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Volume 272, Number 2, Issue of January 10, 1997 pp. 952-960
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

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

(Received for publication, September 8, 1996, and in revised form, October 16, 1996)

Kiyotaka Nishikawa , Alex Toker , Franz-Josef Johannes ^¶ , Zhou Songyang and Lewis C. Cantley

From the  Division of Signal Transduction, Beth Israel Hospital and Department of Cell Biology, Harvard Medical School, Boston Massachusetts 02115 and the ^¶ Institute of Cell Biology and Immunology, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany

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, , , , , µ, 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 PKCµ, 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 -µ 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, PKCµ 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 -µ 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.

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