| Homologues of mammalian Dock180 and ELMO proteins in C. elegans and Drosophila were previously demonstrated to function together, upstream of Rae, during multiple biological processes. Mammalian Dock180/ELMO1 were then observed to stimulate an increase in the levels of activated Rac in cells. However, the molecular mechanisms by which Dock180/ELMO1 (and their homologues) led to Rac activation were unknown. In chapter 3, we identify the DOCKER domain as a novel, highly evolutionarily conserved domain containing residues 1111--1657 of Dock180 that is sufficient to mediate nucleotide-free Rac binding. Versions of Dock180 containing point mutations in the DOCKER domain failed to bind or activate Rac (both in vitro and in vivo), and failed to promote Dock180 mediated phagocytosis. This suggests that the DOCKER domain of Dock180 is a novel guanine nucleotide exchange factor (GEF) domain. Moreover, the presence of ELMO1 in the Dock180/ELMO1 complex augments the interaction of Dock180 with nucleotide-free Rac. In Chapter 4, we observed that Dock180 and ELMO1 functionally synergize to promote Rac-dependent cell migration, both in an in vitro in a Transwell assay and in a whole animal model. Interestingly, N-terminal deletion mutants of ELMO1, which still bind and cooperate with Dock180 in Rac activation, failed to promote migration, and this correlated with inability to localize to lamellipodia. This suggests that Rac activation by the ELMO1/Dock180 complex at discrete intracellular locations mediated by the N-terminus of ELMO1, rather than generalized Rac activation is critical for cell migration. We also present evidence that RhoG may be an important regulator of ELMO1/Dock180 during migration. In chapter 5, we describe a novel interaction of ELMO1 with ERM proteins. ELMO1 directly interacted with ERM proteins, both in vitro and in intact cells at endogenous levels. However, Rac activation by the Dock180/ELMO1 complex was not affected by the presence of radixin in the Dock180/ELMO1 complex. Similarly, ELMO1 had no apparent effect on the radixin interaction with F-actin. Since ERM proteins function as crosslinking proteins between the plasma membrane and the actin cytoskeleton, this suggests that ELMO1/ERM may provide coordinated control over actin cytoskeletal remodeling during dynamic cell processes. |
