Requirements of protein kinase C delta 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 threoni ne (Thr(505)) in the activation loop for catalytic competence (Stempka ct a l, (1997) J, Biol, Chem, 272, 6805-6811). Here, we show that the acidic res idue glutamic acid 500 (Glu(500)) in the activation loop is important for t he catalytic function of PKC delta, A Glu(500) to valine mutant shows 76 an d 73% reduced kinase activity toward autophosphorylation and substrate phos phorylation, respectively. With regard to thermal stability and inhibition by the inhibitors Go6976 and Go6983 the mutant does not differ from the wil d type, indicating that the general conformation of the molecule is not alt ered by the site directed mutagenesis. Thus, Glu(500) in the activation loo p of PKC delta might take over at least part of the role of the phosphate g roups on Thr(497) and Thr(500) Of PKC alpha and beta(II), respectively. Acc ordingly, PKC delta exhibits kinase activity and is able to autophosphoryla te probably without posttranslational modification. Autophosphorylation of PKC delta in vitro occurs on Ser(643), as demonstrated by matrix-assisted l aser desorption ionization mass spectrometry of tryptic peptides of autopho sphorylated PKC delta wild type and mutants. A peptide containing this site is phosphorylated also in vivo, i.e, in recombinant PKC delta purified fro m baculovirus-infected insect cells. A Ser(643) to alanine mutation indicat es that autophosphorylation of Ser(643) is not essential for the kinase act ivity of PKC delta, Probably additional (auto)phosphorylation site(s) exist that have not yet been identified.