Requirements of protein kinase Cδ for catalytic function -: Role of glutamic acid 500 and autophosphorylation on serine 643

Recently, we reported that, in contrast to protein kinase C (PKC) alpha and beta(II), PKC delta does not require phosphorylation of a specific threonine (Thr(505)) in the activation loop for catalytic competence (Stempka ct al, (1997) J, Biol, Chem, 272, 6805-6811). Here, we show that the acidic residue glutamic acid 500 (Glu(500)) in the activation loop is important for the catalytic function of PKC delta, A Glu(500) to valine mutant shows 76 and 73% reduced kinase activity toward autophosphorylation and substrate phosphorylation, respectively. With regard to thermal stability and inhibition by the inhibitors Go6976 and Go6983 the mutant does not differ from the wild type, indicating that the general conformation of the molecule is not altered by the site directed mutagenesis. Thus, Glu(500) in the activation loop of PKC delta might take over at least part of the role of the phosphate groups on Thr(497) and Thr(500) Of PKC alpha and beta(II), respectively. Accordingly, PKC delta exhibits kinase activity and is able to autophosphorylate probably without posttranslational modification. Autophosphorylation of PKC delta in vitro occurs on Ser(643), as demonstrated by matrix-assisted laser desorption ionization mass spectrometry of tryptic peptides of autophosphorylated PKC delta wild type and mutants. A peptide containing this site is phosphorylated also in vivo, i.e, in recombinant PKC delta purified from baculovirus-infected insect cells. A Ser(643) to alanine mutation indicates that autophosphorylation of Ser(643) is not essential for the kinase activity of PKC delta, Probably additional (auto)phosphorylation site(s) exist that have not yet been identified.