CED-5/CED-12 (DOCK/ELMO) can promote and inhibit F-actin formation via distinct motifs that target different GTPases

Abstract

Coordinated activation and inhibition of F-actin supports the movements of morphogenesis. Understanding the proteins that regulate F-actin is important, since these proteins are mis-regulated in diseases like cancer. Our studies of C. elegans embryonic epidermal morphogenesis identified the GTPase CED-10/Rac1 as an essential activator of F-actin. However, we need to identify the GEF, or Guanine-nucleotide Exchange Factor, that activates CED-10/Rac1 during embryonic cell migrations. The two-component GEF, CED-5/CED-12, is known to activate CED-10/Rac1 to promote cell movements that result in the engulfment of dying cells during embryogenesis, and a later cell migration of the larval Distal Tip Cell. It is believed that CED-5/CED-12 powers cellular movements of corpse engulfment and DTC migration by promoting F-actin formation. Therefore, we tested if CED-5/CED-12 was involved in embryonic migrations, and got a contradictory result. CED-5/CED-12 definitely support embryonic migrations, since their loss led to embryos that died due to failed epidermal cell migrations. However, CED-5/CED-12 inhibited F-actin in the migrating epidermis, the opposite of what was expected for a CED-10 GEF. To address how CED-12/CED-5 could have two opposing effects on F-actin, during corpse engulfment and cell migration, we investigated if CED-12 harbors GAP (GTPase Activating Protein) functions. A candidate GAP region in CED-12 faces away from the CED-5 GEF catalytic region. Mutating a candidate catalytic Arginine in the CED-12 GAP region (R537A) altered the epidermal cell migration function, and not the corpse engulfment function. A candidate GEF region on CED-5 faces towards Rac1/CED-10. Mutating Serine-Arginine in CED-5/DOCK predicted to bind and stabilize Rac1 for catalysis, resulted in loss of both ventral enclosure and corpse engulfment. Genetic and expression studies showed the GEF and GAP functions act on different GTPases. Thus, we propose CED-5/CED-12 support the cycling of multiple GTPases, by using distinct domains, to both promote and inhibit F-actin nucleation. Author SummaryGTPases in their active state promote actin nucleation that drives cellular events, from cell migrations, to cell shape changes, to cell-cell interactions. To function correctly, GTPases need to cycle from the active, GTP-bound state, to the inactive, GDP-bound state. This cycle is supported by Guanine-nucleotide Exchange Factors, or GEFs, that support activation as GDP is switched for GTP, and GTPase-Activating Proteins, or GAPs that support hydrolysis back to the GDP bound state. The Rac1/CED-10 GTPase has a well-studied GEF, CED-5/CED-12, that promotes Rac1 activation during cell engulfment of dying cells. Here we tested if CED-5/CED-12 also functioned as the activator, or GEF for Rac1 during embryonic cell migrations. Surprisingly, CED-5/CED-12 behaved completely opposite to what was expected during this cell migration. Therefore, we investigated if CED-5/CED-12 could harbor a GAP function. Comparing models of human and C. elegans protein structures suggested a putative GAP region, which we mutated to show that CED-12 likely functions as a GAP. Genetic and gene expression tests identify other GTPases, CDC-42 and RHO-1, regulated by this newly uncovered CED-12 GAP function. This places CED-5/CED-12 at a central position, where it can support the cycling of multiple GTPases, and both promote and inhibit F-actin nucleation.