title: The weak complex between RhoGAP protein ARHGAP22 and signal regulatory protein 14-3-3 has 1:2 stoichiometry and a single peptide binding mode.
abstract: ARHGAP22 is a RhoGAP protein comprising an N-terminal PH domain, a RhoGAP domain and a C-terminal coiled-coil domain. It has recently been identified as an Akt substrate that binds 14-3-3 proteins in response to treatment with growth factors involved in cell migration. We used a range of biophysical techniques to investigate the weak interaction between 14-3-3 and a truncated form of ARHGAP22 lacking the coiled-coil domain. This weak interaction could be stabilized by chemical cross-linking which we used to show that: a monomer of ARHGAP22 binds a dimer of 14-3-3; the ARHGAP22 PH domain is required for the 14-3-3 interaction; the RhoGAP domain is unlikely to participate in the interaction; Ser16 is the more important of two predicted 14-3-3 binding sites; and, phosphorylation of Ser16 may not be necessary for 14-3-3 interaction under the conditions we used. Small angle X-ray scattering and cross-link information were used to generate solution structures of the isolated proteins and of the cross-linked ARHGAP22:14-3-3 complex, showing that no major rearrangement occurs in either protein upon binding, and supporting a role for the PH domain and N-terminal peptide of ARHGAP22 in the 14-3-3 interaction. Small-angle X-ray scattering measurements of mixtures of ARHGAP22 and 14-3-3 were used to establish that the affinity of the interaction is ∼30 µM.
authors.affiliation: Institute for Molecular Bioscience, Division of Chemistry and Structural Biology, The University of Queensland, Brisbane, Queensland, Australia.
authors.forename: Shu-Hong
authors.initials: SH
authors.lastname: Hu
authors.forename: Andrew E
authors.initials: AE
authors.lastname: Whitten
authors.forename: Gordon J
authors.initials: GJ
authors.lastname: King
authors.forename: Alun
authors.initials: A
authors.lastname: Jones
authors.forename: Alexander F
authors.initials: AF
authors.lastname: Rowland
authors.forename: David E
authors.initials: DE
authors.lastname: James
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Martin
mesh_terms: D048948:14-3-3 Proteins / Q000378:metabolism*; D002465:Cell Movement; D003432:Cross-Linking Reagents / Q000494:pharmacology; D019281:Dimerization; D020690:GTPase-Activating Proteins / Q000737:chemistry / Q000378:metabolism*; D015894:Genome, Human; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry*; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D012542:Scattering, Radiation; D015398:Signal Transduction; D014965:X-Rays
chemical_list: D048948:14-3-3 Proteins; C578338:ARHGAP22 protein, human; D003432:Cross-Linking Reagents; D020690:GTPase-Activating Proteins; D010455:Peptides; D020033:Protein Isoforms; C085379:rho GTPase-activating protein