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The Weak Complex between RhoGAP Protein ARHGAP22 and Signal Regulatory Protein 14-3-3 Has 1:2 Stoichiometry and a Single Peptide Binding Mode

Journal Article


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. © 2012 Hu et al.

UOW Authors


  •   Hu, Shu-Hong (external author)
  •   Whitten, Andrew (external author)
  •   King, Gordon J. (external author)
  •   Jones, Alun (external author)
  •   Rowland, Alexander (external author)
  •   James, David E. (external author)
  •   Martin, Jennifer (external author)

Publication Date


  • 2012

Citation


  • Hu, S., Whitten, A. E., King, G. J., Jones, A., Rowland, A. F., James, D. E. & Martin, J. L. (2012). The Weak Complex between RhoGAP Protein ARHGAP22 and Signal Regulatory Protein 14-3-3 Has 1:2 Stoichiometry and a Single Peptide Binding Mode. PLoS One, 7 (8), e41731-1-e41731-11.

Scopus Eid


  • 2-s2.0-84865466650

Start Page


  • e41731-1

End Page


  • e41731-11

Volume


  • 7

Issue


  • 8

Place Of Publication


  • United States

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. © 2012 Hu et al.

UOW Authors


  •   Hu, Shu-Hong (external author)
  •   Whitten, Andrew (external author)
  •   King, Gordon J. (external author)
  •   Jones, Alun (external author)
  •   Rowland, Alexander (external author)
  •   James, David E. (external author)
  •   Martin, Jennifer (external author)

Publication Date


  • 2012

Citation


  • Hu, S., Whitten, A. E., King, G. J., Jones, A., Rowland, A. F., James, D. E. & Martin, J. L. (2012). The Weak Complex between RhoGAP Protein ARHGAP22 and Signal Regulatory Protein 14-3-3 Has 1:2 Stoichiometry and a Single Peptide Binding Mode. PLoS One, 7 (8), e41731-1-e41731-11.

Scopus Eid


  • 2-s2.0-84865466650

Start Page


  • e41731-1

End Page


  • e41731-11

Volume


  • 7

Issue


  • 8

Place Of Publication


  • United States