Protein kinase C (PKC) subtypes alpha, gamma, delta, epsilon, zeta, and 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 chemically different nontumor- and
tumor-promoting agents, as well as their inhibition of
kinase activity by
staurosporine and two related compounds. An endogenous PKC-like
kinase activity of Sf9 cells was detected and analyzed for cofactor requirements and inhibition.
Protamine sulfate was most efficiently phosphorylated by all of the PKC subtypes tested, although this phosphorylation was independent of
phosphatidylserine (PS) and
diacylglycerol (DAG) or 12-O-tetradecanoylphorbol 13-acetate (TPA). Except for PKC-zeta, all subtypes tested phosphorylated
myelin basic protein (MBP),
histone, or a
peptide derived from the pseudosubstrate region of PKC-alpha in a PS/DAG-dependent manner but to varying extents. Among the various agents tested, TPA most efficiently stimulated the
kinase activities of the PKC subtypes in a
phospholipid-dependent 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 whereas the other subtypes were activated more effectively. The weak
tumor promoter resiniferonol 9,13,14-orthophenyl
acetate (ROPA) mainly stimulated PKC-alpha and
PKC-gamma at 1 microM concentration, whereas
PKC-epsilon and
PKC-eta were much less activated.
Sapintoxin D,
mezerein,
indolactam V, and
resiniferatoxin at concentrations of 1-100 nM preferentially activated PKC-alpha in a DAG-like manner, whereas at 1 microM other subtypes were activated as well. Preferential activation of PKC-alpha 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 inhibited
kinase activity of the PKC subtypes, whereas
K252a and
CGP 41251 were at least 10 times less effective. However,
K252a showed certain specificity for inhibition of PKC-alpha, and
CGP 41251 failed to inhibit
PKC-epsilon and PKC-zeta. Given the different substrate specificities and modes of activation by various
tumor-promoting and nontumor-promoting agents, as well as the different sensitivities towards different inhibitors, our results indicate a divergence of individual PKC subtypes in signal transduction.