Norbin Stimulates the Catalytic Activity and Plasma Membrane Localization of the Guanine-Nucleotide Exchange Factor P-Rex1.

P-Rex1 is a guanine-nucleotide exchange factor (GEF) that activates the small G protein (GTPase) Rac1 to control Rac1-dependent cytoskeletal dynamics, and thus cell morphology. Three mechanisms of P-Rex1 regulation are currently known: (i) binding of the phosphoinositide second messenger PIP3, (ii) binding of the Gßγ subunits of heterotrimeric G proteins, and (iii) phosphorylation of various serine residues. Using recombinant P-Rex1 protein to search for new binding partners, we isolated the G-protein-coupled receptor (GPCR)-adaptor protein Norbin (Neurochondrin, NCDN) from mouse brain fractions. Coimmunoprecipitation confirmed the interaction between overexpressed P-Rex1 and Norbin in COS-7 cells, as well as between endogenous P-Rex1 and Norbin in HEK-293 cells. Binding assays with purified recombinant proteins showed that their interaction is direct, and mutational analysis revealed that the pleckstrin homology domain of P-Rex1 is required. Rac-GEF activity assays with purified recombinant proteins showed that direct interaction with Norbin increases the basal, PIP3- and Gßγ-stimulated Rac-GEF activity of P-Rex1. Pak-CRIB pulldown assays demonstrated that Norbin promotes the P-Rex1-mediated activation of endogenous Rac1 upon stimulation of HEK-293 cells with lysophosphatidic acid. Finally, immunofluorescence microscopy and subcellular fractionation showed that coexpression of P-Rex1 and Norbin induces a robust translocation of both proteins from the cytosol to the plasma membrane, as well as promoting cell spreading, lamellipodia formation, and membrane ruffling, cell morphologies generated by active Rac1. In summary, we have identified a novel mechanism of P-Rex1 regulation through the GPCR-adaptor protein Norbin, a direct P-Rex1 interacting protein that promotes the Rac-GEF activity and membrane localization of P-Rex1.

The guanine-nucleotide-exchange factor P-Rex1 is activated by protein phosphatase 1α.

P-Rex1 is a GEF (guanine-nucleotide-exchange factor) for the small G-protein Rac that is activated by PIP3 (phosphatidylinositol 3,4,5-trisphosphate) and Gßγ subunits and inhibited by PKA (protein kinase A). In the present study we show that PP1α (protein phosphatase 1α) binds P-Rex1 through an RVxF-type docking motif. PP1α activates P-Rex1 directly in vitro, both independently of and additively to PIP3 and Gßγ. PP1α also substantially activates P-Rex1 in vivo, both in basal and PDGF (platelet-derived growth factor)- or LPA (lysophosphatidic acid)-stimulated cells. The phosphatase activity of PP1α is required for P-Rex1 activation. PP1ß, a close homologue of PP1α, is also able to activate P-Rex1, but less effectively. PP1α stimulates P-Rex1-mediated Rac-dependent changes in endothelial cell morphology. MS analysis of wild-type P-Rex1 and a PP1α-binding-deficient mutant revealed that endogenous PP1α dephosphorylates P-Rex1 on at least three residues, Ser834, Ser1001 and Ser1165. Site-directed mutagenesis of Ser1165 to alanine caused activation of P-Rex1 to a similar degree as did PP1α, confirming Ser1165 as a dephosphorylation site important in regulating P-Rex1 Rac-GEF activity. In summary, we have identified a novel mechanism for direct activation of P-Rex1 through PP1α-dependent dephosphorylation.

Membrane translocation of P-Rex1 is mediated by G protein betagamma subunits and phosphoinositide 3-kinase.

P-Rex1 is a guanine-nucleotide exchange factor (GEF) for the small GTPase Rac that is directly activated by the betagamma subunits of heterotrimeric G proteins and by the lipid second messenger phosphatidylinositol (3,4,5)-trisphosphate (PIP(3)), which is generated by phosphoinositide 3-kinase (PI3K). Gbetagamma subunits and PIP(3) are membrane-bound, whereas the intracellular localization of P-Rex1 in basal cells is cytosolic. Activation of PI3K alone is not sufficient to promote significant membrane translocation of P-Rex1. Here we investigated the subcellular localization of P-Rex1 by fractionation of Sf9 cells co-expressing P-Rex1 with Gbetagamma and/or PI3K. In basal, serum-starved cells, P-Rex1 was mainly cytosolic, but 7% of the total was present in the 117,000 x g membrane fraction. Co-expression of P-Rex1 with either Gbetagamma or PI3K caused only an insignificant increase in P-Rex1 membrane localization, whereas Gbetagamma and PI3K together synergistically caused a robust increase in membrane-localized P-Rex1 to 23% of the total. PI3K-driven P-Rex1 membrane recruitment was wortmannin-sensitive. The use of P-Rex1 mutants showed that the isolated Dbl homology/pleckstrin homology domain tandem of P-Rex1 is sufficient for synergistic Gbetagamma- and PI3K-driven membrane localization; that the enzymatic GEF activity of P-Rex1 is not required for membrane translocation; and that the other domains of P-Rex1 (DEP, PDZ, and IP4P) contribute to keeping the enzyme localized in the cytosol of basal cells. In vitro Rac2-GEF activity assays showed that membrane-derived purified P-Rex1 has a higher basal activity than cytosol-derived P-Rex1, but both can be further activated by PIP(3) and Gbetagamma subunits.

PI3K and RAC signalling in leukocyte and cancer cell migration.

Leukocytes and amoeboid-stage cancer cells migrate in a similar manner. Key signalling molecules regulating leukocyte and cancer cell migration are phosphoinositide 3-kinase (PI3K) and the Rho family of small GTPases. While PI3K activity defines the leading edge of the cell, Rho family GTPases regulate the cytoskeletal remodelling during polarisation and migration. We review here briefly the roles of PI3K, of the Rho-family GTPase Rac, and of the guanine-nucleotide exchange factors that activate Rac, in leukocyte and cancer cell migration.