Hic-5 interacts with GIT1 with a different binding mode from paxillin.
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.
Mesh Terms:
3T3 Cells, Adaptor Proteins, Signal Transducing, Amino Acid Sequence, Animals, Binding Sites, Cell Cycle Proteins, Cytoskeletal Proteins, DNA-Binding Proteins, GTPase-Activating Proteins, Guanine Nucleotide Exchange Factors, Humans, Intracellular Signaling Peptides and Proteins, Mice, Molecular Sequence Data, Paxillin, Phosphoproteins, Protein Binding, Protein Structure, Tertiary, Recombinant Fusion Proteins, Sequence Alignment, Signal Transduction, Two-Hybrid System Techniques
3T3 Cells, Adaptor Proteins, Signal Transducing, Amino Acid Sequence, Animals, Binding Sites, Cell Cycle Proteins, Cytoskeletal Proteins, DNA-Binding Proteins, GTPase-Activating Proteins, Guanine Nucleotide Exchange Factors, Humans, Intracellular Signaling Peptides and Proteins, Mice, Molecular Sequence Data, Paxillin, Phosphoproteins, Protein Binding, Protein Structure, Tertiary, Recombinant Fusion Proteins, Sequence Alignment, Signal Transduction, Two-Hybrid System Techniques
J. Biochem.
Date: Aug. 01, 2002
PubMed ID: 12153727
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