Further definition of the substrate specificity of the alpha-herpesvirus protein kinase and comparison with protein kinases A and C.

The pseudorabies virus protein kinase prefers model substrates containing arginyl residues on the amino-terminal side of a target seryl or threonyl residue. We have defined this substrate specificity more precisely in experiments using a new series of synthetic model peptides. When the number of arginyl residues was varied from two to four in substrates of the type RnASVA it was found that peptides with four arginyl residues constituted the best substrates, although the most marked decrease in Km was seen on increasing the number of arginyl residues from two to three. The effect of varying the number of 'spacer' alanyl residues from zero to three was investigated in peptides of the type R4AmSVA, and the peptide with one alanyl residue was found to be the best substrate, making R4X the optimal amino-terminal environment for this enzyme. A similar substrate specificity was observed with the herpes simplex type 1 protein kinase. Protein kinase C was found to have a quite similar substrate preference to the viral enzyme as far as the number and position of the amino-terminal basic residues was concerned; but, unlike the viral protein kinase, it also requires carboxy-terminal basic residues in optimal peptide substrates, and can tolerate the substitution of lysyl for arginyl residues. The cyclic AMP-dependent protein kinase, like the viral enzyme, had favourable kinetic constants for this series of peptides, but differed from the latter in being able to catalyze the phosphorylation of the peptides with two to four arginyl residues with similar efficiency. Studies with the protein, clupeine Y1, as substrate indicated that the pseudorabies virus protein kinase can tolerate arginyl residues on the carboxyl-terminal side of its target residue when there are suitable amino-terminal arginyl determinants. In this respect the virus protein kinase resembled protein kinase C but differed from the cyclic AMP-dependent protein kinase which cannot tolerate such carboxyl-terminal basic residues. The relationship of substrate specificity with model peptides to the ability of the pseudorabies virus protein kinase to phosphorylate proteins in vitro and in vivo is discussed.