An ADP-ribosylation factor GTPase-activating protein Git2-short/KIAA0148 is involved in subcellular localization of paxillin and actin cytoskeletal organization

Paxillin acts as an adaptor protein in integrin signaling. We have shown th at paxillin exists in a relatively large cytoplasmic pool, including perinu clear areas, in addition to focal complexes formed at the cell periphery an d focal adhesions formed underneath the cell. Several ADP-ribosylation fact or (ARF) GTPase-activating proteins (GAPs; ARFGAPs) have been shown to asso ciate with paxillin. We report here that Git2-short/KIAA0148 exhibits prope rties of a paxillin-associated ARFGAP and appears to be colocalized with pa xillin, primarily at perinuclear areas. A fraction of Git2-short was also l ocalized to actin-rich structures at the cell periphery. Unlike paxillin, h owever, Git2-short did not accumulate at focal adhesions underneath the cel l. Git2-short is a short isoform of Git2, which is highly homologous to p95 PKL, another paxillin-binding protein, and showed a weaker binding affinity toward paxillin than that of Git2. The ARFGAP activities of Git2 and Git2- short have been previously demonstrated in vitro, and we provided evidence that at least one ARF isoform, ARF1, is an intracellular substrate for the GAP? activity of Git2-short. We also showed that Git2-short could antagoniz e several known ARF1-mediated phenotypes: overexpression of Git2-short, but not its GAF-inactive mutant, caused the redistribution of Golgi protein be ta -COP and reduced the amounts of paxillin-containing focal adhesions and actin stress fibers. Perinuclear localization of paxillin, which was sensit ive to ARF inactivation, was also affected by Git2-short overexpression. On the other hand, paxillin localization to focal complexes at the cell perip hery was unaffected or even augmented by Git2-short overexpression. Therefo re, an ARFGAP protein weakly interacting with paxillin, Git2-short, exhibit s pleiotropic functions involving the regulation of Golgi organization, act in cytoskeletal organization, and subcellular localization of paxillin, all of which need to be coordinately regulated during integrin-mediated cell a dhesion and intracellular signaling.