Protein kinase C has a crucial role in signal transduction for a variety of biologically active substances which activate cellular functions and proliferation. When cells are stimulated, protein kinase C is transiently activated by diacylglycerol which is produced in the membrane during the signal-induced turnover of inositol phospholipids. Tumour-promoting phorbol esters, when intercalated into the cell membrane, may substitute for diacylglycerol and permanently activate protein kinase C. The enzyme probably serves as a receptor for the tumour promoters. Further exploration of the roles of this enzyme may provide clues for understanding the mechanism of cell growth and differentiation.
Hydrolysis of inositol phospholipids by phospholipase C is initiated by either receptor stimulation or opening of Ca2+ channels. This was once thought to be the sole mechanism to produce the diacylglycerol that links extracellular signals to intracellular events through activation of protein kinase C. It is becoming clear that agonist-induced hydrolysis of other membrane phospholipids, particularly choline phospholipids, by phospholipase D and phospholipase A2 may also take part in cell signaling. The products of hydrolysis of these phospholipids may enhance and prolong the activation of protein kinase C. Such prolonged activation of protein kinase C is essential for long-term cellular responses such as cell proliferation and differentiation.
Protein kinase C, an enzyme that is activated by the receptor-mediated hydrolysis of inositol phospholipids, relays information in the form of a variety of extracellular signals across the membrane to regulate many Ca2+-dependent processes. At an early phase of cellular responses, the enzyme appears to have a dual effect, providing positive forward as well as negative feedback controls over various steps of its own and other signaling pathways, such as the receptors that are coupled to inositol phospholipid hydrolysis and those of some growth factors. In biological systems, a positive signal is frequently followed by immediate negative feedback regulation. Such a novel role of this protein kinase system seems to give a logical basis for clarifying the biochemical mechanism of signal transduction, and to add a new dimension essential to our understanding of cell-to-cell communication.
Protein kinase C is now known to be a large family of proteins, with multiple subspecies that have subtle individual enzymological characteristics. Some members of the family exhibit distinct patterns of tissue expression and intracellular localization; different kinases probably have distinct functions in the processing and modulation of a variety of physiological and pathological responses to external signals.
Since the second messenger role was proposed for the products of inositol phospholipid hydrolysis, considerable progress has been made in our understanding of the biochemical mechanism of the intracellular signaling network. It is now becoming evident that stimulation of a cell surface receptor initiates a degradation cascade of various membrane lipid constituents. Many of their metabolites have potential to induce, intensify, and prolong the activation of protein kinase C that is needed for sustained cellular responses.
Various extracellular informational signals such as those from a group of hormones and some neurotransmitters appear to be passed from the cell surface into the cell interior by two routes, protein kinase C activation and Ca2+ mobilization. Both routes usually become available as the result of an interaction of a single ligand and a receptor and act synergistically to evoke subsequent cellular responses such as release reactions. The signal-dependent breakdown of inositol phospholipids, particularly phosphatidylinositol bisphosphate, now appears to be a key event for initiating these processes.
Protein kinase C (PKC) isoforms, ␣, I, and ␥ of cPKC subgroup, ␦ and of nPKC subgroup, and of aPKC subgroup, were tyrosine phosphorylated in COS-7 cells in response to H 2 O 2 . These isoforms isolated from the H 2 O 2 -treated cells showed enhanced enzyme activity to various extents. The enzymes, PKC ␣ and ␦, recovered from the cells were independent of lipid cofactors for their catalytic activity. Analysis of mutated molecules of PKC ␦ showed that tyrosine residues, which are conserved in the catalytic domain of the PKC family, are critical for PKC activation induced by H 2 O 2 . These results suggest that PKC isoforms can be activated through tyrosine phosphorylation in a manner unrelated to receptor-coupled hydrolysis of inositol phospholipids.
The primary structure of the ; subspecies of rat brain protein kinase C was deduced from its overlapping cDNAs. The ; 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- The physiological importance of protein kinase (PKC) is now widely accepted and well documented (1). Molecular cloning and biochemical analysis has revealed the enzyme to exist as a family of multiple subspecies having closely related structures (1). Initially, four cDNA clones, a, /31, PII, and y were isolated (2-8). The four PKC subspecies all consist of a single polypeptide with four conserved (C1-C4) and five variable (V1-V5) regions. The amino-terminal half, containing regions C1 and C2, is presumably the regulatory domain that interacts with Ca2 , phospholipid, and diacylglycerol or phorbol ester, whereas the carboxyl-terminal half, containing regions C3 and C4, appears to be the protein kinase domain, as it shows large clusters of sequences that resemble many other protein kinases. The region C1 contains a tandem repeat of the characteristic cysteine-rich zinc-finger-like sequence. The structure and genetic identity of these subspecies have been determined by comparison with the enzymes that are separately expressed in mammalian COS-7 cells transfected by the respective cDNA-containing plasmids (9,10) and by immunoblot analysis, using type-specific antibodies, of COS-7 cells transfected with plasmids containing cDNA inserts of the different PKC subspecies (11). Recently, several additional cDNA clones designated 6, E, and {, were isolated from a rat brain library by using a mixture of a, 3II, and y cDNAs as probes under low-stringency conditions (12, 13). A cDNA clone designated nPKC, probably encoding the E subspecies, was also found in a rabbit brain cDNA library (14). Another cDNA clone, RP16, isolated previously from a rat brain library (15) may also encode a part of the E subspecies. The three PKC molecules have a common structure closely related to, but clearly distinct from, the four subspecies initially described. The enzyme encoded by 6, E, and r cDNA all lack the region C2, and the translational products of 6 and e cDNA in COS-7 cells did not show an absolute requirement of Ca2 , phospholipid, and diacylglycerol (13). On the other hand, the structure and enzymatic properties of the {-subspecies remain unknown, because the full length of its cDNA has not been available. This paper will describe the complete structure, expression, and some kinetic properties of the ; subspecies of PKC.* MATERIALS AND METHODSIsolation and Characterization of cDNA Clones. Two cDNA clones, ACKRL;5 and ACKRL;8, both encoding the r subspecies of PKC were isolated from a rat brain cDNA library, which was constructed in AgtlO, by using a 0.4-kilobase (k...
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