RHGF-2 Is an Essential Rho-1 Specific RhoGEF that binds to the Multi-PDZ Domain Scaffold Protein MPZ-1 in Caenorhabditis elegans

Abstract

RhoGEF proteins activate the Rho family of small GTPases and thus play a key role in regulating fundamental cellular processes such as cell morphology and polarity, cell cycle progression and gene transcription. We identified a Caenorhabditis elegans RhoGEF protein, RHGF-2, as a binding partner of the C. elegans multi-PDZ domain scaffold protein MPZ-1 (MUPP1 in mammals). RHGF-2 exhibits significant identity to the mammalian RhoGEFs PLEKHG5/Tech/Syx and contains a class I C-terminal PDZ binding motif (SDV) that interacts most strongly to MPZ-1 PDZ domain eight. RHGF-2 RhoGEF activity is specific to the C. elegans RhoA homolog RHO-1 as determined by direct binding, GDP/GTP exchange and serum response element-driven reporter activity. rhgf-2 is an essential gene since rhgf-2 deletion mutants do not elongate during embryogenesis and hatch as short immobile animals that arrest development. Interestingly, the expression of a functional rhgf-2::gfp transgene appears to be exclusively neuronal and rhgf-2 overexpression results in loopy movement with exaggerated body bends. Transient expression of RHGF-2 in N1E-115 neuroblastoma cells prevents neurite outgrowth similar to constitutive RhoA activation in these cells. Together, these observations indicate neuronally expressed RHGF-2 is an essential RHO-1 specific RhoGEF that binds most strongly to MPZ-1 PDZ domain eight and is required for wild-type C. elegans morphology and growth.

pix-1 Controls Early Elongation in Parallel with mel-11 and let-502 in Caenorhabditis elegans

“…As previously shown, few let-502 ( sb118ts) embryos arrest between the 1.2- and 2-fold stage at restrictive temperature and the vast majority of the animals hatch as non-elongated larvae (Table 2, line 5, Figure 1A) [10]. 89% and 100% of let-502(sb118ts) embryos arrest between 1.2- and 2-fold stages in pix-1(gk416) and pak-1(ok448) backgrounds, respectively at 25.5°C (Table 2, line 6 and 11).…”

“…To assess whether pix-1 also controls early elongation in parallel with the mel-11/let-502 pathway, we tested whether pix-1(gk416) genetically interacts with mel-11(it26) and let-502(sb118ts) temperature sensitive mutants, as reported for pak-1 [3]. At 18°C, 98% of mel-11(it26) mutant embryos rupture during elongation as previously described (Table 2, line 3) [10]. The penetrance of this phenotype is increased to 100% at 25.5°C.…”

“…These kinases act antagonistically with the MEL-11/PP-1M and are organized in two parallel pathways: The let-502/mel-11 pathway including mrck-1 and a second pathway involving pak-1 [3], [8], [10]. Downstream of these pathways, MLC-4/MLC phosphorylation leads to non-muscle myosin filament assembly and contractility, while its dephosphorylation is associated with relaxation.…”

“…The function of these two later proteins in the regulation of MLC-4 phosphorylation and/or PAK-1 function remains unknown [3], [8], [10], [16].…”

RHGF-1/PDZ-RhoGEF and retrograde DLK-1 signaling drive neuronal remodeling on microtubule disassembly

“…The GTP exchange activity of RHGF-1 resides in the DH domain. Previous studies indicated that RHGF-1 participated in neurotransmitter release (26,28). Consistent with its roles in the nervous system, rhgf-1 was expressed in many neurons in C. elegans, including the PLM (Fig.…”