Phosphorylation of P-Rex1 by the cyclic AMP-dependent protein kinase inhibits the phosphatidylinositiol (3,4,5)-trisphosphate and Gbetagamma-mediated regulation of its activity.
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 Gbetagamma 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 Gbetagamma-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 Gbeta(1)gamma(2). Gbeta(1)gamma(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 Gbetagamma 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 GTP-bound 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.
Mayeenuddin
Linnia H
LH
Department of Pharmacology, University of Virginia Health System, Charlottesville, VA 22908, USA. lhm4n@virginia.edu
Garrison
James C
JC
D000818:Animals; D002460:Cell Line; D002633:Chemotaxis; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D019281:Dimerization; D044387:GTP-Binding Protein beta Subunits / Q000737:chemistry* / Q000378:metabolism; D044388:GTP-Binding Protein gamma Subunits / Q000737:chemistry* / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D005982:Glutathione Transferase / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry* / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000737:chemistry; D020962:Heterotrimeric GTP-Binding Proteins / Q000737:chemistry; D006801:Humans; D047468:Immunoprecipitation; D007313:Insecta; D007545:Isoproterenol / Q000494:pharmacology; D013058:Mass Spectrometry; D010716:Phosphatidylinositols / Q000737:chemistry*; D010766:Phosphorylation; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D020744:rac GTP-Binding Proteins / Q000378:metabolism
C118981:G-protein Beta gamma; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D020662:Guanine Nucleotide Exchange Factors; C453280:PREX1 protein, human; D010716:Phosphatidylinositols; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C118303:phosphoinositide-3,4,5-triphosphate; D006160:Guanosine Triphosphate; D005982:Glutathione Transferase; D017868:Cyclic AMP-Dependent Protein Kinases; D020962:Heterotrimeric GTP-Binding Proteins; D020744:rac GTP-Binding Proteins; D007545:Isoproterenol