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 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 beta-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.
Mazaki
Y
Y
Department of Molecular Biology, Osaka Bioscience Institute, Suita, Osaka 565-0874, Japan.
Hashimoto
S
S
Okawa
K
K
Tsubouchi
A
A
Nakamura
K
K
Yagi
R
R
Yano
H
H
Kondo
A
A
Iwamatsu
A
A
Mizoguchi
A
A
Sabe
H
H
D020823:ADP-Ribosylation Factor 1 / Q000378:metabolism; D020727:ADP-Ribosylation Factors / Q000235:genetics / Q000378:metabolism*; D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002352:Carrier Proteins / Q000235:genetics; D002460:Cell Line; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D003599:Cytoskeleton / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D020690:GTPase-Activating Proteins / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008856:Microscopy, Fluorescence; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D051419:Paxillin; D010750:Phosphoproteins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions / Q000378:metabolism
D000199:Actins; D002352:Carrier Proteins; D003598:Cytoskeletal Proteins; D017931:DNA Primers; C430213:GIT2 protein, human; D020690:GTPase-Activating Proteins; C496061:PXN protein, human; D051419:Paxillin; D010750:Phosphoproteins; D011994:Recombinant Proteins; D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors