title: Characterization of novel calmodulin binding domains within IQ motifs of IQGAP1.
abstract: IQ motif-containing GTPase-activating protein 1 (IQGAP1), which is a well-known calmodulin (CaM) binding protein, is involved in a wide range of cellular processes including cell proliferation, tumorigenesis, adhesion, and migration. Interaction of IQGAP1 with CaM is important for its cellular functions. Although each IQ domain of IQGAP1 for CaM binding has been characterized in a Ca(2+)-dependent or -independent manner, it was not clear which IQ motifs are physiologically relevant for CaM binding in the cells. In this study, we performed immunoprecipitation using 3xFLAGhCaM in mammalian cell lines to characterize the domains of IQGAP1 that are key for CaM binding under physiological conditions. Interestingly, using this method, we identified two novel domains, IQ(2.7-3) and IQ(3.5-4.4), within IQGAP1 that were involved in Ca(2+)-independent or -dependent CaM binding, respectively. Mutant analysis clearly showed that the hydrophobic regions within IQ(2.7-3) were mainly involved in apoCaM binding, while the basic amino acids and hydrophobic region of IQ(3.5-4.4) were required for Ca(2+)/CaM binding. Finally, we showed that IQ(2.7-3) was the main apoCaM binding domain and both IQ(2.7-3) and IQ(3.5-4.4) were required for Ca(2+)/CaM binding within IQ(1-2-3-4). Thus, we identified and characterized novel direct CaM binding motifs essential for IQGAP1. This finding indicates that IQGAP1 plays a dynamic role via direct interactions with CaM in a Ca(2+)-dependent or -independent manner.
authors.affiliation: Department of Applied Biology, College of Ecology and Environment, Kyungpook National University, Daegu 742-711, Korea. jangdj@knu.ac.kr
authors.forename: Deok-Jin
authors.initials: DJ
authors.lastname: Jang
authors.forename: Byungkwan
authors.initials: B
authors.lastname: Ban
authors.forename: Jin-A
authors.initials: JA
authors.lastname: Lee
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002147:Calmodulin / Q000378:metabolism*; D003001:Cloning, Molecular; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D020703:ras GTPase-Activating Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D002147:Calmodulin; C101203:IQ motif containing GTPase activating protein 1; D011993:Recombinant Fusion Proteins; D020703:ras GTPase-Activating Proteins
references.citation: J Biol Chem. 1999 Jan 1;274(1):464-70
references.pmid: 9867866
references.citation: Proc Natl Acad Sci U S A. 2005 Apr 26;102(17):5969-74
references.pmid: 15840729
references.citation: EMBO J. 1995 Sep 15;14(18):4450-9
references.pmid: 7556088
references.citation: J Biol Chem. 2002 Apr 5;277(14):12324-33
references.pmid: 11809768
references.citation: J Biol Chem. 2003 Feb 7;278(6):4347-52
references.pmid: 12446675
references.citation: J Biol Chem. 1999 Dec 31;274(53):37885-92
references.pmid: 10608854
references.citation: Mol Cell Biol. 2005 Sep;25(18):7940-52
references.pmid: 16135787
references.citation: J Biol Chem. 1991 Jan 5;266(1):229-37
references.pmid: 1824695
references.citation: J Biol Chem. 2004 Apr 23;279(17):17329-37
references.pmid: 14970219
references.citation: J Biol Chem. 1996 Oct 25;271(43):26646-52
references.pmid: 8900139
references.citation: J Biol Chem. 2002 Mar 1;277(9):7453-65
references.pmid: 11734550
references.citation: Proc Natl Acad Sci U S A. 1985 May;82(10):3187-91
references.pmid: 3858814
references.citation: J Invest Dermatol. 1985 Nov;85(5):450-2
references.pmid: 2997334
references.citation: J Biol Chem. 1997 Jun 13;272(24):15419-25
references.pmid: 9182573
references.citation: FEBS Lett. 2009 Jun 18;583(12):1817-24
references.pmid: 19433088
references.citation: Pflugers Arch. 1989 Jul;414(3):282-5
references.pmid: 2780212
references.citation: J Proteome Res. 2007 Sep;6(9):3718-28
references.pmid: 17696464
references.citation: Mol Cells. 2009 May 31;27(5):563-70
references.pmid: 19466605
references.citation: J Cell Sci. 2005 May 15;118(Pt 10):2085-92
references.pmid: 15890984
references.citation: J Biol Chem. 1987 May 5;262(13):6108-13
references.pmid: 2952648
references.citation: FEBS Lett. 2003 May 8;542(1-3):7-11
references.pmid: 12729888
references.citation: FEBS Lett. 2002 Feb 20;513(1):107-13
references.pmid: 11911888
references.citation: FASEB J. 1997 Apr;11(5):331-40
references.pmid: 9141499
references.citation: J Biol Chem. 2002 Jul 5;277(27):24753-63
references.pmid: 11948177
references.citation: Anal Biochem. 1997 Oct 1;252(1):96-105
references.pmid: 9324946