Rap1 and Ral signaling networks in C. elegans and mammals

In ~15% of all human tumors, any one of the Ras-proteins are mutated. More recent focus on two other small Ras-like GTPases, Rap1 and Ral, revealed their important function in regulation of cellular dynamics. Studying model organisms has provided insights on the function of the Rap1 and Ral signaling networks. In Chapter 1, these have been summarized and related to biochemical data. In a genome-wide RNAi screen for synthetic lethality with rap-1 null C. elegans mutants, RAL-1 and the previously described RAL-1 effectors, SEC-5 and EXOC-8, emerged (Chapter 2). SEC-5 and EXOC-8 are members of the exocyst complex members. Progeny of rap-1 null mutants subjected to either ral-1, sec-5 or exoc-8(RNAi) arrested during embryonic development with a similar phenotype: hypodermal cell organization was disturbed leading to failure of ventral enclosure and HMP-1/alpha-catenin was lost from the cell-cell contacts. Together, these data show that the RAP-1 and RAL-1/exocyst pathways act synergistically in C. elegans development. To investigate whether this synergy was also present in other model systems, we turned to human A549 and DLD1 cell lines (Chapter 3). E-cadherin levels at the adherens junctions were not much affected in Rap1- and RalA-depleted DLD1 cells, but strikingly E-cadherin levels were largely reduced upon depletion of RalA in A549 cells. Moreover, depletion of the exocyst members Sec8 and Exoc8 and of the Ral activator RalGDS also led loss of E-cadherin. Interestingly, depletion of RalA did not lead to loss of N-cadherin and adherens junctions were still present. In the aforementioned screen with the rap-1 null mutant, we also identified phi-24, sur-6, vhp-1, him-3 and C01B7. In chapter 4, we investigated the phenotype of rap-1 null mutants subjected to sur-6, phi-24 or vhp-1(RNAi). Progeny of these animals arrested at different stages than observed for rap-1 null animals on ral-1(RNAi). Next, we investigated the synthetic lethality of RAP-1 and VHP-1, a phosphatase for KGB-1 and PMK-1, which are involved in stress signaling. rap-1 null mutants do not only require VHP-1 but also the opposing kinases MEK-1, SEK-1 and KGB-1. Finally, we used the mek-1 and kgb-1 null mutants in a revertant screen to identify new players in the RAP-1 signaling network, but we did not obtain synthetic lethal genes with the mek-1 and kgb-1 null mutants. In chapter 5, we addressed the question if in C. elegans, MIG-10 is an effector protein of RAP-1. The mammalian homologs for MIG-10, Riam and Lamellipodin regulate actin dynamics and Riam has been shown to bind to Rap1. Mutants for mig-10 appear viable but require the presence of UNC-34/Ena. The data presented in chapter 5 show that rap-1 null mutants also require unc-34 for survival. Interestingly, rap-1;mig-10 double mutants appear sick, indicating that RAP-1 and MIG-10 do not operate in a single pathway. This is further supported by the little overlap of the synthetic lethal profiles of rap-1 and mig-10 null mutants. The findings described in this thesis and their implications for the understanding of the role of Rap1 and Ral in regulating cellular dynamics are discussed (chapter 6).