The dominant negative Ras mutant, N17Ras, can inhibit signaling independently of blocking Ras activation

Ras plays an important role in a variety of cellular functions, including g rowth, differentiation, and oncogenic transformation. For instance, Ras par ticipates in the activation of Raf, which phosphorylates and activates mito gen-activated protein kinase kinase (MEK), which then phosphorylates and ac tivates extracellular signal-regulated kinase (ERK), a mitogen-activated pr otein (MAP) kinase. Activation of MAP kinase appears to be essential for pr opagating a wide variety of extracellular signals from the plasma membrane to the nucleus. N17Ras, a GDP-bound dominant negative mutant, is used widel y as an interfering mutant to assess Ras function in vivo, Surprisingly, me observed that expression of N17Ras inhibited the activity and phosphorylat ion of Elk-1, a physiological substrate of MAP kinases, in response to phor bol myristate acetate, The activity and phosphorylation of the MAP kinase h emagglutinin epitope (HA)-ERK1 were not affected by N17Ras in response to t he same stimulus, Additionally, expression of N17Ras, but not L61S186Ras, a GTP-bound interfering mutant, inhibited MEK-induced Elk-1 phosphorylation, suggesting that inhibition of Elk-1 may be unique to GDP-bound Res mutants , Finally, we observed that V12Ras-induced focus formation in NIH3T3 cells is inhibited by coexpression of GDP-bound Ras mutants, such as N17, A15, an d N17N69. Therefore, N17Ras and V12 Ras may be codominant with respect to E lk-1 activation and cellular transformation. These results indicate that N1 7Ras appears to have at least two distinguishable functions: interference w ith endogenous Ras activation and inhibition of Elk-1 and transfomation, Fu rthermore, our data imply the possibility that GDP-bound Ras, Like N17Ras, may have a direct role in signal transduction.