Increased expression of a high molecular weight (130 KD) protein kinase C isoform in a differentiation-defective ras-transfected keratinocyte line
Abstract
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...Continue Reading
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Mode of activation and kinetic properties of three distinct forms of protein kinase C from rat brain
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