Determination of the Specific Substrate Sequence Motifs of Protein Kinase C Isozymes

Abstract

Protein kinase C (PKC) family members play significant roles in a variety of intracellular signal transduction processes, but information about the substrate specificities of each PKC family member is quite limited. In this study, we have determined the optimal peptide substrate sequence for each of nine human PKC isozymes (␣, ␤I, ␤II, ␥, ␦, ⑀, , , and ) by using an oriented peptide library. All PKC isozymes preferentially phosphorylated peptides with hydrophobic amino acids at position ؉1 carboxyl-terminal of the phosphorylated Ser and basic residues at position ؊3. All isozymes, except PKC, selected peptides with basic amino acids at positions ؊6, ؊4, and ؊2. PKC␣, -␤I, -␤II, -␥, and -selected peptides with basic amino acid at positions ؉2, ؉3, and ؉4, but PKC␦, -⑀, -, and -preferred peptides with hydrophobic amino acid at these positions. At position ؊5, the selectivity was quite different among the various isozymes; PKC␣, -␥, and -␦ selected peptides with Arg at this position while other PKC isozymes selected hydrophobic amino acids such as Phe, Leu, or Val. Interestingly, PKC showed extreme selectivity for peptides with Leu at this position. The predicted optimal sequences from position ؊3 to ؉2 for PKC␣, -␤I, -␤II, -␥, -␦, and -were very similar to the endogenous pseudosubstrate sequences of these PKC isozymes, indicating that these core regions may be important to the binding of corresponding substrate peptides. Synthetic peptides based on the predicted optimal sequences for PKC␣, -␤I, -␦, -, and -were prepared and used for the determination of K m and V max for these isozymes. As judged by V max /K m values, these peptides were in general better substrates of the corresponding isozymes than those of the other PKC isozymes, supporting the idea that individual PKC isozymes have distinct optimal substrates. The structural basis for the selectivity of PKC isozymes is discussed based on residues predicted to form the catalytic cleft. Protein kinase C (PKC)1 family members play crucial roles in the signal transduction of a variety of extracellular stimuli, such as hormones and growth factors (1). To date, twelve isozymes of PKC have been identified in mammalian tissues and subdivided into conventional PKC (cPKC) members comprising ␣, ␤I, ␤II, and ␥ isoforms (activated by calcium, acidic phospholipid, and diacylglycerol (DAG)), novel PKCs (nPKC) comprising ␦, ⑀, , and (activated by DAG and acidic phospholipid but insensitive to calcium), and atypical PKCs (aPKC) / and (mechanism of regulation not clear) (1-6). Another subgroup of PKCs may be defined by PKC, which has a potential signal peptide and transmembrane domain (7). Since these PKC isozymes differ in their expression in different tissues and in their mode of activation (1), each isozyme may play some specific role in signal transduction processes. Recent investigations using various approaches such as overexpression and down-regulation of specific isozymes support this idea (1,5). A large number of proteins have been shown to be phosphorylated by PKC in ...

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