MEMBRANE-TARGETING POTENTIATES GUANINE-NUCLEOTIDE EXCHANGE FACTOR CDC25 AND SOS1 ACTIVATION OF RAS TRANSFORMING ACTIVITY

Growth factor-triggered activation of Ras proteins is believed to be m ediated by guanine nucleotide exchange factors (CDC25/GRF and SOS1/2) that promote formation of the active Ras GTP-bound state. Although the mechanism(s) of guanine nucleotide exchange factor regulation is uncl ear, recent studies suggest that translocation of SOS1 to the plasma m embrane, where Ras is located, might be responsible for Ras activation . To evaluate this model, we generated constructs that encode the cata lytic domains of human CDC25 or mouse SOS1, either alone (designated c CDC25 and cSOS1, respectively) or terminating in the carboxyl-terminal CAAX membrane-targeting sequence from K-Ras4B (designated cCDC25-CAAX and cSOS1-CAAX, respectively; in CAAX, C is Cys, A is an aliphatic am ino acid, and X is Ser or Met). We then compared the transforming pote ntial of cCDC25 and cSOS1 with their membrane-targeted counterparts. W e observed that addition of the Ras plasma membrane-targeting sequence to the catalytic domains of CDC25 and SOS1 greatly enhanced their foc us-forming activity (10- to 50-fold) in NIH 3T3 transfection assays. S imilarly, we observed that the membrane-targeted versions showed a 5- to 10-fold enhanced ability to induce transcriptional activation from the Ets/AP-1 Ras-responsive element. Furthermore, whereas cells that s tably expressed cCDC25 or cSOS1 exhibited the same morphologies as unt ransformed NLH 3T3 cells, cells expressing cCDC25-CAAX or cSOS1-CAAX d isplayed transformed morphologies that were indistinguishable from the elongated and refractile morphology of oncogenic Ras-transformed cell s. Thus, these results suggest that membrane translocation alone is su fficient to potentiate guanine nucleotide exchange factor activation o f Ras.