An ADP-Ribosylation Factor GTPase-activating Protein Git2-short/KIAA0148 Is Involved in Subcellular Localization of Paxillin and Actin Cytoskeletal Organization

Abstract

Paxillin acts as an adaptor protein in integrin signaling. We have shown that paxillin exists in a relatively large cytoplasmic pool, including perinuclear areas, in addition to focal complexes formed at the cell periphery and focal adhesions formed underneath the cell. Several ADP-ribosylation factor (ARF) GTPase-activating proteins (GAPs; ARFGAPs) have been shown to associate with paxillin. We report here that Git2-short/KIAA0148 exhibits properties of a paxillin-associated ARFGAP and appears to be colocalized with paxillin, primarily at perinuclear areas. A fraction of Git2-short was also localized to actin-rich structures at the cell periphery. Unlike paxillin, however, Git2-short did not accumulate at focal adhesions underneath the cell. Git2-short is a short isoform of Git2, which is highly homologous to p95PKL, 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 antagonize several known ARF1-mediated phenotypes: overexpression of Git2-short, but not its GAP-inactive mutant, caused the redistribution of Golgi protein ␤-COP and reduced the amounts of paxillin-containing focal adhesions and actin stress fibers. Perinuclear localization of paxillin, which was sensitive to ARF inactivation, was also affected by Git2-short overexpression. On the other hand, paxillin localization to focal complexes at the cell periphery was unaffected or even augmented by Git2-short overexpression. Therefore, an ARFGAP protein weakly interacting with paxillin, Git2-short, exhibits pleiotropic functions involving the regulation of Golgi organization, actin cytoskeletal organization, and subcellular localization of paxillin, all of which need to be coordinately regulated during integrin-mediated cell adhesion and intracellular signaling. INTRODUCTIONIntegrins play an essential role in a number of dynamic aspects of cell regulation, including adhesion and migration. A number of different cytoplasmic proteins, with scaffolding as well as signaling properties, must assemble on the cytoplasmic tails of integrins for proper integrin functioning (Hynes, 1992;Clark and Brugge, 1995;Burridge and Chrzanowska-Wodnicka, 1996). Formation of integrin-mediated ¶ Corresponding author. E-mail address: sabe@obi.or.jp. Abbreviations used: AlF, aluminum fluoride; ARF, ADP-ribosylation factor; BFA, brefeldin A; BSA, bovine serum albumin; COP, coat protein; ECM, extracellular matrix; EGFP, enhanced green fluorescent protein; ER, endoplasmic reticulum; GAP, GTPase-activating protein; GFP, green fluorescent protein; Git2, G protein-coupled receptor kinase interactor 2; GST, glutathione S-transferase; HA, influenza hemagglutinin; NP-40, Nonidet P-40; PAG, paxillin-associated protein with ARFGAP activity; PBS, phosphate-buffered saline; PCR, polymerase ...

Differential proteome profile of skin mucus of gilthead seabream (Sparus aurata) after probiotic intake and/or overcrowding stress

“…ARFGAP has been associated with Golgi organization and actin cytoskeletal organization, mediating cell adhesion [49]. In relation with Overall, our data show some important regulation of seabream skin mucus metabolic proteins after feeding a probiotic and/or after overcrowding stress.…”

Paxillin: Adapting to Change

“…The individual paxillin (and Hic-5) LD motifs are flanked by proline-and glycine-rich segments (average composition of 25%), which may also contribute to the global folding, presentation, regulation, and function of the individual paxillin protein binding domains. This is reflected in the ␤-and ␥-paxillin isoforms exhibiting reduced affinity for the LD4 binding proteins FAK and GIT (175,176). Also interspersed between the LD motifs are potential SH3-binding domains and numerous phosphorylation sites, including SH2 binding domains, that may also regulate the activities and context of the LD binding partners, thereby imparting an additional means of temporal-spatial regulation of the adaptor functions of paxillin.…”

“…Importantly, Arf1-dependent paxillin shuttling between the Golgi and focal adhesions has been described (191), with the ArfGAPs, GIT2short, and ASAP2/PAP␣ implicated in this process (128,175). Other critical mediators of Golgi reorientation/polarization are Src (120) and p120RasGAP (135), which are paxillin binding partners (112,302).…”

Crk Associates with a Multimolecular Paxillin/GIT2/β-PIX Complex and Promotes Rac-dependent Relocalization of Paxillin to Focal Contacts

“…In further support of the regulation of actin reorganization and cell migration by ARF GTPases, ARF guanine nucleotide exchange factors and ARF-GTPase activating proteins (ARF-GAP) regulate these processes as well (Franco et al, 1999;Turner et al, 1999;Di Cesare et al, 2000;Jackson et al, 2000;Kondo et al, 2000;Randazzo et al, 2000;Mazaki et al, 2001;Santy and Casanova, 2001;Uchida et al, 2001;West et al, 2001;Brown et al, 2002;Liu et al, 2002a;Manabe Ri et al, 2002). For example, the overexpression of various ARF-GAP proteins modulates the formation and/or turnover of focal adhesions (Di Cesare et al, 2000;Jackson et al, 2000;Kondo et al, 2000;Randazzo et al, 2000;Mazaki et al, 2001;Liu et al, 2002a) and the overexpression of an ARF guanine nucleotide exchange factor, ARNO, enhances the spreading and dispersal of epithelial cells (Santy and Casanova, 2001). In addition to their role as GAPs, ARF-GAP proteins may also influence signaling pathways through additional proteinprotein interactions.…”