Mutagenesis of the regulatory domain of rat protein kinase C-eta. A molecular basis for restricted histone kinase activity.

Protein kinase C-theta (PKC-eta) is a member of the protein kinase C family that is characterized by Ca2+ independence and restricted histone kinase activity (Dekker, L. V., Parker, P. J., and McIntyre, P. (1992) FEBS Lett. 312, 195-199). Here we have investigated the molecular basis of this low histone kinase activity by limited proteolysis and site-directed mutagenesis. It is shown that a 46-kDa C-terminal tryptic fragment, representing the catalytic domain of PKC-eta, can phosphorylate histone. The Km value for histone of this catalytic fragment is 25-fold lower than that of intact PKC-eta. Thus, sites in the N-terminal regulatory domain upstream of the trypsin cleavage site (near residue 320) restrict histone kinase activity of intact PKC-eta. Deletion of the "Vo domain" (residues 2-137) generates a PKC-eta mutant that shows the same cofactor dependence and substrate phosphorylation as wild-type PKC-eta, indicating that the relevant sites do not appear to lie in the Vo domain but between amino acid 137 and the start of the catalytic domain. Deletion of the pseudosubstrate region (residue 155-171) generates a cofactor-independent kinase that has high histone kinase activity. A pseudosubstrate site point mutation in which the alanine residue at position 161 is replaced with a glutamic acid residue shows the same properties as the pseudosubstrate site deletion mutant. Km values for histone for both mutants are similar to that observed for the catalytic fragment. Therefore, in addition to its role in conferring cofactor dependence, the pseudosubstrate site also mediates the low histone kinase activity of wild-type PKC-eta. The data are discussed in the light of current models for PKC activation.