Ha-ras p21-GTP levels remain constant during primary keratinocyte differentiation

Several lines of evidence that indicate that mutation of the Ha-ras oncogene is the initiating event in mouse skin carcinogenesis. Keratinocytes known to possess a mutated Ha-ras have been shown to be resistant to differentiation. Thus, overstimulation of the Ha-ras signaling pathway appears to block normal keratinocyte differentiation, and we hypothesized that for normal keratinocytes to terminally differentiate, the Ha-ras signaling cascade must be turned off. In the present studies, we measured the level and activity state of Ha-ras p21 protein in cultured keratinocytes undergoing calcium-induced differentiation. We have employed Western blot analysis to demonstrate that Ha-ras p21 protein levels remain constant during primary newborn and adult keratinocyte differentiation. The overall level of Ha-ras p21 was higher in immortalized, benign, and malignant mouse keratinocyte cell lines than in normal keratinocytes but did not change within each cell type when subjected to differentiating conditions. The percentage of Ha-ras p21 protein in its active, GTP-bound form also remained unchanged during primary adult keratinocyte differentiation and in immortalized, benign, and malignant keratinocytes subjected to differentiating conditions. Our results indicate that terminal differentiation of primary adult mouse keratinocytes occurred in the presence of constant levels of Ha-ras p21-GTP, suggesting that the Ha-ras signaling pathway may be blocked at a point distal to a step involving the Ha-ras p21 protein itself.