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 activation loop for full enzyme activity, Previous studies by several groups have indicated that the isotypes alpha, beta(I), and beta(II) of protein kinase C (PKC) are synthesized as inactive precursors and require phosphorylation by a putative ''PKC kinase'' for permissive activation, Expression of PKC alpha in bacteria resulted in a nonfunctional enzyme, apparently due to lack of this kinase, The phosphorylation sites for 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 bacteria in a functional form, The activity of the recombinant enzyme regarding 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 delta expressed in baculovirus infected insect cells. By site directed mutagenesis we were able to show that Thr(505), corresponding to Thr(497) and Thr(500) of PKC alpha and PKC beta(II), respectively, is not essential for obtaining a catalytically competent conformation of PKC delta, The mutant Ala(505) can be activated and does not differ from the wild type regarding activity and several other features, Ser(504) can not take over the role of Thr(505) and is not prerequisite for the kinase 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 formation of functional PKC delta and that at least this PKC isoenzyme differs from the isotypes alpha, beta(I), and beta(II) regarding the permissive activation by a PKC kinase.