PHOSPHORYLATION OF PROTEIN-KINASE-C-DELTA (PKC-DELTA) AT THREONINE-505 IS NOT A PREREQUISITE FOR ENZYMATIC-ACTIVITY - EXPRESSION OF RAT PKC-DELTA AND AN ALANINE-505 MUTANT IN BACTERIA IN A FUNCTIONAL FORM

A structural feature shared by many protein kinases is the requirement for phosphorylation of threonine or tyrosine in the so called activat ion loop for full enzyme activity, Previous studies by several groups have indicated that the isotypes alpha, beta(I), and beta(II) of prote in kinase C (PKC) are synthesized as inactive precursors and require p hosphorylation by a putative ''PKC kinase'' for permissive activation, Expression of PKC alpha in bacteria resulted in a nonfunctional enzym e, apparently due to lack of this kinase, The phosphorylation sites fo r the PKC kinase in the activation loop of PKC alpha and PKC beta(II) could be identified as Thr(497) and Thr(500), respectively, We report here that PKC delta, contrary to PKC alpha, can be expressed in bacter ia in a functional form, The activity of the recombinant enzyme regard ing substrate phosphorylation, autophosphorylation, and dependence an activation by 12-O-tetradecanoylphorbol-13-acetate as well as the K-m values for two substrates are comparable to those of recombinant PKC d elta expressed in baculovirus infected insect cells. By site directed mutagenesis we were able to show that Thr(505), corresponding to Thr(4 97) and Thr(500) of PKC alpha and PKC beta(II), respectively, is not e ssential for obtaining a catalytically competent conformation of PKC d elta, The mutant Ala(505) can be activated and does not differ from th e wild type regarding activity and several other features, Ser(504) ca n not take over the role of Thr(505) and is not prerequisite for the k inase to become activated, as proven by the unaffected enzyme activity of respective mutants (Ala(504) and Ala(504)/Ala(505)), These results indicate that phosphorylation of Thr(505) is not required for the for mation of functional PKC delta and that at least this PKC isoenzyme di ffers from the isotypes alpha, beta(I), and beta(II) regarding the per missive activation by a PKC kinase.