In mammalian oncogenesis, Ras signals through three main canonical effector pathways: Raf kinase, phosphoinositide-3 (PI3) kinase, and Ral-specific guanine nucleotide exchange factor (Ral-GDS). Mechanisms of Ras>Raf signaling in cancer have been studied extensively, but recent studies suggest that signaling through RalGDS>Ral is equally important as Ras>Raf. However, little is known about the in vivo functions of Ral. The C. elegans vulva is an ideal system for detailed analysis of Ras signaling. EGF signaling through the LET-60/Ras > LIN-45/Raf > MPK-1/MAP kinase cascade induces vulval precursor cells to assume a vulval cell fate. Suppressor and enhancer analyses of the
let-60(
n1046) activating mutation have identified a large number of positive and negative regulators of this pathway. To ask whether
ral-1 functions in vulval induction, we injected
ral-1-directed RNAi into
let-60(gf) and wild-type backgrounds. Based on the positive effector role of Ral in human cells, we expected
ral-1(RNAi) to suppress the
let-60(
n1046gf) multivulva (Muv) phenotype. Surprisingly, we found that
ral-1(RNAi) enhanced
let-60(gf), causing a stronger Muv phenotype. This experiment was reproduced with feeding RNAi of
ral-1 and its exchange factor, RalGDS. These results corroborate Brightbill, Klampert, and Wightman's (ECWM 1998) findings that the deletion of an X-chromosome region including RalGDS and approximately 8 neighboring genes enhances Ras gain-of-function mutations.
ral-1(RNAi) in a wild-type background caused no Muv phenotype. We conclude that
ral-1 inhibits rather than promotes vulval cell fates.To test whether activated Ral alone can over-induce vulval tissue, a Q61L constitutively activated Ral construct driven by the
lin-31 promoter (pB255, expressed in the vulval precursor cells) was injected into wild-type worms. However, no phenotype from the activated RAL-1 was seen. The fact that
ral-1 knockdown and activation in a wild-type background did not cause a vulval phenotype suggests that Ral is not part of the core pathway, but rather a modifier. We are currently testing the
ral-1(gf) construct as well as a dominant negative
ral-1 construct in the
let-60(
n1046gf) background. Double mutant analysis will be used to test at which level of the LET-60>LIN-45>MPK-1 pathway Ral regulation occurs. Finally, in vitro biochemical analysis and mammalian cell biology will be used to corroborate the physical interactions among LET-60, RALGDS and RAL-1.