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 two keratinocyte cell lines. Increasing the level of extracellular calcium 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 also 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 revealed the major isoform expressed in HaCaT to be an 80 KD species recognized by the anti-PKC alpha antibody. In ras-HaCaT, the major expressed isoform is a 130 KD species recognized by the PKC beta antibody. The 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 by immunoblotting using affinity-purified antibodies against PKC alpha, beta, delta, epsilon and zeta revealed a 130 KD band reacting with the PKC delta antibody. Increased expression of this high molecular weight protein was observed in ras-HaCaT. Immunoprecipitation of PKC in ras-HaCaT using the PKC delta antibody also revealed a 130 KD species. Analysis of the PKC delta immunoprecipitate demonstrated a phospholipid, but not calcium-dependent kinase which autophosphorylated. These results suggest that the 130 KD protein may be a novel (calcium-independent) PKC (nPKC) isoform and increased expression in the ras-transfected HaCaT may be a consequence of oncogenic ras expression. This 130 KD species may also play a role in the ras-associated inhibition of differentiation in HaCaT.
Rosales
J L
JL
School of Medicine, Department of Dermatology, University of California, Davis 95616, USA.
Isseroff
R R
RR
D015153:Blotting, Western; D029742:Caenorhabditis elegans Proteins; D002118:Calcium / Q000494:pharmacology; D002352:Carrier Proteins; D002454:Cell Differentiation; D002462:Cell Membrane / Q000201:enzymology; D011905:Genes, ras; D006801:Humans; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D015603:Keratinocytes / Q000166:cytology / Q000201:enzymology*; D007633:Keratins / Q000096:biosynthesis; D008970:Molecular Weight; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism; D011493:Protein Kinase C / Q000737:chemistry / Q000378:metabolism*; D016283:Proto-Oncogene Proteins p21(ras) / Q000737:chemistry; D011955:Receptors, Drug / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection; D011503:Transglutaminases / Q000378:metabolism; D014212:Tretinoin / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D029742:Caenorhabditis elegans Proteins; D002352:Carrier Proteins; D007527:Isoenzymes; D010750:Phosphoproteins; D011955:Receptors, Drug; C083141:phorbol ester binding protein; C027776:phorbol ester receptor; D014212:Tretinoin; D007633:Keratins; D011503:Transglutaminases; D011493:Protein Kinase C; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras); D002118:Calcium