The weak complex between RhoGAP protein ARHGAP22 and signal regulatory protein 14-3-3 has 1:2 stoichiometry and a single peptide binding mode
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 approximately 30 microM. PLoS One. 2012;7(8):e41731. doi: 10.1371/journal.pone.0041731. Epub 2012 Aug 28.
|ISBN||1932-6203 (Electronic) 1932-6203 (Linking)|
|Authors||Hu, S. H.; Whitten, A. E.; King, G. J.; Jones, A.; Rowland, A. F.; James, D. E.; Martin, J. L.;|
|Publisher Name||PLoS One|
|URL link to publisher's version||http://www.ncbi.nlm.nih.gov/pubmed/22952583|
|OpenAccess link to author's accepted manuscript version||https://publications.gimr.garvan.org.au/open-access/11632|