title: Hic-5 interacts with GIT1 with a different binding mode from paxillin.
abstract: Hic-5, a member of the paxillin family of adaptor molecules, is localized at focal adhesion and implicated in integrin-mediated signaling. Hic-5 and paxillin exhibit structural homology and share interacting factors, however, diverse functions are suggested for them. In this study, we carried out yeast two-hybrid screening to identify Hic-5 interacting factors using its LD3-4 region, which includes the Hic-5-specific amino acid sequence, as a bait. Through the screening, we identified GIT1, an Arf GTPase-activating protein, as a Hic-5 binding protein. The interaction of these two proteins was mediated by the LD3 motif of Hic-5 and the C-terminal region, which includes a paxillin-binding subdomain, of GIT1. Although GIT1 is known as a paxillin-binding protein, we only observed weak association of paxillin with GIT1 in the overexpression system. In contrast, Hic-5 firmly bound to GIT1 under the same conditions. In addition, the paxillin/GIT1 complex contained PIX, a guanine nucleotide exchange factor, whereas the Hic-5/GIT1 complex contained a smaller amount of PIX. These results suggested that paxillin and Hic-5 associate with GIT1 with different binding modes, and that the Hic-5 complex possesses static features compared with the paxillin complex, which contains both positive and negative regulators of GTPases involved in actin dynamics. Moreover, Hic-5-mediated inhibition of cell spreading was restored by co-expression of the C-terminal fragment of GIT1, which perturbs the interaction of Hic-5 with endogenous GIT1. Thus, it was demonstrated that Hic-5 and GIT1 interact functionally in addition to showing a physical association.
authors.affiliation: Department of Microbiology, Showa University School of Pharmaceutical Sciences, Hatanodai Shinagawa-ku, Tokyo 142-8555, Japan.
authors.forename: Naoyuki
authors.initials: N
authors.lastname: Nishiya
authors.forename: Takeshi
authors.initials: T
authors.lastname: Shirai
authors.forename: Wataru
authors.initials: W
authors.lastname: Suzuki
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Nose
mesh_terms: D016475:3T3 Cells; D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D018797:Cell Cycle Proteins / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D020690:GTPase-Activating Proteins / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D060588:LIM Domain Proteins; D051379:Mice; D008969:Molecular Sequence Data; D051419:Paxillin; D010750:Phosphoproteins / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D064067:Rho Guanine Nucleotide Exchange Factors; D016415:Sequence Alignment; D015398:Signal Transduction / Q000502:physiology; D020798:Two-Hybrid System Techniques
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D018797:Cell Cycle Proteins; D003598:Cytoskeletal Proteins; D004268:DNA-Binding Proteins; C485647:GIT1 protein, human; D020690:GTPase-Activating Proteins; C120766:Git1 protein, mouse; D020662:Guanine Nucleotide Exchange Factors; D047908:Intracellular Signaling Peptides and Proteins; D060588:LIM Domain Proteins; C496061:PXN protein, human; D051419:Paxillin; D010750:Phosphoproteins; C496062:Pxn protein, mouse; D011993:Recombinant Fusion Proteins; D064067:Rho Guanine Nucleotide Exchange Factors; C490351:TGFB1I1 protein, human; C090090:Tgfb1i1 protein, mouse