p115RhoGEF is a member of a family of Rho-specific guanine nucleotide exchange factors that also contains a regulator of G protein signaling homology domain (RH-RhoGEFs) that serves as a link between Gα13 signaling and RhoA activation. While the mechanism of regulation of p115RhoGEF by Gα13 is becoming well-known, the role of other regulatory mechanisms, such as post-translational modification or autoinhibition, in mediating p115RhoGEF activity is less well-characterized. Here, putative phosphorylation sites on p115RhoGEF are identified and characterized. Mutation of Ser(330) leads to a decrease in serum response element-mediated transcription as well as decreased activation by Gα13 in vitro. Additionally, this study provides the first report of the binding kinetics between full-length p115RhoGEF and RhoA in its various nucleotide states and examines the binding kinetics of phospho-mutant p115RhoGEF to RhoA. These data, together with other recent reports on regulatory mechanisms of p115RhoGEF, suggest that this putative phosphorylation site serves as a means for initiation or relief of autoinhibition of p115RhoGEF, providing further insight into the regulation of its activity.
Keywords: ATRA; All-trans retinoic acid; DH; DMEM; Dbl homology; Dulbecco's modified Eagle's medium; FBS; Fetal bovine serum; GAP; GTPase activating protein; Galpha13; HPLC–MS/MS; High pressure/tandem mass spectrometry; LARG; LC/MS/MS; LPA; Leukemia-associated RhoGEF; Liquid chromatography/tandem mass spectrometry; Lysophosphatidic acid; Nα-p-tosyl-l-lysine phenylalanine chloromethyl ketone; Nα-tosyl-l-lysine chloromethyl ketone; PAR-1; PDZ; PH; PMA; Phorbol 12-myristate 13-acetate; Pleckstrin homology; Post-synaptic density protein, discs large protein, zonula occludens-1; Protease-activated receptor 1; RH-RhoGEF; RU; Regulator of G protein signaling homology domain-containing Rho guanine nucleotide exchange factor; Response units; RhoA; S1P; SPR; Sf9; Signal transduction; Sphingosine-1-phosphate; Spodoptera frugiperda; Surface plasmon resonance; TLCK; TPCK; p115RhoGEF.