MUTAGENESIS OF THE REGULATORY DOMAIN OF RAT PROTEIN KINASE-C-ETA - A MOLECULAR-BASIS FOR RESTRICTED HISTONE KINASE-ACTIVITY

Protein kinase C-eta (PKC-eta) is a member of the protein kinase C fam ily 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 bas is of this low histone kinase activity by limited proteolysis and site -directed mutagenesis. It is shown that a 46-kDa C-terminal tryptic fr agment, representing the catalytic domain of PKC-eta, can phosphorylat e histone. The K(m) 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 ''V(o) domain'' (residues 2-137) generates a PKC-eta mutant that s hows the same cofactor dependence and substrate phosphorylation as wil d-type PKC-eta, indicating that the relevant sites do not appear to li e in the V(o) domain but between amino acid 137 and the start of the c atalytic domain. Deletion of the pseudosubstrate region (residue 155-1 71) generates a cofactor-independent kinase that has high histone kina se activity. A pseudosubstrate site point mutation in which the alanin e residue at position 161 is replaced with a glutamic acid residue sho ws the same properties as the pseudosubstrate site deletion mutant. K( m) values for histone for both mutants are similar to that observed fo r the catalytic fragment. Therefore, in addition to its role in confer ring cofactor dependence, the pseudosubstrate site also mediates the l ow histone kinase activity of wild-type PKC-eta. The data are discusse d in the light of current models for PKC activation.