Self-association of IQGAP1: characterization and functional sequelae.
The scaffolding protein IQGAP1 participates in numerous cellular functions by binding to target proteins such as actin, calmodulin, E-cadherin, beta-catenin, Cdc42, Rac1, and CLIP-170. IQGAP1 regulates the cytoskeleton, promotes cell motility, and modulates E-cadherin-mediated cell-cell adhesion. However, how IQGAP1 exerts its functions in vivo is still unclear. In this study we investigate the self-association of IQGAP1 and its role in IQGAP1 function. Endogenous IQGAP1 co-immunoprecipitated from MCF-7 cells with IQGAP1 tagged with enhanced green fluorescent protein, indicating that IQGAP1 self-associates in cells. In vitro assays confirmed that IQGAP1 can self-associate and that this effect is mediated by the N-terminal half of the protein. Gel filtration analysis suggested that full-length IQGAP1 exists as a combination of monomers, dimers, and larger oligomers. Analysis performed with multiple fragments of IQGAP1 narrowed the self-association region to amino acids 763-863. In support of this observation, a peptide comprising residues 763-863 disrupted self-association of full-length IQGAP1 in a dose-dependent manner. Similarly, deleting this sequence from IQGAP1 abolished binding to full-length IQGAP1. In addition, the ability of IQGAP1 to increase the amount of active Cdc42 in cells is abrogated upon removal of this region. Consistent with these findings, transfection into cells of a peptide containing the self-association domain significantly reduced the amount of active Cdc42 in cell lysates. These observations define a sequence of IQGAP1 that is necessary for its oligomerization and demonstrate that self-association is required for the normal cellular function of IQGAP1.
Ren
Jian-Guo
JG
Department of Pathology, Brigham and Women's Hospital, and Harvard Medical School, Boston, Massachusetts 02115, USA.
Li
Zhigang
Z
Crimmins
Dan L
DL
Sacks
David B
DB
D001665:Binding Sites; D002460:Cell Line; D045744:Cell Line, Tumor; D002850:Chromatography, Gel; D004305:Dose-Response Relationship, Drug; D017353:Gene Deletion; D005982:Glutathione Transferase / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism; D020703:ras GTPase-Activating Proteins / Q000737:chemistry* / Q000378:metabolism
C101203:IQ motif containing GTPase activating protein 1; D010455:Peptides; D011993:Recombinant Fusion Proteins; D020703:ras GTPase-Activating Proteins; D049452:Green Fluorescent Proteins; D005982:Glutathione Transferase; D020764:cdc42 GTP-Binding Protein