Kouji Ogita

Three distinct forms of rat brain protein kinase C: differential response to unsaturated fatty acids.

Although the three distinct forms of protein kinase C isolated from rat brain soluble fraction are structurally very similar, they respond differently to free unsaturated fatty acids such as arachidonic acid to exhibit their catalytic activity. Type I enzyme encoded by gamma-sequence, as predicted by cDNA clone analysis, responds to these fatty acids only slightly, whereas Type III enzyme determined by alpha-sequence is activated by free unsaturated fatty acids in the presence of Ca2+ in a comparable manner to phosphatidylserine plus diacylglycerol. Type II, a mixture of two enzymes encoded by beta I- and beta II-sequence, resulting from alternative splicing, shows properties in between those of Type I and Type III. Some of these forms of protein kinase C may function at a relatively later phase of cellular responses when large amounts of unsaturated fatty acids and Ca2+ are mobilized.

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.

Identification of the structures of multiple subspecies of protein kinase C expressed in rat brain.

Rat brain protein kinase C purified to apparent homogeneity [(1986) Biochem. Biophys. Res. Commun. 135, 636–643] was resolved into three distinct fractions, type I, II and III, upon chromatography on a hydroxyapatite column connected to high‐performance liquid chromatography. Comparison of each fraction with the four subspecies of protein kinase C, that were separately expressed in COS cells transfected by the respective cDNAs, α, βI,βII an d γ, identified the primary structures of these three fractions of protein kinase C. Type I corresponded to the enzyme encoded by the γ‐sequence; type II was a mixture of the two subspecies determined by the βI‐ and βII‐sequences; and type III had the structure encoded by the α‐sequence. The structures and properties of these subspecies of protein kinase C were similar to each other.

Two types of complementary DNAs of rat brain protein kinase C. Heterogeneity determined by alternative splicing.

Two types of complementary DNA clones for rat brain protein kinase C were isolated. These clones encode 671 and 673 amino acid sequences, which differ from each other only in the carboxyl‐terminal regions of approx. 50 amino acid residues. This difference seems to result from alternative splicing. Elucidation of the sequences of these cDNA clones as well as some peptides from the purified rat brain enzyme suggests the existence of an additional species of protein kinase C in this tissue. It is attractive to imagine that the heterogeneity of protein kinase C may reflect diverse pathways of signal transduction into the cell.

Identification of three additional members of rat protein kinase C family: gd-, ϵ- and ξ-subspecies

Three types of cDNA clone of the protein kinase C family termed δ, ϵ and ξ were newly identified by molecular cloning and sequence analysis. These members have a common structure that is closely related to, but clearly different from the other four known members of the family which have α‐, βI‐, βII‐ and γ‐sequences, although the ξ‐cDNA available at present does not contain a complete reading frame for a protein kinase C molecule. The diverse heterogeneity of the enzyme seems to be an important factor in determining the mode of response of many tissues and cell types to a variety of external stimuli.

Cellular and intracellular localization of ε-subspecies of protein kinase C in the rat brain; presynaptic localization of the ε-subspecies

Abstract The cellular and intracellular localization of the e-subspecies of protein kinase C (PKC) in the rat brain was demonstrated by immunocytochemistry using specific antibodies against e-PKC. The e-PKC-specific immunochemistry was most abundant in the hippocampal formation, olfactory tubercle and Callejas islands, was moderate in the cerebral cortex, anterior olfactory nuclei, accumbens nucleus, lateral septal nuclei and caudate-putamen and low in the thalamus and medulla. The e-PKC-immunoreacctivity was scanty in the perikarya, except for the pyramidal cells of CA3 region of the hippocampus and the immunoreactivity was mainly present in neuropils and nerve fibers. The distribution of e-PKC immunoreactive neurons was consistent with that obtained by in situ hybridization histochemistry. Electron microscopic observations of the hippocampus revealed that the e-PKC is predominantly present in the cytoplasm of axon and nerve terminals and that this enzyme is assiciated with mitochondrial membrane and vesicles. These results suggested that e-PKC is probably involved in presynaptic functions in CNS, perhaps even neurotransmitter release.

The common structure and activities of four subspecies of rat brain protein kinase C family

Elucidation of the complete sequences of four cDNA clones (α, βI, βII, and γ) of the rat brain protein kinase C family has revealed their common structure composed of a single polypeptide chain with four constant (C1‐C4) and five variable (V1‐V5) regions. Although these sequences are highly homologous and closely related to one another V3‐, V4‐, and V5‐regions of γ‐subspecies are slightly bigger than the corresponding regions of the other three subspecies. The first constant region, C1, contains a tandem repeat of cysteine‐rich sequence (6, total 12 cysteine residues). The third constant region, C3, has an ATP‐binding sequence which is found in many protein kinases. In adult rat whole brain, the relative activities of α‐, βI, βII, and γ‐subspecies are roughly 16, 8, 55, and 21%, respectively. γ‐Subspecies is expressed after birth apparently only in the central nervous tissue, implying its role in the regulation of specific neuronal functions.

Cloning of rat brain protein kinase C complementary DNA

Four peptides derived from rat brain protein kinase C were partially sequenced. Using synthetic oligonucleotides deduced from the amino acid sequences as probes, a clone of complementary DNA (cDNA) was isolated from a cDNA library prepared from the same tissue. The nucleotide sequence of this cDNA clone revealed the primary structure of the carboxyl‐terminal region as having 224 amino acids, with significant sequence homology with cyclic AMP‐dependent and cyclic GMP‐dependent protein kinases.

Chapter 9: Protein kinase C family and nervous function

Publisher Summary Protein kinase C (PKC) is involved in many aspects of neuronal function such as the short-term modulation of membrane excitability and neurotransmitter release. In areas such as the hippocampus, it also appears, together with other protein kinases and possibly proteases, to play a part in the long-term, use-dependent potentiation of synaptic transmission. Although the extent of the diversity of PKC family is not fully clarified at present, this chapter discusses some aspects of the present studies and prospective of this enzyme. It is becoming clear that PKC plays a crucial role in the cross-talk of cell signaling pathways that can be seen between various levels of receptors, coupling factors such as G-proteins, effectors such as adenylate cyclase and phospholipases, second messengers such as cyclic AMP, diacylglycerol and IP 3 , Ca 2+ , and many protein kinases including protein kinase cascade, as well as between various types of ion channels. At present, such intra- and intercellular events have not yet been fully substantiated on a firm biochemical basis, but it is extremely important to clarify the molecular mechanism of such cross-talk among cell-signaling pathways for better understanding the role of PKC in nervous tissues.

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, δ‐, ϵ‐, and ζ‐PKC were detected in both normal melanocytes and the four melanoma cell lines. In contrast, both α‐PKC and β‐PKC were expressed in normal melanocytes, whereas either α‐PKC or β‐PKC was detected in melanoma cells. Specifically, HM3KO, MeWo, and HMV‐1 cells were shown to contain α‐PKC but not β‐PKC, while G361 cells expressed β‐PKC but not α‐PKC. The growth of these melanoma cells was suppressed by TPA treatment, and the growth of the G361 cells lacking α‐PKC was inhibited more efficiently than the other melanoma cell lines which lacked β‐PKC. It was further shown that β‐PKC was not detected in freshly isolated human primary or metastatic melanoma tissues. These results suggest that the expression of α‐PKC or β‐PKC may be altered during the malignant transformation of normal melanocytes and that loss of α‐PKC or β‐PKC may be related to the inhibitory effect of TPA on the growth of melanoma cells.