No abstract

We examined the structure of protein kinase C in an attempt to understand the molecular events connecting protein kinase C activation with the cellular response. Rabbit complementary DNA clones coding for three distinct types of protein kinase C, named alpha, beta and gamma, have been identified and sequenced. The deduced amino acid sequence for alpha, beta and gamma (673, 671 and 672 amino acids, respectively) are closely related. Kinases alpha and beta share an identical N-terminal sequence of 621 amino acid residues and their messenger RNAs arise from a single gene. The C-terminal halves of alpha, beta and gamma are protein kinase domains and are highly homologous to other protein kinases. The mRNAs for alpha, beta and gamma are expressed in various tissues with strikingly different tissue specificities. The one for gamma is found ubiquitously among various tissues, while those for alpha and beta predominate in the brain.

Calcium-dependent protease (calcium protease) is apparently involved in a variety of cellular processes. Here we have attempted to clarify the role and regulatory mechanism of calcium protease by analysing its structure. The complete primary structure of calcium protease (relative molecular mass (Mr) 80,000 (80K), 705 amino acids) was deduced from the nucleotide sequence of cloned complementary DNA. The protein contains four distinct domains, and we have observed a marked similarity between the second and fourth domains and the papain-like thiol proteases and calmodulin-like calcium-binding proteins, respectively. This finding suggests that calcium protease arose from the fusion of genes for proteins of completely different function and evolutionary origin. Further, it provides functional insight into cellular regulatory mechanisms mediated by Ca2+ through calcium-binding proteins.

The structures of calcium-activated neutral protease (CANP) and its endogenous inhibitor elucidated recently have revealed novel features with respect to their structure-function relationship and enzyme activity regulation. The protease is regarded as a proenzyme which can be activated at the cell membrane in the presence of Ca 2 + and phospholipid, and presumably regulates the functions of proteins, especially membrane-associated proteins, by limited proteolysis. Protein kinase C is hydrolysed and activated by CANP at the cell membrane to a cofactor-independent form. These results are reviewed and the possible involvement of CANP in signal transduction is discussed.

A cDNA encoding an endogenous inhibitor, termed calpastatin, for calcium-dependent cysteine protease (calpain, EC 3.4.22.17) was cloned by screening rabbit cDNA libraries with a synthetic oligodeoxynudeotide probe based on the partial amino acid sequence of the purified protein. The deduced amino acid sequence contains 718 amino acid residues (Mr, 76,964), and the mature protein corresponds to the deduced sequence from the 80th residue of the primary translation product (resultant Mr, 68,113). This deduced molecular weight is significantly lower than that determined by NaDodSO4/polyacrylamide gel electrophoresis, suggesting the possibility that the inhibitor is post-translationally modified. The sequence of the mature inhibitor contains four consecutive internal repeats 140 amino acid residues long, each of which might be responsible for the inhibitory activity. Calpastatin is apparently different from a typical cysteine protease inhibitor (cystatin), suggesting that the mechanism of inhibition of calcium-dependent cysteine protease by the inhibitor might be different from that of other cysteine proteases by cystatin.Calcium-dependent cysteine protease (calpain, EC 3.4.22.17) is a widely distributed intracellular cysteine protease involved in a variety of cellular processes mediated by Ca2'(1-3). The enzyme catalyzes the limited proteolysis of various proteins and its activity, which is absolutely dependent on Ca2+, is controlled by a specific endogenous inhibitor, calpastatin (1-3).The structure of calpain has been well characterized; its large subunit has four domains, including a papain-like cysteine protease domain and a calmodulin-like calciumbinding domain (4-6), and the small subunit consists of two domains, a glycine-rich hydrophobic domain and a calmodulin-like calcium-binding domain (7-9). However, the structure of calpastatin has not been determined, and various values have been reported for its molecular weight (10-15). The molecular weights estimated by NaDodSO4/polyacrylamide gel electrophoresis were -110,000 for the human liver (13), rabbit skeletal muscle (14), and porcine heart (15) inhibitor and -70,000 for the porcine erythrocyte inhibitor (15). The existence of multiple reactive sites for inhibition has also been reported: 1 mol of calpastatin inhibits several moles of calpain, although the precise mechanism of inhibition is not known (13, 15).As a step toward elucidation of the inhibition mechanism of calpastatin, its structure was determined by analysis of its cDNA sequence. The complete derived amino acid sequence of the inhibitor is presented with a discussion on the structure-function relationship. MATERIALS AND METHODSPurification of Calpastatin from Rabbit Liver and the Designing of an Oligodeoxynucleotide Probe. Calpastatin was purified from rabbit liver essentially as described (13). The purified inhibitor gave a single band on NaDodSO4/polyacrylamide gel electrophoresis, and its various properties were similar to those of the human liver inhibitor (13). Purified calpastat...