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U Kikkawa

Publications and source records attributed to U Kikkawa.

At least 55 records · Page 3Linked to original sources
PubMed1997

Distinct specificity in the binding of inositol phosphates by pleckstrin homology domains of pleckstrin, RAC-protein kinase, diacylglycerol kinase and a new 130 kDa protein.

The pleckstrin homology domains (PH domains) derived from four different proteins, the N-terminal part of pleckstrin, RAC-protein kinase, diacylglycerol kinase and the 130 kDa protein originally cloned as an inositol 1,4,5-trisphosphate binding protein, were analysed for binding of inositol phosphates and derivatives of inositol lipids. The PH domain from pleckstrin bound inositol phosphates according to a number of phosphates on the inositol ring, i.e. more phosphate groups, stronger the binding, but a very limited specificity due to the 2-phosphate was also observed. On the other hand, the PH domains from RAC-protein kinase and diacylglycerol kinase specifically bound inositol 1,3,4,5,6-pentakisphosphate and inositol 1,4,5,6-tetrakisphosphate most strongly. The PH domain from the 130 kDa protein, however, had a preference for inositol 1,4,5-trisphosphate and 1,4,5,6-tetrakisphosphate. Comparison was also made between binding of inositol 1,4,5-trisphosphate, inositol 1,3,4,5-tetrakisphosphate and soluble derivatives of their corresponding phospholipids. The PH domains examined, except that from pleckstrin, showed a 8- to 42-times higher affinity for inositol 1,4,5-trisphosphate than that for corresponding phosphoinositide derivative. However, all PH domains had similar affinity for inositol 1,3,4,5-tetrakisphosphate compared to the corresponding lipid derivative. The present study supports our previous proposal that inositol phosphates and/or inositol lipids could be important ligands for the PH domain, and therefore inositol phosphates/inositol lipids may have the considerable versatility in the control of diverse cellular function. Which of these potential ligands are physiologically relevant would depend on the binding affinities and their cellular abundance.

Amino Acid Sequence↗
PubMed1997

Activation of protein kinase C by tyrosine phosphorylation in response to H2O2.

Protein kinase C (PKC) isoforms, alpha, betaI, and gamma of cPKC subgroup, delta and epsilon of nPKC subgroup, and zeta of aPKC subgroup, were tyrosine phosphorylated in COS-7 cells in response to H2O2. These isoforms isolated from the H2O2-treated cells showed enhanced enzyme activity to various extents. The enzymes, PKC alpha and delta, recovered from the cells were independent of lipid cofactors for their catalytic activity. Analysis of mutated molecules of PKC delta showed that tyrosine residues, which are conserved in the catalytic domain of the PKC family, are critical for PKC activation induced by H2O2. These results suggest that PKC isoforms can be activated through tyrosine phosphorylation in a manner unrelated to receptor-coupled hydrolysis of inositol phospholipids.

Amino Acid Sequence↗
PubMed1997

Activation of protein kinase B (Akt/RAC-protein kinase) by cellular stress and its association with heat shock protein Hsp27.

Protein kinase B (PKB, also named as Akt or RAC-protein kinase), that is activated by cellular stress such as heat shock and hyperosmotic treatment, was revealed to be activated by oxidative stress and by chemical stressors of CdCl2 and NaAsO2 by measuring the activity of the enzyme immunoprecipitated from the transfected COS-7 cells. Upon stress treatment, a 30-kDa phosphoprotein was co-immunoprecipitated with PKB from the cells metabolic labeled with [32P]orthophosphate. The phosphoprotein was identified as Hsp27, a small heat shock protein, by immunoblot analysis and co-immunoprecipitation. The association of Hsp27 was specific to PKB as the heat shock protein was not co-immunoprecipitated with other protein kinases such as protein kinase C and PKN. When the cells were treated with H2O2, PKB was activated gradually and the association of Hsp27 with PKB increased concurrently with the enhancement of PKB activity. In heat-shocked cells, activation of PKB and the association of Hsp27 were detected immediately after the treatment, and the association of the heat shock protein decreased while PKB kept stimulated activity when the cells were further incubated at 37 degrees C. These results suggest that Hsp27 is involved in the activation process of PKB in the signal transduction pathway of various forms of stress.

Animals↗
PubMed1997

Expression of a constitutively active phosphatidylinositol 3-kinase induces process formation in rat PC12 cells. Use of Cre/loxP recombination system.

It has been shown that inhibition of phosphatidylinositol (PI) 3-kinase blocks neurite outgrowth of PC12 cells stimulated with nerve growth factor. To further assess the role of PI 3-kinase, the active form of PI 3-kinase was expressed in PC12 cells by the adenovirus mediated introduction of a site-specific recombinase, Cre. After expression of the active PI 3-kinase, elevation of the levels of PI 3,4-diphosphate and PI 3,4,5-trisphosphate as well as formation of neurite-like processes was observed. The process formation was inhibited by wortmannin, a selective inhibitor of PI 3-kinase, which suggests that a high activity of PI 3-kinase was responsible for the formation of these processes. The processes lacked accumulation of F-actin and GAP43 at the growth cone, which suggests that the processes were incomplete compared with neurites. Instead, the bundling of microtubules was enhanced, which suggests that organization of the microtubules might be driving the process of elongation in the cells expressing the active PI 3-kinase. Induction of active PI 3-kinase resulted in activation of Jun N-terminal kinase but not of mitogen-activated protein kinase or protein kinase B/Rac protein kinase/Akt. These results suggest that PI 3-kinase is involved in neurite outgrowth in PC12 cells and that activation of Jun N-terminal kinase cascade may be involved in the cell response.

Animals↗
PubMed1997

Comparison of chemical characteristics of the first and the second cysteine-rich domains of protein kinase C gamma.

Protein kinase C (PKC) is a key enzyme family involved in cellular signal transduction. The binding of endogenous diacyl glycerol (DAG) to the cysteine-rich domain (CRD) of PKC is associated with normal cell signaling and function. In contrast, the binding of exogenous phorbol esters to the CRD of PKC is considered to be a key initiating event in tumor promotion. Conventional PKC isozymes (PKC alpha, beta I, beta II, and gamma) contain two CRDs, both of which are candidates for the phorbol ester binding site. In order to elucidate the binding requirements of phorbol esters and to obtain information on the phorbol ester binding site in native PKC gamma, several key chemical characteristics of the first and the second CRDs consisting of ca. 50 amino acids of rat PKC gamma (gamma-CRD1 and gamma-CRD2) were examined. In the presence of Zn2+ and phosphatidylserine (PS), both CRDs gave similar Kd values (65.3 nM for gamma-CRD1, 44.1 nM for gamma-CRD2) in phorbol 12,13-dibutyrate (PDBu) binding assays. In comparison, the binding affinity of PDBu for native rat PKC gamma was found to be 6.8 nM. Zn2+ was shown to play an important role in the folding and PDBu binding of both CRDs. A Zn(2+)-induced conformational change was observed for the first time by CD spectroscopic analysis of the complexed and uncomplexed CRDs. Relative to the pronounced Zn2+ effect, most divalent first row transition metal ions along with Ca2+, Mg2+, and Al3+ were ineffective in folding either CRD. Notably, however, Co2+ exhibited a gamma-CRD1-selective effect, suggesting that metal ions, not unlike extensively used organic probes, might also become effective tools for controlling isozyme selective activation of PKC. Moreover, group Ib (Cu2+ and Ag+) and group IIb element ions other than Zn2+ (Cd2+ and Hg2+) were found to abolish PDBu binding of both CRDs. Importantly, these inhibitory effects of Cu2+, Ag+, and Cd2+, and Hg2+ were also observed with native PKC gamma. These results indicate that recent reports on the modulation of conventional PKC by heavy metal ions could be explained by their coordination to the CRDs. While the similar affinities of gamma-CRD1 and gamma-CRD2 for PDBu suggest that either site qualifies as the PDBu binding site, new molecular probes of these CRD3 have now been identified that provide information on the preferred site. These novel ligands (5a and 5b) were synthesized by aza-Claisen rearrangement of (-)-N13-desmethyl-N13-allylindolactam-G (4). These compounds did not significantly affect the specific PDBu binding of gamma-CRD1 but did inhibit that of gamma-CRD2 with similar potency to (-)-indolactam-V. Moreover, these new probes did not significantly inhibit the PDBu binding of native PKC gamma. (-)-Indolactam-V itself bound almost equally to gamma-CRD1, gamma-CRD2, and native PKC gamma. These results suggest that the major PDBu binding site in native PKC gamma is the first CRD, not the second CRD, unlike the novel PKCs.

Amino Acid Sequence↗
PubMed1997

Characterization of tyrosine-phosphorylated delta isoform of protein kinase C isolated from Chinese hamster ovary cells.

Phorbol ester treatment of Chinese hamster ovary cells stably overexpressing the delta isoform of protein kinase C induced the association of the isoform with the particulate fraction and the tyrosine phosphorylation of a small portion of the delta isoform. The delta isoform without tyrosine phosphorylation was recovered as an enzyme dependent on phospholipid and diacylglycerol, whereas the tyrosine-phosphorylated delta isoform was recovered in two fractions, one dependent on, and the other independent of, phospholipid and diacylglycerol. The tyrosine-phosphorylated delta isoform independent of lipid activators might be associated with phorbol ester and phospholipids. Immunoblot analysis revealed that the delta isoform is a doublet protein of 76 and 78 kDa, and that the delta isoform fraction without tyrosine phosphorylation contained 76- and 78-kDa proteins, whereas the tyrosine-phosphorylated delta isoform contained the 78-kDa protein but not the 76-kDa protein. In vitro analysis showed that the 78-kDa protein of the delta isoform without tyrosine phosphorylation is an efficient substrate of tyrosine kinase only when phosphatidylserine and either diacylglycerol or phorbol ester are present; however, the 76-kDa protein can not be tyrosine-phosphorylated even in the presence of these lipid activators. The phospholipid and diacylglycerol-dependent form of the tyrosine-phosphorylated enzyme isolated from the cell line required lower concentrations of phosphatidylserine and phorbol ester for its activity in vitro as compared with the enzyme without tyrosine phosphorylation. These results suggest that the tyrosine-phosphorylated enzyme generated upon stimulation of the cells may associate with membranes and exert its full activity even with the lower concentrations of the lipid activators.

Animals↗
PubMed1996

Protein-protein interaction of zinc finger LIM domains with protein kinase C.

The LIM domain comprising two zinc-finger motifs is found in a variety of proteins and has been proposed to direct protein-protein interactions. During the identification of protein kinase C (PKC)-interacting proteins by a yeast two-hybrid assay, a novel protein containing three LIM domains, designated ENH, was shown to associate with PKC in an isoform-specific manner. Deletion analysis demonstrated that any single LIM domain of ENH associates with the NH2-terminal region of PKC. ENH associated with PKC in COS-7 cells and was phosphorylated by PKC in vitro. Upon treatment of the cells with phorbol ester, ENH in the membrane fraction was translocated to the cytosol fraction in vivo. Other LIM domain-containing proteins, such as Enigma and LIM-kinase 1, also interacted with PKC through their LIM domains. These results suggest that the LIM domain is one of the targets of PKC and that the LIM-PKC interaction may shed light on undefined roles of LIM domain-containing proteins.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Isolation of the active form of RAC-protein kinase (PKB/Akt) from transfected COS-7 cells treated with heat shock stress and effects of phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 4,5-bisphosphate on its enzyme activity.

RAC-protein kinase (PKB/Akt) has been shown to be activated by growth factor stimulation as a downstream target of phosphatidylinositol 3-kinase and also by heat shock through a pathway independent of phosphatidylinositol 3-kinase. RAC-protein kinase was purified by antibody affinity chromatography from COS-7 cells transfected with the epitope-tagged expression plasmid. The protein kinase activity of RAC-protein kinase purified from heat-treated cells was 9-fold higher than the enzyme isolated from untreated control cells. Phosphatidylinositol 3,4,5-trisphosphate did not enhance the activity of RAC-protein kinase purified from either heat-treated cells or control cells, whereas phosphatidylinositol 4,5-bisphosphate suppressed the enzyme isolated from heat-treated cells. These results indicate that RAC-protein kinase may interact with phosphoinositides, however, it could not be activated by simple association with the product of phosphatidylinositol 3-kinase reaction.

Animals↗
PubMed1996

Ser-27, Tyr-10 and Tyr-7 in the alpha-chain of pig stomach H+,K(+)-ATPase as Ca(2+)-dependent phosphorylatable sites by intrinsic and extrinsic protein kinases.

When pig stomach membrane H+,K(+)-ATPase preparations were incubated with [gamma-32P]ATP, Mg2+ and Ca2+, reversible phosphorylation of specific Tyr and Ser residues in the N-terminal alpha-chain of H+,K(+)-ATPase occurred without any detectable phosphorylation in other regions of the alpha-chain. Mild tosylphenylalanyl chloromethyl ketone trypsin treatment followed by reverse-phase column chromatography yielded three radioactive peptide peaks. The first peak contained both Tyr10(32P) and Tyr7(32P) and the second peak contained Tyr10(32P). The third peak contained Ser27(32P) which was also obtained after trypsin treatment of partially purified H+,K(+)-ATPase preparations phosphorylated with protein kinase-C + Ca2+ or protein kinase-A. This is the first demonstration of Ca2(+)-dependent phosphorylation of the alpha-chain of H+,K(+)-ATPase by protein kinases.

Amino Acid Sequence↗
PubMed1996

Activation of RAC-protein kinase by heat shock and hyperosmolarity stress through a pathway independent of phosphatidylinositol 3-kinase.

RAC protein kinase (RAC-PK), a serine/threonine protein kinase containing a pleckstrin homology (PH) domain, was activated by cellular stress such as heat shock and hyperosmolarity. Wortmannin, which is known as a potent inhibitor of phosphatidylinositol 3-kinase and normally inhibits growth factor-induced activation of RAC-PK, did not suppress heat-shock induced activation of RAC-PK, indicating that this stress-induced activation of the kinase is not mediated by phosphatidylinositol 3-kinase. The PH domain was indispensable for stress-induced activation of RAC PK. In heat-treated cells, PKC delta, a member of the protein kinase C family, was found to associate with the PH domain of RAC-PK. This PKC subspecies was phosphorylated in vitro by RAC-PK. The results suggest that RAC-PK may play a role in the cellular response to stress through its PH domain.

3T3 Cells↗
PubMed1996

Phosphorylation of human m1 muscarinic acetylcholine receptors by G protein-coupled receptor kinase 2 and protein kinase C.

Human muscarinic acetylcholine receptor m1 subtypes (m1 receptors) were expressed in and purified from insect Sf9 cells and then subjected to phosphorylation by G protein-coupled receptor kinase 2 (GRK2) expressed in and purified from Sf9 cells and by protein kinase C purified from rat brain (a mixture of alpha, beta, and gamma types, PKC). The m1 receptor was phosphorylated by either GRK2 or PKC in an agonist-dependent or independent manner, respectively. G protein beta gamma subunits stimulated the phosphorylation by GRK2 but did not affect the phosphorylation by PKC. The number of incorporated phosphates was 4.6 and 2.8 mol/mol of receptor for phoshorylation by GRK2 and PKC, respectively. The number of incorporated phosphates was 7.5 mol/mol receptor for phosphorylation by GRK2 followed by PKC, but was 5.8 mol/mol of receptor for the phosphorylation by PKC followed by GRK2. Major sites phosphorylated by GRK2 and PKC were located in the third intracellular loop and the carboxyl-terminal tail, respectively. These results indicate that GRK2 and PKC phosphorylate different sites of m1 receptors and that the phosphorylation by PKC partially inhibits the phosphorylation by GRK2, probably by affecting activation of GRK2 by agonist-bound receptors.

Amino Acid Sequence↗
PubMed1996

Deletion of specific protein kinase C subspecies in human melanoma cells.

It has been shown that tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), stimulates the proliferation of normal human melanocytes, whereas it inhibits the growth of human melanoma cell lines. The expression of protein kinase C (PKC) subspecies, the major intracellular receptors for TPA, was examined in normal melanocytes and the four melanoma cell lines HM3KO, MeWo, HMV-1, and G361. PKC was partially purified and then separated into subspecies by column chromatography on Mono Q and hydroxyapatite successively, and finally subjected to immunoblot analysis using antibodies specific for the PKC subspecies. Of the PKC subspecies examined, delta-, epsilon-, and zeta-PKC were detected in both normal melanocytes and the four melanoma cell lines. In contrast, both alpha-PKC and beta-PKC were expressed in normal melanocytes, whereas either alpha-PKC or beta-PKC was detected in melanoma cells. Specifically, HM3KO, MeWo, and HMV-1 cells were shown to contain alpha-PKC but not beta-PKC, while G361 cells expressed beta-PKC but not alpha-PKC. The growth of these melanoma cells was suppressed by TPA treatment, and the growth of the G361 cells lacking alpha-PKC was inhibited more efficiently than the other melanoma cell lines which lacked beta-PKC. It was further shown that beta-PKC was not detected in freshly isolated human primary or metastatic melanoma tissues. These results suggest that the expression of alpha-PKC or beta-PKC may be altered during the malignant transformation of normal melanocytes and that loss of alpha-PKC or beta-PKC may be related to the inhibitory effect of TPA on the growth of melanoma cells.

Cell Division↗
PubMed1995

A novel phosphatidylserine-binding peptide motif defined by an anti-idiotypic monoclonal antibody. Localization of phosphatidylserine-specific binding sites on protein kinase C and phosphatidylserine decarboxylase.

A monoclonal anti-idiotypic antibody, Id8F7, previously shown to bind to a phosphatidylserine (PS)-specific binding site on protein kinase C (PKC) has been used to identify a 12-amino acid consensus sequence shared by PKC and phosphatidylserine decarboxylase (PSD). The 14-amino acid synthetic peptide derived from the corresponding region of PSD (amino acids 351-364 of the enzyme from Chinese hamster ovary cells) bound effectively and specifically to PS, and that derived from rat PKC gamma (amino acids 227-240) bound weakly but specifically to PS. Analysis of binding of Id8F7 to various synthetic peptides revealed that the consensus sequence motif, FXFXLKXXXKXR, is responsible for the interaction with both Id8F7 and PS. The results suggest that the conserved amino acid residues represent a basic structural motif for the specific interaction with PS, and the corresponding regions of PKC and PSD form the PS-specific binding sites of these enzymes.

Amino Acid Sequence↗
PubMed1995

Molecular cloning and characterization of a new member of the RAC protein kinase family: association of the pleckstrin homology domain of three types of RAC protein kinase with protein kinase C subspecies and beta gamma subunits of G proteins.

cDNA clones encoding the third member of the RAC protein kinase family, termed RAC-PK gamma, were isolated from a rat brain cDNA library. The deduced amino acid sequence of RAC-PK gamma was highly related to those of previously identified family members, RAC-PK alpha and beta, that have a pleckstrin homology domain and a protein-serine/threonine kinase catalytic domain at the amino- and carboxyl-terminal regions, respectively. Northern blot analysis indicated that RAC-PK gamma was expressed abundantly in brain and testis. Specific activities of RAC-PK alpha, beta, and gamma purified from transfected COS-7 cells were similar when measured by using myelin basic protein as a phosphate acceptor. Analysis using fusion proteins of glutathione S-transferase revealed that the pleckstrin homology domain of the three subtypes of RAC-PK associate with both protein kinase C subspecies and beta gamma subunits of G proteins. These results suggest that the pleckstrin homology domains of RAC protein kinase family could associate more than one protein to regulate the activity and/or intracellular distribution of this enzyme family by different ways.

Amino Acid Sequence↗
PubMed1995

Functional characterization of the promoter region of the mouse protein kinase C gamma gene.

Promoter activity of protein kinase C (PKC) gamma gene was analysed by chloramphenicol acetyltransferase (CAT) assay using extracts from the cells transfected with various fusion constructs containing the 5'-flanking region of the mouse PKC gamma gene and CAT gene. Transient expression experiments in PC12 cells revealed that the upstream region of 87 bp from the transcriptional initiation site was sufficient for promoter activity. The region containing nucleotides 87 upstream from the transcriptional initiation site was shown to silence CAT activity in Balb/c3T3 cells, in which mRNA of PKC gamma was not detected, suggesting that this region might contain a transcriptional regulatory element for the cell type-specific expression of the PKC gamma gene.

Animals↗
PubMed1995

Characterization of Gq family G proteins GL1 alpha (G14 alpha), GL2 alpha (G11 alpha), and Gq alpha expressed in the baculovirus-insect cell system.

The alpha subunits of Gq family G proteins, GL1 alpha (G14 alpha), GL2 alpha(G11 alpha), and Gq alpha were expressed with G protein beta 1 and gamma 2 subunits in insect cells using a baculovirus system. The trimeric forms of G proteins, GL1 (GL1 alpha beta gamma), GL2 (GL2 alpha beta gamma), and Gq (Gq alpha beta gamma), were solubilized by 1% sodium cholate and purified by sequential chromatography on three kinds of columns. GL1, GL2, and Gq activated phospholipase C-beta purified from bovine brain in the presence of aluminum fluoride to the same extent. Muscarinic acetylcholine receptor m1 subtype stimulated the guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) binding to GL1, GL2, and Gq in the presence of similar concentrations of carbamylcholine. When m1 receptor, G protein, and phospholipase C-beta were reconstituted in lipid vesicles, each subtype of Gq family G proteins mediated the activation of phospholipase C-beta by carbamylcholine in the presence of either 1 microM GTP gamma S or 1 mM GTP. Phospholipase C-beta stimulated the GTPase activity of GL1, GL2, and Gq in the presence of m1 receptor and carbamylcholine but did not stimulate the GTPase activity of GO. Protein kinase C phosphorylated m1 receptor and phospholipase C-beta, but the phosphorylation did not significantly affect the ability of the m1 receptor to stimulate phospholipase C-beta in the reconstitution system of purified proteins.

Animals↗
PubMed1995

Distinct effects of saturated fatty acids on protein kinase C subspecies.

The effects of saturated fatty acids on the protein kinase C family were studied. Saturated fatty acids with carbon chain lengths of C12 to C14 activated the alpha-, beta-, gamma-, and epsilon-subspecies, and this activation was synergistic with that by diacylglycerol. Tridecanoic acid (C13) was most effective among the saturated fatty acids examined. Saturated fatty acids having carbon chain lengths of less than C10 were inert, and fatty acids with longer carbon chain lengths, such as C16 and C18, showed little effect. In contrast, fatty acids were almost inactive toward the delta-subspecies, and rather inhibited its activity in the presence of phosphatidylserine and diacylglycerol. Phorbol ester enhanced the activity of the alpha-, beta-, and gamma-subspecies when added with fatty acids; however, it suppressed the activity of the epsilon-subspecies in the presence of fatty acids. Saturated fatty acids having different effects on protein kinase C subspecies may be useful as tools for studying the mechanism and role of protein kinase C activation.

Animals↗