Two cytosolic proteins of murine epidermis or porcine spleen with molecular masses of 37 kDa (p37) and 50 kDa (p50) are differentially phosphorylated in vitro by the purified protein kinase C (PKC) isoenzymes , , (cPKC) and PKC. p37, identified as annexin I, is preferentially phosphorylated by cPKC, whereas p50, identified as elongation factor eEF-1, is phosphorylated with much greater efficacy by PKC than by cPKC. Using the recombinant PKC isoenzymes , , , , , , and , we could show that purified eEF-1 is indeed a specific substrate of PKC. It is not significantly phosphorylated by PKC, -, and - and only slightly by PKC, -, and -. PKC phosphorylates eEF-1 at Thr-431 (based on the murine amino acid sequence). The peptide RFAVRDMRQTVAVGVIKAVDKK with a sequence corresponding to that of 422-443 from murine eEF-1 and containing Thr-431 is an absolutely specific substrate for the -type of PKC. The single basic amino acid close to Thr-431 (Arg-429) is essential for recognition of the peptide as a substrate by PKC and for the selectivity of this recognition. Substitution of Arg-429 by alanine abolishes the ability of PKC to phosphorylate the peptide, and insertion of additional basic amino acids in the vicinity of Thr-431 causes a complete loss of selectivity.

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