GIT1 functions in a motile, multi-molecular signaling complex that regulates protrusive activity and cell migration

Abstract

GIT1 is a multidomain protein that is thought to function as an integrator of signaling pathways controlling vesicle trafficking, adhesion and cytoskeletal organization. It regulates ARF GTPases and has binding domains for paxillin and PIX, which is a PAK-binding protein and an exchange factor for Rac. We show that GIT1 cycles between at least three distinct subcellular compartments, including adhesion-like structures, the leading edge and cytoplasmic complexes. The cytoplasmic structures, which also contain paxillin, PAK and PIX, do not detectably co-localize with endosomal Golgi or membrane markers, suggesting that they represent a novel supramolecular complex. The GIT1 cytoplasmic complexes are motile and tended to move toward the cell periphery where they joined existing adhesions. In retracting regions of the cells, the GIT1 complexes moved away from the disassembling adhesions toward the cell body. Using deletion mutants, we have identified domains that target GIT1 to each of the compartments. Localization to adhesions and the leading edge requires the paxillin-binding domain, which comprises the C-terminal 140 residues (cGIT1), whereas targeting to the cytoplasmic complexes requires the central region that contains ankyrin repeats and the PIX-binding domain. Expression of GIT1 or cGIT, but not nGIT1 in which the paxillin-binding domain is deleted, increases the rate of migration and the size and number of protrusions. The latter are inhibited when GIT1 is co-expressed with a kinase-dead PAK, suggesting that the GIT1 interaction with PAK is required for enhanced migration and protrusive activity. Furthermore,GIT1 targets constitutively activated PAK to adhesions and the leading edge via its interaction with paxillin. Since expression of cGIT targets endogenous GIT1 to the leading edge, it appears that the leading edge is the location of GIT1 responsible for these activities. Thus, GIT1 is a component of a motile,multimolecular complex that traffics a set of signaling components to specific locations in the cell where they regulate localized activities.