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 growth, differentiation, and oncogenic transformation. For instance, Ras participates in the activation of Raf, which phosphorylates and activates mitogen-activated protein kinase kinase (MEK), which then phosphorylates and activates extracellular signal-regulated kinase (ERK), a mitogen-activated protein (MAP) kinase. Activation of MAP kinase appears to be essential for propagating a wide variety of extracellular signals from the plasma membrane to the nucleus. N17Ras, a GDP-bound dominant negative mutant, is used widely as an interfering mutant to assess Ras function in vivo. Surprisingly, we observed that expression of N17Ras inhibited the activity and phosphorylation of Elk-1, a physiological substrate of MAP kinases, in response to phorbol myristate acetate. The activity and phosphorylation of the MAP kinase hemagglutinin epitope (HA)-ERK1 were not affected by N17Ras in response to the 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 Ras 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, and N17N69. Therefore, N17Ras and V12 Ras may be codominant with respect to Elk-1 activation and cellular transformation. These results indicate that N17Ras appears to have at least two distinguishable functions: interference with endogenous Ras activation and inhibition of Elk-1 and transfomation. Furthermore, our data imply the possibility that GDP-bound Ras, like N17Ras, may have a direct role in signal transduction.