Published by Portland Press on behalf of the Biochemical Society
Biochemical Journal vol:443 pages:173-183
P‑Rex1 is a guanine-nucleotide exchange factor (GEF) for the small G protein Rac that is activated by PIP3 and Gbg subunits and inhibited by PKA. Here, we show that Protein Phosphatase 1a (PP1a) binds P‑Rex1 through an RVxF-type docking motif. PP1a activates P‑Rex1 directly in vitro, both independently of and additively to PIP3 and Gbg. PP1a also substantially activates P‑Rex1 in vivo, both in basal and PDGF- or LPA-stimulated cells. The phosphatase activity of PP1a is required for P‑Rex1 activation. PP1b, a close homologue of PP1a, is also able to activate P‑Rex1, but less effectively. PP1a stimulates P-Rex1-mediated, Rac-dependent changes in endothelial cell morphology. Mass spectrometric analysis of wild-type P‑Rex1 and a PP1a-binding deficient mutant revealed that endogenous PP1a dephosphorylates P‑Rex1 on at least three residues, S834, S1001 and S1165. Site-directed mutagenesis of S1165 to alanine caused activation of P-Rex1 to a similar degree as did PP1a, confirming S1165 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 PP1a-dependent dephosphorylation.