Phosphorylation of P-Rex1 by the Cyclic AMP-dependent Protein Kinase Inhibits the Phosphatidylinositiol (3,4,5)-Trisphosphate and Gβγ-mediated Regulation of Its Activity

Abstract

Rac activation is a key step in chemotaxis of hematopoietic cells, which is both positively and negatively regulated by receptors coupled to heterotrimeric G proteins. P-Rex1, a Rac-specific guanine nucleotide exchange factor, is dually activated by phosphatidylinositol (3,4,5)-trisphosphate (PIP 3 ) and the G␤␥ subunits of heterotrimeric G proteins. This study explored the regulation of P-Rex1 by phosphorylation with the cAMP-dependent protein kinase (protein kinase A) in vitro and by G i -and G s -coupled receptors in HEK293T cells. P-Rex1 isolated from Sf9 and HEK293T cells migrates as two distinct bands that are partially phosphorylated. Phosphorylation of P-Rex1 with protein kinase A (PKA) inhibits the PIP 3 -and G␤␥-stimulated P-Rex1 guanine nucleotide exchange activity on Rac. The guanine nucleotide exchange factor activity of three different forms of P-Rex1 (native Sf9, de-phosphorylated, and phosphorylated) was examined in the presence of PIP 3 and varying concentrations of G␤ 1 ␥ 2 . G␤ 1 ␥ 2 was 47-fold less potent in activating the phosphorylated form of P-Rex1 compared with the de-phosphorylated form. HEK293T cells expressing P-Rex1 were labeled with 32 P and stimulated with lysophosphatidic acid (LPA) to release G␤␥ or isoproterenol to activate PKA. Treatment with isoproterenol or S p -cAMPS, a potent activator of PKA, increased the incorporation of 32 P into P-Rex1. LPA increased the amount of GTP-bound Rac in the cells and isoproterenol reduced basal levels of GTPbound Rac and blunted the effect of LPA. Treatment of the cells with S p -cAMPS also reduced the levels of GTP-bound Rac. These results outline a novel mechanism for G s -linked receptors to regulate the function of P-Rex1 and inhibit its function in cells.P-Rex1 is a Rac-specific guanine nucleotide exchange factor (Rac-GEF) 2 that is dually modulated by heterotrimeric G protein ␤␥ subunits and PIP 3 (1), a phospholipid messenger produced in cells via the activation of phosphatidylinositol 3-kinases (2). P-Rex1, P-Rex2a, and P-Rex2b are known members of the P-Rex family; all are multimodular proteins containing a tandem DH (Dbl homology), a PH (pleckstrin homology) domain, and two DEP and PDZ domains (1, 3, 4). Although there is an inositol polyphosphate 4-phosphatase (InsP x 4-phosphatase) domain contained within P-Rex1 and P-Rex2a, neither of these proteins demonstrate InsP x 4-phosphatase activity (1, 3) and this domain is not needed for the protein to act as a Rac-GEF (5).Like the P-Rex family of GEFs, all GEFs are multimodular proteins containing at least the DH/PH tandem domain and various other functional domains, such as SH2, SH3, Ser/Thr, or tyrosine kinase, Ras-GEF, Rho-GAP, Ran-GEF, PDZ and/or additional PH domains (6 -8). Their structural complexity allows for a number of distinct modes of regulation although no universal mode of GEF regulation exists. Thus far, at least five different modes of regulation have been identified for the known proteins exhibiting GEF activity: (a) regulation by localization; (b) regulation by int...

PREX1 Protein Function Is Negatively Regulated Downstream of Receptor Tyrosine Kinase Activation by p21-activated Kinases (PAKs)

“…3D). This is consistent with previous reports showing that dephosphorylation of PREX1 by protein phosphatases activates GEF activity (30,31). In addition, PREX1 that was purified from cells co-expressing PAK1 showed reduced GEF activity at multiple PIP 3 concentrations compared with dephosphorylated PREX1, with a significant difference in GEF activation in the presence of 0.1 M PIP 3 .…”

“…PREX1 is phosphorylated downstream of neuregulin and IGF1 (18,19), and multiple studies have demonstrated that phosphorylation causes an electrophoretic mobility shift in PREX1 (18,30,31). In MCF7 breast cancer cells, we found that insulin also caused a PREX1 mobility shift (Fig.…”

“…Kinases-PREX proteins are also activated by G␤␥, and PREX1 is both activated and phosphorylated after GPCR activation with the ␤-adrenergic receptor agonist isoproterenol (31,35). Thus far, we have established that RTK-dependent phosphorylation of PREX1 is PAK-dependent, and next we sought to determine whether certain GPCR-mediated phosphorylation events on PREX1 also require PAKs.…”

“…Importantly, PREX1 is phosphorylated after the activation of specific RTKs and GPCRs, indicating that the regulation of PREX1 by phosphorylation has a role in these signaling pathways. The stimulation of ␤-adrenergic GPCRs with isoproterenol results in the phosphorylation of PREX1, which is likely due to the activation of PKA downstream of the ␤-adrenergic receptor (31). Similarly, downstream of sphingosine 1-phosphate, PKA phosphorylates PREX1 to regulate an intramolecular interaction that reduces PREX1 GEF activity (49).…”

“…It is known that PREX1 is phosphorylated in the cell, demonstrated by the fact that after gel electrophoresis, it is detected as multiple bands that are collapsed by both -phosphatase and protein phosphatase 1␣ (PP1␣) (30,31). -Phosphatase and PP1␣ dephosphorylation of PREX1 increase GEF activity, which is consistent with reports showing that PKA phosphorylation of PREX1 leads to a reduction in its in vitro GEF activity both in vitro and in cells (31,32,49). Importantly, PREX1 is phosphorylated after the activation of specific RTKs and GPCRs, indicating that the regulation of PREX1 by phosphorylation has a role in these signaling pathways.…”