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...

Several lines of CHO cells stably overexpressing protein kinase C (PKC) subspecies to various extents were established by the DNA-mediated transfer. Upon treatment with phorbol 12-myristate 13-acetate, the growth of the cells expressing the PKC-6 subspecies was markedl inhibited, whereas cell lines expressing PKC-a, PKC-IH, and PKC-C subspecies were not sign tly affected. Flow cytometric analysis indicated that all cell les overexpressing PKC-8 subspecies accumulated in G2/M phase in response to phorbol 12-myristate 13-acetate. In these arrete cells, dikaryons were predominant, implying that phorbol ester-induced inhibition of cell division is specific to telophase. These results suggest PKC-6 subspecies may play a role in the normal cell cycle progression.Protein kinase C (PKC) appears to play crucial roles in signal transduction leading to cell growth and differentiation. This enzyme was first shown to be activated by diacylglycerol generated by the receptor-mediated hydrolysis of inositol phospholipids and has been identified as a major receptor of tumor-promoting phorbol esters (1). Molecular cloning and biochemical studies have revealed that the PKC family is a large family, consisting ofat least eight subspecies, a, 3PI, /11, y, 8, E, A, and q (PKC-L) in mammalian'tissues (2-5). These subspecies show subtly different enzymological properties and distinct tissue distribution. Molecular genetic approaches have also been used to explore the role of this PKC family in cell proliferation by expressing defined enzyme subspecies, such as PKC-a, PKC-P, or PKC-y, in mammalian cells (6-11). In some cell lines stably overproducing PKC subspecies, growth promotion such as enhanced growth rates, increased saturation densities, and anchorageindependent growth has been observed (6,7,9,10 MATERIALS AND METHODS Construction of Expression Plasmids. cDNAs for rat PKC-a, -/311, -8, and -C subspecies were excised from plasmids pTB755, pTB708, pTB808, and pTB949, respectively (14-17), by complete or partial digestion with EcoP. These cDNAs were then separately inserted downstream of the Abelson murine leukemia virus long terminal repeat in plasmid pTB399 (18) by replacing the interleukin 2 sequence. The long terminal repeat-PKC cDNA segments between Sal I and Cla I sites ofthese plasmids were transferred to the same sites of plasmid pTB348 (18), which carried the hamster dihydrofolate reductase (DHFR) cDNA under the control of the simian virus 40 early promoter. The resulting expression plasmids for the a, . 3II,8, and ; subspecies are referred to as pTB789, pTB705, pTB1322, and pTB1324, respectively. All the procedures were done as described (19).Transfection, Selection, and Gene Amplification. DHFR-CHO cells (20) were transfected with each of the expression constructs by the calcium phosphate-DNA coprecipitation method (21). DHFR+ transformants were selected by colony formation in Dulbecco's modified Eagle's medium (DMEM) containing 10%6 (vol/vol) dialyzed fetal bovine serum and proline (35 ,Ag/ml). Gene amplificatio...

Accession nos.X07286 and X07287 Recent molecular cloning analysis has shown that protein kinase C is a family of multiple subspecies having closely related structures (1-7). The structures of four subspecies a,

Two complementary DNA's, encoding the complete sequences of 671 and 673 amino acids for subspecies of rat brain protein kinase C, were expressed in COS 7 cells. The complementary DNA sequence analysis predicted that the two enzymes are derived from different ways of splicing and differ from each other only in the short ranges of their carboxyl-terminal regions. Both enzymes showed typical characteristics of protein kinase C that responded to Ca2+, phospholipid, and diacylglycerol. The enzymes showed practically identical physical and kinetic properties and were indistinguishable from one of the several subspecies of protein kinase C that occurs in rat brain but not in untransfected COS 7 cells. Partial analysis of the genomic structure confirmed that these two subspecies of protein kinase C resulted indeed from alternative splicing of a single gene.

The £ subspecies of protein kinase C (ePKC) 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 EPKC cDNA expression plasmid showed the same elution profile. The purified enzyme from the brain was a doublet (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 EPKC. When treated with potato acid phosphatase, both doublet proteins disappeared with the concomitant appearance of a single protein at 90 kDa, suggesting that EPKC 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 a-, f11-, 1311-, and yPKC, EPKC was independent of Ca2+ but absolutely required phosphatidylserine and diacylglycerol for its activation; a tumor-promoting phorbol ester could replace diacylglycerol. EPKC showed enzymological properties similar to those of 6PKC, except that EPKC but not 6PKC was greatly activated by free arachidonic acid. Immunoblot analysis revealed that, in marked contrast to