title: Autoinhibition of GEF activity in Intersectin 1 is mediated by the short SH3-DH domain linker.
abstract: Intersectin 1L (ITSN1L) acts as a specific guanine nucleotide exchange factor (GEF) for the small guanine nucleotide binding protein Cdc42 via its C-terminal DH domain. Interestingly, constructs of ITSN1L that comprise additional domains, for instance the five SH3 domains amino-terminal of the DH domain, were shown to be inhibited in their exchange factor activity. Here, we investigate the inhibitory mechanism of ITSN1L in detail and identify a novel short amino acid motif which mediates autoinhibition. We found this motif to be located in the linker region between the SH3 domains and the DH domain, and we show that within this motif W1221 acts as key residue in establishing the inhibitory interaction. This assigns ITSN1L to a growing class of GEFs that are regulated by a short amino acid motif inhibiting GEF activity by an intramolecular interaction. Moreover, we quantify the interaction between the ITSN1L SH3 domains and the Cdc42 effector N-WASP using fluorescence anisotropy binding experiments. As the SH3 domains are not involved in autoinhibition, binding of N-WASP does not release inhibition of nucleotide exchange activity in kinetic experiments, in contrast to earlier observations.
authors.affiliation: Department of Protein Evolution, Max Planck Institute for Developmental Biology, Tuebingen D-72076, Germany.
authors.forename: Carsten
authors.initials: C
authors.lastname: Kintscher
authors.forename: Silvia
authors.initials: S
authors.lastname: Wuertenberger
authors.forename: Roy
authors.initials: R
authors.lastname: Eylenstein
authors.forename: Theresia
authors.initials: T
authors.lastname: Uhlendorf
authors.forename: Yvonne
authors.initials: Y
authors.lastname: Groemping
mesh_terms: D033942:Adaptor Proteins, Vesicular Transport / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D005454:Fluorescence Polarization; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D058977:Molecular Sequence Annotation; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D014364:Tryptophan / Q000737:chemistry / Q000378:metabolism; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal / Q000737:chemistry / Q000378:metabolism; D018909:src Homology Domains
chemical_list: D033942:Adaptor Proteins, Vesicular Transport; D020662:Guanine Nucleotide Exchange Factors; C485568:ITSN1 protein, human; C495771:WASL protein, human; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal; D014364:Tryptophan