Increased activation of Ras in psoriatic lesions

Ras functions as an essential upstream regulator of growth-factor-receptor- coupled signal transduction pathways. Ras is converted from an inactive GDP -bound state to an active GTP-bound state in response to receptor activatio n. Thus, the ratio of GTP/GDP bound to Ras is a measure of its state of act ivation. Mutations that stabilize the GTP-bound form of Ras result in const itutive activation and cellular transformation. The most widely used method for measuring Ras activation utilizes [P-32]PO4 to label cellular nucleoti de pools and is therefore limited to use with cultured cells. We have modif ied and adapted an enzyme-based method for rapid, precise measurement of Ra s-bound GTP and GDP in normal and psoriatic human skin. This method does no t require radiolabeling of cellular nucleotides, In cultured fibroblasts, t he enzymatic and [P-32]PO4 incorporation methods yielded similar results. A pplication of the enzymatic method to human skin revealed that 6% of Ras wa s in the active GTP-bound state in normal skin, compared to 15.4% of Ras in psoriatic lesions. The total amount of Ras normalized to protein content w as similar in normal and psoriatic skin. Enhanced activation of Ras is like ly a critical mediator of the increased cell growth characteristic of psori atic lesions.