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
D. Geiges et al., 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, Biochemical pharmacology, 53(6), 1997, pp. 865-875
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.