Activation of protein kinase C subtypes alpha, gamma, delta, epsilon, zeta, and eta by tumor-promoting and nontumor-promoting agents.

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.

Loss of protein kinase C delta from human HaCaT keratinocytes upon ras transfection is mediated by TGF alpha.

The spontaneously immortalized human skin keratinocytes HaCaT contain protein kinase C (PKC) alpha, -delta, -epsilon, and -zeta. All PKC isoenzymes except PKC zeta are down-regulated by TPA as well as by bryostatin. However, with PKC delta, bryostatin but not TPA was found to be much less effective at high concentrations than at low ones. PKC delta expression at the protein and mRNA level is significantly suppressed in HaCaT cells I-7 and II-4, which are transfected with mutated c-Ha-ras. The expression of the other isoenzymes remains essentially unchanged in the ras-transfected cells compared to normal ones. PKC delta is lost when growing HaCaT cells in a medium obtained from the cultivation of ras-transfected cells ("ras-conditioned" medium). The factor secreted into the medium by the ras-transfected cells that is responsible for this effect appears to be TGF alpha, since the action of ras-conditioned medium on PKC delta expression can be overcome by the addition of an anti-TGF alpha antibody. Moreover, treatment of HaCaT cells with TGF alpha suppresses selectively the expression of the PKC isoenzyme delta.