INCREASED EXPRESSION OF A HIGH-MOLECULAR-WEIGHT (130 KD) PROTEIN-KINASE-C ISOFORM IN A DIFFERENTIATION-DEFECTIVE RAS-TRANSFECTED KERATINOCYTE LINE

The role of ras on protein kinase C (PKC) signaling was examined in tw o keratinocyte cell lines. Increasing the level of extracellular calci um from 0.15 mM to 1.0 mM induces some features of differentiation in the spontaneously immortalized HaCaT line, but fails to do so in a c-H -ras-transfected subline (ras-HaCaT). Raising extracellular calcium al so induced a transient increase in membrane-associated PKC activity 5 min after calcium addition, in HaCaT, but not in the ras-HaCaT cells. Partial purification of PKC from the membrane/particulate fraction rev ealed the major isoform expressed in HaCaT to be an 80 KD species reco gnized by the anti-PKC alpha antibody. In ras-HaCaT, the major express ed isoform is a 130 KD species recognized by the PKC beta antibody. Th e kinase activity of the partially purified high molecular weight PKC is phospholipid dependent but calcium independent. Further evaluation of PKC in the HaCaT and ras-HaCaT membrane/particulate cell fraction b y immunoblotting using affinity-purified antibodies against PKC alpha, beta, delta, epsilon and zeta revealed a 130 KD band reacting with th e PKC delta antibody. Increased expression of this high molecular weig ht protein was observed in ras-HaCaT. Immunoprecipitation of PKC in ra s-HaCaT using the PKC delta antibody also revealed a 130 KD species. A nalysis of the PKC delta immunoprecipitate demonstrated a phospholipid , but not calcium-dependent kinase which autophosphorylated. These res ults suggest that the 130 KD protein may be a novel (calcium-independe nt) PKC (nPKC) isoform and increased expression in the ras-transfected HaCaT may be a consequence of oncogenic ras expression. This 130 KD s pecies may also play a role in the ras-associated inhibition of differ entiation in HaCaT. (C) 1995 Wiley-Liss, Inc.