No abstract

Native protein kinase C_~ from porcine spleen is phosphorylated in vitro by the tyrosine kinase src and to a much smaller extent by fyn. The tyrosine phosphorylation of PKC~ is restricted to the activated state of the enzyme, i.e. it occurs only in the presence of an activator, such as TPA or bryostatin. Upon phosphorylation at tyrosine, the apparent molecular weight of PKC~ increases by 6 kDa. Phosphorylation by src induces a stimulation of PKC~ activity apparently exhibiting some substrate selectivity. Other PKC isoenzymes, such as cPKC (g~,7), are not phosphorylated by src or only to a very small extent. This phosphorylation is not dependent on TPA and does not cause an increase in activity and molecular weight of the enzyme.

No abstract

Two cytosolic proteins of murine epidermis or porcine spleen with molecular masses of 37 kDa (p37) and 50 kDa (p50) are differentially phosphorylated in vitro by the purified protein kinase C (PKC) isoenzymes alpha, beta, gamma (cPKC) and PKC delta. p37, identified as annexin I, is preferentially phosphorylated by cPKC, whereas p50, identified as elongation factor eEF-1 alpha, is phosphorylated with much greater efficacy by PKC delta than by cPKC. Using the recombinant PKC isoenzymes alpha, beta, gamma, delta, epsilon, eta, and zeta, we could show that purified eEF-1 alpha is indeed a specific substrate of PKC delta. It is not significantly phosphorylated by PKC epsilon, -eta, and -zeta and only slightly by PKC alpha, -beta, and -gamma. PKC delta phosphorylates eEF-1 alpha at Thr-431 (based on the murine amino acid sequence). The peptide RFAVRDMRQTVAVGVIKAVDKK with a sequence corresponding to that of 422-443 from murine eEF-1 alpha and containing Thr-431 is an absolutely specific substrate for the delta-type of PKC. The single basic amino acid close to Thr-431 (Arg-429) is essential for recognition of the peptide as a substrate by PKC delta and for the selectivity of this recognition. Substitution of Arg-429 by alanine abolishes the ability of PKC delta to phosphorylate the peptide, and insertion of additional basic amino acids in the vicinity of Thr-431 causes a complete loss of selectivity.