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

Abstract

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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