scholarly journals A New Paxillin-binding Protein, PAG3/Papα/KIAA0400, Bearing an ADP-Ribosylation Factor GTPase-activating Protein Activity, Is Involved in Paxillin Recruitment to Focal Adhesions and Cell Migration

2000 ◽  
Vol 11 (4) ◽  
pp. 1315-1327 ◽  
Author(s):  
Akiko Kondo ◽  
Shigeru Hashimoto ◽  
Hajime Yano ◽  
Kuniaki Nagayama ◽  
Yuichi Mazaki ◽  
...  
Keyword(s):  
Subcellular Localization ◽  
Binding Protein ◽  
Focal Adhesions ◽  
Cell Periphery ◽  
Gtpase Activating Protein ◽  
Protein Activity ◽  
Adp Ribosylation ◽  
Focal Contacts ◽  
Adp Ribosylation Factor

Paxillin acts as an adaptor molecule in integrin signaling. Paxillin is localized to focal contacts but seems to also exist in a relatively large cytoplasmic pool. Here, we report the identification of a new paxillin-binding protein, PAG3 (paxillin-associated protein with ADP-ribosylation factor [ARF] GTPase-activating protein [GAP] activity, number 3), which is involved in regulation of the subcellular localization of paxillin. PAG3 bound to all paxillin isoforms and was induced during monocyte maturation, at which time paxillin expression is also increased and integrins are activated. PAG3 was diffusely distributed in the cytoplasm in premature monocytes but became localized at cell periphery in mature monocytes, a fraction of which then colocalized with paxillin. PAG3, on the other hand, did not accumulate at focal adhesion plaques, suggesting that PAG3 is not an integrin assembly protein. PAG3 was identical to KIAA0400/Papα, which was previously identified as a Pyk2-binding protein bearing a GAP activity toward several ARFs in vitro. Mammalian ARFs fall into three classes, and we showed that all classes could affect subcellular localization of paxillin. We also examined possible interaction of PAG3 with ARFs and showed evidence that at least one of them, ARF6, seems to be an intracellular substrate for GAP activity of PAG3. Moreover, overexpression of PAG3, but not its GAP-inactive mutant, inhibited paxillin recruitment to focal contacts and hampered cell migratory activities, whereas cell adhesion activities were almost unaffected. Therefore, our results demonstrate that paxillin recruitment to focal adhesions is not mediated by simple cytoplasmic diffusion; rather, PAG3 appears to be involved in this process, possibly through its GAP activity toward ARF proteins. Our result thus delineates a new aspect of regulation of cell migratory activities.

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

2001 ◽  
Vol 12 (3) ◽  
pp. 645-662 ◽  
Author(s):  
Yuichi Mazaki ◽  
Shigeru Hashimoto ◽  
Katsuya Okawa ◽  
Asako Tsubouchi ◽  
Kuniaki Nakamura ◽  
...  
Keyword(s):  
Subcellular Localization ◽  
Intracellular Signaling ◽  
Focal Adhesions ◽  
Adaptor Protein ◽  
Cell Periphery ◽  
Cytoskeletal Organization ◽  
Adp Ribosylation ◽  
Pleiotropic Functions ◽  
Adp Ribosylation Factor

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 β-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.

scholarly journals Identification of centaurin-α1 as a potential in vivo phosphatidylinositol 3,4,5-trisphosphate-binding protein that is functionally homologous to the yeast ADP-ribosylation factor (ARF) GTPase-activating protein, Gcs1

1999 ◽  
Vol 340 (2) ◽  
pp. 359-363 ◽  
Author(s):  
Kanamarlapudi VENKATESWARLU ◽  
Paru B. OATEY ◽  
Jeremy M. TAVARÉ ◽  
Trevor R. JACKSON ◽  
Peter J. CULLEN
Keyword(s):  
Plasma Membrane ◽  
Growth Factor ◽  
Fluorescent Protein ◽  
Binding Protein ◽  
Dominant Negative ◽  
Gtpase Activating Protein ◽  
Adp Ribosylation ◽  
Adp Ribosylation Factor

Centaurin-α is a 46 kDa in vitro binding protein for the lipid second messenger PtdIns(3,4,5)P3. In this report we have addressed whether centaurin-α1, a human homologue of centaurin-α, binds PtdIns(3,4,5)P3in vivo and furthermore, identified a potential physiological function for centaurin-α1. Using confocal microscopy of live PC12 cells, transiently transfected with a chimera of green fluorescent protein (GFP) fused to the N-terminus of centaurin-α1 (GFP-centaurin-α1), we demonstrated the rapid plasma membrane recruitment of cytosolic GFP-centaurin-α1 following stimulation with either nerve growth factor or epidermal growth factor. This recruitment was dependent on the centaurin-α1 pleckstrin homology domains and was blocked by the PtdIns(4,5)P2 3-kinase (PI 3-kinase) inhibitors wortmannin (100 nM) and LY294002 (50 μM), and also by co-expression with a dominant negative p85. Functionally, we demonstrated that centaurin-α1 could complement a yeast strain deficient in the ADP-ribosylation factor (ARF) GTPase-activating protein Gcs1; a complementation that was blocked by mutagenesis of conserved cysteine residues within the ARF GTPase-activating protein analogous domain of centaurin-α1. Taken together, our data demonstrated that centaurin-α1 could potentially function as an ARF GTPase-activating protein that, on agonist stimulation, was recruited to the plasma membrane possibly through an ability to interact with PtdIns(3,4,5)P3.

scholarly journals Adp Ribosylation Factor-like Protein 2 (Arl2) Regulates the Interaction of Tubulin-Folding Cofactor D with Native Tubulin

2000 ◽  
Vol 149 (5) ◽  
pp. 1087-1096 ◽  
Author(s):  
Arunashree Bhamidipati ◽  
Sally A. Lewis ◽  
Nicholas J. Cowan
Keyword(s):  
Cultured Cells ◽  
Gtpase Activating Protein ◽  
Adp Ribosylation ◽  
Chaperone Protein ◽  
Ras Family ◽  
Mutant Forms ◽  
Adp Ribosylation Factor ◽  
Β Tubulin

The ADP ribosylation factor-like proteins (Arls) are a family of small monomeric G proteins of unknown function. Here, we show that Arl2 interacts with the tubulin-specific chaperone protein known as cofactor D. Cofactors C, D, and E assemble the α/β- tubulin heterodimer and also interact with native tubulin, stimulating it to hydrolyze GTP and thus acting together as a β-tubulin GTPase activating protein (GAP). We find that Arl2 downregulates the tubulin GAP activity of C, D, and E, and inhibits the binding of D to native tubulin in vitro. We also find that overexpression of cofactors D or E in cultured cells results in the destruction of the tubulin heterodimer and of microtubules. Arl2 specifically prevents destruction of tubulin and microtubules by cofactor D, but not by cofactor E. We generated mutant forms of Arl2 based on the known properties of classical Ras-family mutations. Experiments using these altered forms of Arl2 in vitro and in vivo demonstrate that it is GDP-bound Arl2 that interacts with cofactor D, thereby averting tubulin and microtubule destruction. These data establish a role for Arl2 in modulating the interaction of tubulin-folding cofactors with native tubulin in vivo.

scholarly journals The Association of ASAP1, an ADP Ribosylation Factor-GTPase Activating Protein, with Focal Adhesion Kinase Contributes to the Process of Focal Adhesion Assembly

2002 ◽  
Vol 13 (6) ◽  
pp. 2147-2156 ◽  
Author(s):  
Yunhao Liu ◽  
Joost C. Loijens ◽  
Karen H. Martin ◽  
Andrei V. Karginov ◽  
J. Thomas Parsons
Keyword(s):  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Focal Adhesions ◽  
Cell Spreading ◽  
Ph Domain ◽  
Wild Type ◽  
Gtpase Activating Protein ◽  
Adp Ribosylation ◽  
Transient Overexpression ◽  
Adp Ribosylation Factor

ASAP1 (ADP ribosylation factor [ARF]- GTPase-activating protein [GAP] containing SH3, ANK repeats, and PH domain) is a phospholipid-dependent ARF-GAP that binds to and is phosphorylated by pp60Src. Using affinity chromatography and yeast two-hybrid interaction screens, we identified ASAP1 as a major binding partner of protein tyrosine kinase focal adhesion kinase (FAK). GlutathioneS-transferase pull-down and coimmunoprecipitation assays showed the binding of ASAP1 to FAK is mediated by an interaction between the C-terminal SH3 domain of ASAP1 with the second proline-rich motif in the C-terminal region of FAK. Transient overexpression of wild-type ASAP1 significantly retarded the spreading of REF52 cells plated on fibronectin. In contrast, overexpression of a truncated variant of ASAP1 that failed to bind FAK or a catalytically inactive variant of ASAP1 lacking GAP activity resulted in a less pronounced inhibition of cell spreading. Transient overexpression of wild-type ASAP1 prevented the efficient organization of paxillin and FAK in focal adhesions during cell spreading, while failing to significantly alter vinculin localization and organization. We conclude from these studies that modulation of ARF activity by ASAP1 is important for the regulation of focal adhesion assembly and/or organization by influencing the mechanisms responsible for the recruitment and organization of selected focal adhesion proteins such as paxillin and FAK.

scholarly journals Involvement of a novel ADP-ribosylation factor GTPase-activating protein, SMAP, in membrane trafficking: Implications in cancer cell biology

2006 ◽  
Vol 97 (9) ◽  
pp. 801-806 ◽  
Author(s):  
Kenji Tanabe ◽  
Shunsuke Kon ◽  
Waka Natsume ◽  
Tetsuo Torii ◽  
Toshio Watanabe ◽  
...  
Keyword(s):  
Cancer Cell ◽  
Membrane Trafficking ◽  
Cell Biology ◽  
Gtpase Activating Protein ◽  
Adp Ribosylation ◽  
Adp Ribosylation Factor

scholarly journals A Class I ADP-Ribosylation Factor GTPase-Activating Protein Is Critical for Maintaining Directional Root Hair Growth in Arabidopsis

2008 ◽  
Vol 147 (4) ◽  
pp. 1659-1674 ◽  
Author(s):  
Cheol-Min Yoo ◽  
Jiangqi Wen ◽  
Christy M. Motes ◽  
J. Alan Sparks ◽  
Elison B. Blancaflor
Keyword(s):  
Root Hair ◽  
Hair Growth ◽  
Class I ◽  
Gtpase Activating Protein ◽  
Adp Ribosylation ◽  
Root Hair Growth ◽  
Adp Ribosylation Factor

ADP ribosylation factor is an essential protein in Saccharomyces cerevisiae and is encoded by two genes

1990 ◽  
Vol 10 (12) ◽  
pp. 6690-6699
Author(s):  
T Stearns ◽  
R A Kahn ◽  
D Botstein ◽  
M A Hoyt
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cold Sensitivity ◽  
Animal Cells ◽  
Gene Encoding ◽  
Phenotypic Differences ◽  
Adp Ribosylation ◽  
Protein Factor ◽  
Adp Ribosylation Factor ◽  
Sublethal Concentrations

ADP ribosylation factor (ARF) is a ubiquitous 21-kDa GTP-binding protein in eucaryotes. ARF was first identified in animal cells as the protein factor required for the efficient ADP-ribosylation of the mammalian G protein Gs by cholera toxin in vitro. A gene (ARF1) encoding a protein homologous to mammalian ARF was recently cloned from Saccharomyces cerevisiae (Sewell and Kahn, Proc. Natl. Acad. Sci. USA, 85:4620-4624, 1988). We have found a second gene encoding ARF in S. cerevisiae, ARF2. The two ARF genes are within 28 centimorgans of each other on chromosome IV, and the proteins encoded by them are 96% identical. Disruption of ARF1 causes slow growth, cold sensitivity, and sensitivity to normally sublethal concentrations of fluoride ion in the medium. Disruption of ARF2 causes no detectable phenotype. Disruption of both genes is lethal; thus, ARF is essential for mitotic growth. The ARF1 and ARF2 proteins are functionally homologous, and the phenotypic differences between mutations in the two genes can be accounted for by the level of expression; ARF1 produces approximately 90% of total ARF. Among revertants of the fluoride sensitivity of an arf1 null mutation were ARF1-ARF2 fusion genes created by a gene conversion event in which the deleted ARF1 sequences were repaired by recombination with ARF2.

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