Regulation of P-Rex1 by Phosphatidylinositol (3,4,5)-Trisphosphate and Gβγ Subunits

Kirsti Hill,Sonja Krugmann,Simon R Andrews,W John Coadwell,Peter Finan,Heidi C.E Welch,Phillip T Hawkins,Len R Stephens

doi:10.1074/jbc.m411262200

Kirsti Hill, Sonja Krugmann + Show 6 more

Open Access

https://doi.org/10.1074/jbc.m411262200

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Abstract

P-Rex1 is a guanine-nucleotide exchange factor (GEF) for the small GTPase Rac. We have investigated here the mechanisms of stimulation of P-Rex1 Rac-GEF activity by the lipid second messenger phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) and the Gbetagamma subunits of heterotrimeric G proteins. We show that a P-Rex1 mutant lacking the PH domain (DeltaPH) cannot be stimulated by PtdIns(3,4,5)P3, which implies that the PH domain confers PtdIns(3,4,5)P3 regulation of P-Rex1 Rac-GEF activity. Consistent with this, we found that PtdIns(3,4,5)P3 binds to the PH domain of P-Rex1 and that the DH/PH domain tandem is sufficient for PtdIns(3,4,5)P3-stimulated P-Rex1 activity. The Rac-GEF activities of the DeltaPH mutant and the DH/PH domain tandem can both be stimulated by Gbetagamma subunits, which infers that Gbetagamma subunits regulate P-Rex1 activity by binding to the catalytic DH domain. Deletion of the DEP, PDZ, or inositol polyphosphate 4-phosphatase homology domains has no major consequences on the abilities of either PtdIns(3,4,5)P3 or Gbetagamma subunits to stimulate P-Rex1 Rac-GEF activity. However, the presence of any of these domains impacts on the levels of basal and/or stimulated P-Rex1 Rac-GEF activity, suggesting that there are important functional interactions between the DH/PH domain tandem and the DEP, PDZ, and inositol polyphosphate 4-phosphatase homology domains of P-Rex1.

Highlights

P-Rex[1] is a guanine-nucleotide exchange factor (GEF) for the small GTPase Rac
We show that a PRex[1] mutant lacking the pleckstrin homology (PH) domain (⌬PH) cannot be stimulated by PtdIns(3,4,5)P3, which implies that the PH domain confers PtdIns(3,4,5)P3 regulation of P-Rex[1] Rac-GEF activity
The RacGEF activities of the ⌬PH mutant and the Dbl homology (DH)/PH domain tandem can both be stimulated by G␤␥ subunits, which infers that G␤␥ subunits regulate P-Rex[1] activity by binding to the catalytic DH domain

Summary

Introduction

P-Rex[1] is a guanine-nucleotide exchange factor (GEF) for the small GTPase Rac. We have investigated here the mechanisms of stimulation of P-Rex[1] Rac-GEF activity by the lipid second messenger phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) and the G␤␥ subunits of heterotrimeric G proteins. The RacGEF activities of the ⌬PH mutant and the DH/PH domain tandem can both be stimulated by G␤␥ subunits, which infers that G␤␥ subunits regulate P-Rex[1] activity by binding to the catalytic DH domain. Deletion of the DEP, PDZ, or inositol polyphosphate 4-phosphatase homology domains has no major consequences on the abilities of either PtdIns(3,4,5)P3 or G␤␥ subunits to stimulate P-Rex[1] Rac-GEF activity.

Results

Conclusion