The small GTPase Ras is the most mutated oncoprotein. Its close relative Rap1 shares 100% identity in the core effector-binding region. Yet the role of Rap1 in Ras-dependent signaling remains unclear, particularly relative to their potentially shared effector, Raf. C. elegans encodes a single inessential RAP-1 isoform, so we are using vulva cell fate specification to ascertain the role of RAP-1 relative to LET-60/Ras and LIN-45/Raf. LIN-3/EGF induces the six equipotent vulval precursor cells (VPCs), resulting in a distinct and highly reproducible pattern of 3 deg -3 deg -2 deg -1 deg -2 deg -3 deg . 1 deg fate is induced via the canonical LET-60/Ras-LIN-45/Raf-MEK-2/MEK-MPK-1/ERK MAP kinase cascade, while 2 deg fate is induced by LIN-12/Notch. We hypothesize that initial cell fate specification is accompanied by partial re-wiring of signaling mechanisms, resulting in increased pattern fidelity via reinforcement of the initial pattern and suppression of contradictory signals. Fluorescently tagged endogenous RAP-1 localized to the plasma membrane of the six VPCs. The endogenous RAP-1(G12V) activating mutation was sufficient to induce ectopic 1 deg VPCs as well as excretory duct cell duplications, both hallmarks of activated LET-60/Ras. Conversely,
rap-1-directed RNAi or deletion reduced excess 1 deg induction in sensitized backgrounds. We are currently testing whether endogenous RAP-1(G12V) can rescue the absent 1 deg VPCs and duct cells in the putative Ras null mutant,
let-60(
dx16). If successful, we will similarly test the ability of endogenous RAP-1(G12V) to rescue the absence of these cells in the putative Raf null mutant,
lin-45(
dx84). We have tagged endogenous MPK-1/ERK to measure activation-dependent nuclear translocation as a biomarker for in vivo LET-60/Ras and RAP-1 activation. We analyzed a transgenic promoter::GFP fusion of the Rap GEF, PXF-1, a potential upstream activator of RAP-1. Prior to LIN-3/EGF signal, GFP was expressed at low levels in all six VPCs. After LIN-3/EGF signal, GFP expression was increased in presumptive 1 deg but decreased in presumptive 2 deg s. Accordingly, we found that vulval-specific
pxf-1-directed RNAi reduced excess 1 deg s in a sensitized genetic background, consistent with PXF-1 functioning to activate RAP-1 in presumptive 1 deg VPCs. We speculate that Ras>Raf>MEK>ERK signaling in presumptive 1 deg VPCs increases PXF-1 expression and hence RAP-1 activation, while exclusion of PXF-1 expression in presumptive 2 deg VPCs reduces RAP-1 activation in these cells. Thus, we hypothesize that the initial LET-60/Ras>LIN-45/Raf signal is reinforced by a positive feedback loop using RAP-1. Our findings establish a novel regulatory feature of the vulval signaling network.