ACTIVATION OF PROTEIN-KINASE-C SUBTYPE-ALPHA, SUBTYPE-GAMMA, SUBTYPE-DELTA, SUBTYPE-EPSILON, SUBTYPE-ZETA AND SUBTYPE-ETA BY TUMOR-PROMOTING AND NONTUMOR-PROMOTING AGENTS

Protein kinase C (PKC) subtypes alpha, gamma, delta, epsilon, zeta, an d eta have been expressed using the baculovirus expression system. The partially purified PKC subtypes have been studied for their substrate specificities and phospholipid-independent activation by various chem ically different nontumor- and tumor-promoting agents, as well as thei r inhibition of kinase activity by staurosporine and two related compo unds. An endogenous PKC-like kinase activity of Sf9 cells was detected and analyzed for cofactor requirements and inhibition. Protamine sulf ate was most efficiently phosphorylated by all of the PKC subtypes tes ted, although this phosphorylation was independent of phosphatidylseri ne (PS) and diacylglycerol (DAG) or 12-O-tetradecanoylphorbol 13-aceta te (TPA). Except for PKC-zeta, all subtypes tested phosphorylated myel in basic protein (MBP), histone, or a peptide derived from the pseudos ubstrate region of PKC-alpha in a PS/DAG-dependent manner but to varyi ng extents. Among the various agents tested, TPA most efficiently stim ulated the kinase activities of the PKC subtypes in a phospholipid-dep endent manner. Phorbol 12,13-dibutyrate (PDBu) was less effective than TPA but displayed no major difference among the subtypes. Activation of PKC-alpha by bryostatin-1 reached only half of the TPA response whe reas the other subtypes were activated more effectively, The weak tumo r promoter resiniferonol 9,13,14-orthophenyl acetate (ROPA) mainly sti mulated PKC-alpha and PKC-gamma at 1 mu M concentration, whereas PKC-e psilon and PKC-eta were much less activated. Sapintoxin D, mezerein, i ndolactam V, and resiniferatoxin at concentrations of 1-100 nM prefere ntially activated PKC-alpha in a DAG-like manner, whereas at 1 mu M ot her subtypes were activated as well. Preferential activation of PKC-al pha was also noted for tinyatoxin and thapsigargin, but their mode of activation is unclear because these two compounds did not compete for the phorbol ester binding of the PKC subtypes as the other agents did. Of the three PKC inhibitors tested, staurosporine most efficiently in hibited kinase activity of the PKC subtypes, whereas K252a and CGP 412 51 were at least 10 times less effective. However, K252a showed certai n specificity for inhibition of PKC-alpha, and CGP 41251 failed to inh ibit PKC-epsilon and PKC-zeta. Given the different substrate specifici ties and modes of activation by various tumor-promoting and nontumor-p romoting agents, as well as the different sensitivities towards differ ent inhibitors, our results indicate a divergence of individual PKC su btypes in signal transduction. (C) 1997 Elsevier Science Inc.