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Biomedical subjects

U Kikkawa

Publications and source records attributed to U Kikkawa.

At least 91 records · Page 5Linked to original sources
PubMed1989

Phorbol ester binding to protein kinase C requires a cysteine-rich zinc-finger-like sequence.

Protein kinase C normally has a tandem repeat of a characteristic cysteine-rich sequence in C1, the conserved region of the regulatory domain. These sequences resemble the DNA-binding zinc finger domain. For the gamma subspecies of rat brain protein kinase C, various deletion and point mutants in this domain were constructed, and the mutated proteins were expressed in Escherichia coli by using the T7 expression system. Radioactive phorbol 12,13-dibutyrate binding analysis indicated that a cysteine-rich zinc-finger-like sequence was essential for protein kinase C to bind phorbol ester and that one of two sequences was sufficient for the phorbol ester binding. Conserved region C2, another region in the regulatory domain, was apparently needed for the enzyme to require Ca2+ for phorbol ester binding activity.

Amino Acid Sequence↗
PubMed1989

Immunocytochemical localization of the beta I subspecies of protein kinase C in rat brain.

Polyclonal antibodies were raised against an oligopeptide containing an amino acid sequence specific for beta I protein kinase C (PKC) in order to localize this subspecies of PKC in the rat brain. beta I PKC immunoreactivity was widely distributed but discretely localized in particular brain regions. The immunoreactivity was associated with neurons but not glial cells and was generally weak in the neutrophils with the exception of areas such as the triangular septal nucleus, pontine nuclei, superficial layer of the superior colliculus, and gray matter of the spinal cord. The largest number of beta I PKC-immunoreactive cells was seen in the triangular septal nucleus and pontine nuclei. In these areas of dense beta I immunoreactivity, there was no or little gamma PKC immunoreactivity. The beta I PKC immunoreactivity was observed in perikarya, dendrites, and axons but not in nuclei or nucleoli. There were various patterns of cytoplasmic immunoreactivity, but the immunoreactivity was seen frequently in the periphery of the perikarya and dendrites. The intraneuronal localization of beta I PKC immunoreactivity differs from that of the subspecies that is coded by the gamma gene sequence. The results suggest that beta I PKC may play a role in the regulation of cell membrane responsiveness in particular neurons.

Animals↗
PubMed1989

Protein kinase C zeta subspecies from rat brain: its structure, expression, and properties.

The primary structure of the zeta subspecies of rat brain protein kinase C was deduced from its overlapping cDNAs. The zeta subspecies of protein kinase C consists of 592 amino acid residues with the calculated molecular mass of 67,740 Da and has regulatory and protein kinase domains in its amino- and carboxyl-terminal halves, respectively. Although all members of the protein kinase C family so far identified have a tandem repeat of the characteristic cysteine-rich zinc-finger-like sequence in the regulatory domain, the zeta subspecies contains only one set of this sequence. Northern (RNA)-blot hybridization analysis indicated that two major RNA transcripts of the zeta subspecies with different lengths may be generated by the use of different polyadenylylational signals. The enzyme was expressed in COS-7 cells by transfection with the cDNA construct encoding its whole sequence. It showed an approximate molecular mass of 64,000 Da upon SDS/PAGE. The enzyme activity was significantly dependent on phospholipid but was independent of the presence of Ca2+ or diacylglycerol, when assayed with calf thymus H1 histone as a phosphate acceptor protein. The zeta subspecies expressed in COS-7 cells did not appear to show binding activity of phorbol ester. The structural and biochemical properties indicate that the zeta subspecies is related to, but distinct from, other subspecies of protein kinase C. Perhaps, this subspecies belongs to another entity of the enzyme family.

Amino Acid Sequence↗
PubMed1989

Immunocytochemical localization of beta II subspecies of protein kinase C in rat brain.

The distribution of a subspecies of protein kinase C (PKC) encoded by the beta II sequence in rat central nervous tissue was demonstrated immunocytochemically by using antibodies raised against an oligopeptide having a partial sequence specific for the beta II PKC. The beta II PKC immunoreactivity was widely but discretely distributed in the brain. The distribution of the beta II PKC immunoreactivity differed from that of the beta I and gamma PKC subspecies. The beta II PKC immunoreactivity was found in the perikarya, dendrites, and axons of neuronal cells. Few if any glial cells were stained. Immunoreactive neurons were present in the anterior olfactory nucleus, olfactory tubercle, amygdaloid complex, caudate-putamen, accumbens nucleus, claustrum, dorsal part of the lateral septal nucleus, CA1 region of the hippocampus, subiculum, medial habenular nucleus, cerebral cortex, nucleus of the spinal tract of the trigeminal nerve, nucleus of the solitary tract, and substantia gelatinosa of the spinal cord. In these neurons, the beta II PKC immunoreactivity was seen mainly in the form of cytoplasmic dots and, in some cases, diffusely in the cytoplasm. Under electron microscopy, these immunoreactive large dots appeared to be associated with the Golgi complex, suggesting that the beta II PKC plays a specialized function at the Golgi complex in certain neuronal cell types.

Animals↗
PubMed1989

Localization of beta II subspecies of protein kinase C in beta-cells.

Protein kinase C (PKC) has been generally accepted to play a key role in stimulus-response coupling in various secretory cells, including pancreatic islets and pancreatic acini. The enzyme exists as a large family of multiple subspecies with highly related structures (alpha-, beta I-, beta II-, gamma-, delta-, epsilon-, and zeta-PKC). With an immunocytochemical procedure with subspecies-specific antibodies, beta II-PKC-like immunoreactivity was identified to localize in the beta-cells of the rat pancreatic islets. Neither beta I- nor gamma-PKC-like immunoreactivity was found in both islets and acini. Biochemical analysis confirmed that the rat whole pancreatic tissues contain a significant amount of alpha-PKC and a minute amount of beta II-PKC but neither beta I- nor gamma-PKC. On the other hand, beta II-PKC-like immunoreactivity was not detected in rat insulinoma cells, in which insulin secretion was induced in response to 12-O-tetradecanoylphorbol-13-acetate and carbachol but not in response to glucose. These findings suggest that beta II-PKC is the major, if not the sole, subspecies of PKC in beta-cells of rat pancreatic islets and a possible candidate for involvement in glucose-induced insulin secretion.

Animals↗
PubMed1988

Properties of protein kinase C subspecies in human platelets.

Protein kinase C (PKC) from human platelets was resolved into two fractions by hydroxyapatite column chromatography. One of the enzymes was indistinguishable from the brain type III PKC having alpha-sequence in its kinetic and immunological properties. The other enzyme was kinetically different from any of the brain PKC subspecies so far isolated, although it resembled the brain type II PKC having beta-sequence. With H1 histone as substrate, this platelet enzyme was not very sensitive to Ca2+, and activated partly by phosphatidylserine plus diacylglycerol or by free arachidonic acid. Both platelet enzymes could phosphorylate the P47 protein in vitro, but the enzyme physiologically responsible for the P47 protein phosphorylation in the activated platelets remains to be identified.

Amino Acid Sequence↗
PubMed1988

Differential down-regulation of protein kinase C subspecies in KM3 cells.

The down-regulation of protein kinase C (PKC) subspecies in KM3 cells (a pre-B, pre-T cell line) has been examined. The PKC from KM3 cells was resolved into two subspecies, type II (mainly beta II) and type III (alpha), upon hydroxyapatite column chromatography. Biochemical and immunocytochemical analysis revealed that, when these cells were treated with 12-O-tetradecanoylphorbol 13-acetate (TPA), the time course of down-regulation of the PKC subspecies was different; type II PKC was translocated and depleted from the cell more quickly than type III enzyme. The results suggest that each PKC subspecies plays a different role in the cellular response to TPA and probably to other external stimuli.

Cell Compartmentation↗
PubMed1988

The heterogeneity and differential expression of protein kinase C in nervous tissues.

Protein kinase C exists as a large family of multiple subspecies with subtle individual characteristics. This heterogeneity comes from different genes as well as from different splicings of a single RNA transcript. The members of this family have closely related structures with a high degree of homology. Biochemical studies have shown that their mode of activation and kinetic and catalytic properties differ slightly from one another. By using a combination of biochemical and immunocytochemical techniques, their differential regional and cellular expression have been shown in the nervous tissues. Each member of this enzyme family may have a specialized function in transducing various physiological and pathological signals into different cell types.

Amino Acid Sequence↗
PubMed1988

Isolation of protein kinase C subspecies from a preparation of human T lymphocytes.

Using a preparation of purified human T lymphocytes, we were able to resolve a partially purified protein kinase C (PKC) enzyme fraction into two distinct subspecies, of approximately equal activity. Biochemical and immunocytochemical analysis revealed that these fractions closely resembled the type II (beta) and type III(alpha) PKC subspecies previously identified and characterised from brain tissue. These results provide valuable information for further studies on the role of individual PKC subspecies in T lymphocyte proliferation.

Calcium Chloride↗
PubMed1988

The structure, expression, and properties of additional members of the protein kinase C family.

In rat brain three members of the protein kinase C family encoded by cDNAs termed delta, epsilon, and zeta were newly identified by molecular cloning and sequence analysis. The new members have a common structure that is closely related to but clearly distinct from the four members of the family previously isolated having alpha-, beta I-, beta II-, and gamma-sequences, although the zeta-cDNA available at present does not appear to contain a complete reading frame for protein kinase C. The delta-, epsilon-, and zeta-cDNAs all encode a characteristic cysteine-rich sequence and protein kinase domain sequence, both of which are highly homologous among the protein kinase C family. However, the new members lack one of the conserved regions that is present in alpha-, beta I-, beta II-, and gamma-sequences. An additional cDNA clone termed epsilon' was isolated, which is identical with epsilon-cDNA except for a short sequence at the 5'-terminal end region. The two members having delta- and epsilon-sequences were expressed in COS 7 cells, and partially purified and characterized. The enzymes having delta- and epsilon-sequences depend on phospholipid and diacylglycerol for the enzymatic activity, but their properties slightly differ from the previously known members of protein kinase C. Northern blot analysis suggests that the new members of protein kinase C exist in the brain and some other tissues.

Amino Acid Sequence↗
PubMed1988

Activation of a brain-specific protein kinase C subspecies in the presence of phosphatidylethanol.

Protein kinase C (PKC) is normally activated by diacylglycerol in the presence of Ca2+ and phosphatidylserine. At physiological concentrations of Ca2+, however, phosphatidylethanol, a product of the phospholipase D-catalyzed transphosphatidylation reaction between membrane phospholipids and ethanol, can replace phosphatidylserine, and activate PKC. This mode of activation is most effective for the gamma-subspecies, which is expressed only in central nervous tissue. Phosphatidylmethanol is also effective to some extent. Consideration of these results suggests the possibility that ethanol may exert some effect on signal transduction in this tissue via changes in protein phosphorylation.

Animals↗
PubMed1988

The heterogeneity of protein kinase C in various rat tissues.

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

Animals↗