Abstract
The induction of epidermal differentiation by Ca^2+^ in vitro is associated with enhanced activity of phosphatidylinositol‐specific phospholipase C (PLC). Neoplastic keratinocyte cell lines expressing a mutant c‐Ha‐ras gene and normal keratinocytes transformed to the neoplastic phenotype by transduction with the v‐Ha‐ras gene (v‐Ha‐ras keratinocytes) have elevated constitutive activity of PLC that increases further in response to Ca^2+^, but the cells do not differentiate normally. PLC‐γ1 (145 kDa) is the major isoform detected by immuno‐blotting of extracts from control, v‐Ha‐ras, and neoplastic keratinocyte cell lines cultured in 0.05 mM Ca^2+^ medium. The amount of PLC‐γ1 protein was higher in neoplastic cell lines than in normal and v‐Ha‐ras keratinocytes that had similar PLC‐γ1 protein levels. Thus, higher PLC‐γ1 protein levels cannot account for the elevated constitutive activity PLC in v‐Ha‐ras keratinocytes. After induction of differentiation by Ca^2+^, the amount of PLC‐γ1 protein increased in all cell types, and PLC‐δ1 (85 kDa), barely detectable in 0.05 mM Ca^2+^, increased. PLC‐β1 was not detected at any Ca^2+^ concentration. PLC‐γ1 and PLC‐δ1 mRNA did not increase after elevation of extracellular Ca^2+^, suggesting that posttranscriptional mechanisms can regulate PLC‐γ1 and PLC‐δ1 protein levels in normal and neoplastic keratinocytes. Activation of protein kinase C by treatment with 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) inhibited the stimulation of inositol phosphate (InsP) formation by Ca^2+^ but did not alter basal InsP levels in normal keratinocytes. In contrast, TPA treatment reduced both Ca^2+^‐stimulated and basal InsP formation in neoplastic cells lines and v‐Ha‐ras keratinocytes. In both normal and v‐Ha‐ras keratinocytes labeled with [^32^P]orthophosphate, antibodies against PLC‐γ1 immunoprecipitated a complex of ^32^ P‐labeled proteins. The relative labeling of the PLC‐γ1 band was greater in normal than in v‐Ha‐ras keratinocytes. Furthermore, treatment with TPA specifically increased the relative phosphorylation of PLC‐γ1 in v‐Ha‐ras keratinocytes but not in normal keratinocytes. These results suggest that the negative regulation of constitutive activity of PLC by protein kinase C differs in normal and neoplastic keratinocytes and that this could be the mechanism of increased PLC activity produced by an oncogenic ras gene in keratinocytes. © 1994 Wiley‐Liss, Inc.