Regulation of P-Rex1 by phosphatidylinositol (3,4,5)-trisphosphate and Gbetagamma subunits.
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.
Hill
Kirsti
K
Inositide Laboratory, The Babraham Institute, Babraham Research Campus, Cambridge CB2 4AT, United Kingdom.
Krugmann
Sonja
S
Andrews
Simon R
SR
Coadwell
W John
WJ
Finan
Peter
P
Welch
Heidi C E
HC
Hawkins
Phillip T
PT
Stephens
Len R
LR
D000818:Animals; D002460:Cell Line; D000939:Epitopes / Q000737:chemistry; D044387:GTP-Binding Protein beta Subunits / Q000737:chemistry*; D044388:GTP-Binding Protein gamma Subunits / Q000737:chemistry*; D017353:Gene Deletion; D005786:Gene Expression Regulation; D020662:Guanine Nucleotide Exchange Factors / Q000096:biosynthesis* / Q000737:chemistry* / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000737:chemistry; D006801:Humans; D007313:Insecta; D008055:Lipids / Q000737:chemistry; D016296:Mutagenesis; D009154:Mutation; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry; D017354:Point Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D020744:rac GTP-Binding Proteins / Q000737:chemistry / Q000502:physiology
D000939:Epitopes; C118981:G-protein Beta gamma; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D020662:Guanine Nucleotide Exchange Factors; D008055:Lipids; C453280:PREX1 protein, human; D011994:Recombinant Proteins; D006153:Guanosine Diphosphate; D010744:Phosphoric Monoester Hydrolases; C053153:phosphatidylinositol-3,4-bisphosphate 4-phosphatase; D020744:rac GTP-Binding Proteins