PgmNr W4099: Novel reinforcement of Ras signaling by Rap1 in C. elegans vulval patterning.

Authors:
N. R. Rasmussen 1 ; D. Dickinson 2 ; D. Reiner 1


Institutes
1) TAMHSC Institute of Biosciences and Technology, Houston, TX; 2) University of North Carolina, Chapel Hill, NC.


Keyword: Other ( Cell patterning )

Abstract:

The small GTPase Ras is notorious as the most mutated oncoprotein in cancer. C. elegans and other systems were instrumental in defining the key components of Ras-Raf-MEK-ERK MAP kinase signaling cascade. However, the signaling activity of the small GTPase Ras is more convoluted than this. Decreased genetic redundancy and the anatomical simplicity of C. elegans provides an ideal setting to further dissect complicated Ras signaling during vulva cell fate specification. EGF induces Ras-mediated patterning of the six equipotent vulval precursor cells (VPCs) to generate the distinctive and highly reproducible pattern of 3˚-3˚-2˚-1˚-2˚-3 cell fates; presumptive 1˚ and 2˚ cells go on to form the vulva while 3˚ cells are non-induced and quiescent. Initial cell fate specification of the pattern is thought to be promptly followed by a reinforcement phase to enact final commitment, all prior to the first VPC division. We hypothesize that additional signaling mechanisms are executed to reinforce initial Ras-directed cell patterning and overcome contradictory signals within presumptive 1˚ VPCs. The small GTPase Rap1 (Ras proximal) is 100% identical to Ras in the core effector-binding region. Yet previous studies were inconclusive about Rap1 and Ras sharing effectors, particularly the S/T kinase Raf. Since C. elegans encodes a single Rap1 isoform that is not required for viability or fertility, we are using this system to define the role of Rap1 in the EGF-Ras signaling system. We N-terminally tagged endogenous Rap1 using CRISPR/Cas9. Tagged Rap1 localized to the plasma membrane of the six VPCs, consistent with published data of Ras promoter being active in VPCs, but providing more cell biological detail. Vulval-specific expression of mutationally-activated Rap1 was sufficient to hyper-induce VPCs. Consistent with Rap1 acting cooperatively with Ras, Rap1(RNAi) or deletion decreased 1˚ hyperinduction in a sensitized hyper-induced background. We also found that vulval-specific RNAi targeting the RapGEF, PXF-1, reduced hyper-induction, consistent with PXF-1 functioning to activate Rap1 in the vulva. We analyzed a transgenic transcriptional promoter::GFP fusion of the RapGEF, PXF-1. Prior to EGF signal, GFP was expressed at low levels in all six of the VPCs. After EGF signal, GFP expression was increased in presumptive 1˚ but decreased in presumptive 2˚ VPCs. Therefore, we hypothesize that Ras-Raf-MEK-ERK in presumptive 1˚ VPCs increases PXF-1 expression, and hence Rap1 activation, specifically in these cells while Rap1 signaling is excluded from presumptive 2˚ VPCs. Thus the Ras-Raf signal is reinforced by a positive feedback loop. Our findings establish a novel aspect of Ras signaling, pointing the way to unconventional treatments for Ras-driven cancers.



Wormbase Genetic Index
1. let-60
2. rap-1
3. pxf-1
4. mpk-1
5. lin-45