the conditions where diacylglycerol and Ca2+ are available. It is likely that all of the immediate products of signal-induced degradation of inositol and choline phospholipids are involved directly in concert in the transmembrane control of cellular functions.

Animals↗
PubMed1990

The p53 tumour suppressor protein is phosphorylated at serine 389 by casein kinase II.

The entire coding sequence of wild-type mouse p53 was expressed in Escherichia coli under control of the PL promoter of bacteriophage lambda. The bacterial p53 protein had identical mobility to p53 from SV3T3 cells on SDS polyacrylamide gels and was recognized in bacterial lysates by three p53-specific monoclonal antibodies, including PAb246 which is specific for wild-type mouse p53. Immunoprecipitates of the bacterial p53 were phosphorylated by a highly purified preparation of rat casein kinase II; the stoichiometry of incorporation was approximately 1 mol of phosphate per mol of p53. The phosphorylated residue was identified by phosphopeptide mapping as serine 389, which is a major site of p53 phosphorylation in vivo. p53 (serine 389) kinase activity was detected on lysates of SV3T3 cells; this activity co-purified with casein kinase II on phosphocellulose and Mono Q columns and was inhibited by heparin. Immunoprecipitates of the p53-T antigen complex from SV3T3 cells also had associated serine 389 kinase activity. Phosphorylation of serine 389 by this kinase was potently inhibited by heparin and quenched by excess unlabelled GTP. The data indicate that p53 is a physiological substrate of casein kinase II, which is stimulated in response to mitogens, phosphorylates nuclear oncoproteins, and may play a role in the transduction of extracellular signals to the nucleus.

Amino Acid Sequence↗
PubMed1989

Human T cell activation by phorbol esters and diacylglycerol analogues.

Activation of protein kinase C (PKC), by the phorbol ester PMA, or the membrane-permeable diacylglycerol 1-oleoyl 2-acetylglycerol (OAG), had different effects on the proliferation-associated responses of a more than 99% pure population of human T cells. Treatment with PMA or OAG caused down-regulation of the TCR-CD3 complex, but only PMA, in combination with ionomycin, was capable of stimulating IL-2R expression and proliferation. Immunocytochemical staining with antisera specific for the PKC subspecies alpha, beta I, beta II, and gamma showed that untreated resting T cells normally coexpress alpha, beta I, and beta II PKC subspecies, which are distributed diffusely throughout the cell, with some localization around the periphery of the nucleus. There was no difference between the responses of these PKC subspecies to OAG and PMA, redistributing, after 10 min of treatment, to a discrete focal area within the cell. Treatment with OAG resulted in transient redistribution of PKC, maximal at 10 min, while in PMA-stimulated cells, the PKC redistribution was prolonged, persisting for at least 24 h. The results suggest that the difference in cellular response to treatment with PMA and OAG is not a consequence of differential activation of various PKC subspecies.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1989

Expression of protein kinase C subspecies in human leukemia-lymphoma cell lines.

Expression of protein kinase C (PKC) subspecies was studied in various human leukemia-lymphoma cell lines. The PKC in most cell lines examined was resolved into two major fractions corresponding to type II (beta-sequence) and type III (alpha-sequence) PKC of the rat brain. The amounts of these two subspecies greatly varied among the cell lines. Type I PKC (gamma-sequence) was expressed in none of the cell lines tested, but PKCs with undefined structures were frequently detected. The differential co-expression of several PKC subspecies is presumably related to the state of cell differentiation.

Animals↗