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• W2024430147 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 (PIP3) and the Gβγ 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 Gi- and Gs-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 PIP3- and Gβγ-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 PIP3 and varying concentrations of Gβ1γ2. Gβ1γ2 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 32P and stimulated with lysophosphatidic acid (LPA) to release Gβγ or isoproterenol to activate PKA. Treatment with isoproterenol or Sp-cAMPS, a potent activator of PKA, increased the incorporation of 32P 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 Sp-cAMPS also reduced the levels of GTP-bound Rac. These results outline a novel mechanism for Gs-linked receptors to regulate the function of P-Rex1 and inhibit its function in cells. 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 (PIP3) and the Gβγ 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 Gi- and Gs-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 PIP3- and Gβγ-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 PIP3 and varying concentrations of Gβ1γ2. Gβ1γ2 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 32P and stimulated with lysophosphatidic acid (LPA) to release Gβγ or isoproterenol to activate PKA. Treatment with isoproterenol or Sp-cAMPS, a potent activator of PKA, increased the incorporation of 32P 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 Sp-cAMPS also reduced the levels of GTP-bound Rac. These results outline a novel mechanism for Gs-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) 2The abbreviations used are: GEF, guanine nucleotide exchange factor; G proteins, guanine nucleotide-binding regulatory proteins; PIP3, phosphatidylinositol 3,4,5-trisphosphate; Sf9 cells, Spondoptera frugiperda cells; Sp-cAMPS, Sp-adenosine-3′,5′-cyclic monophosphorothioate; rolipram, 4-[3-(cyclopentyloxy)-4-methyoxyphenyl]-2-pyrrolidinone; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; DH, Dbl homology; PH, pleckstrin homology; SH, Src homology domain; PKA, protein kinase A; LPA, lysophosphatidic acid; GTPγS, guanosine 5′-3-O-(thio)-triphosphate; PBD, p21-binding domain; GST, glutathione S-transferase; DTT, dithiothreitol; HEK, human embryonic kidney; γ-PPase, λ-protein phosphatase; PAK1, p21-activated kinase 1. that is dually modulated by heterotrimeric G protein βγ subunits and PIP3 (1Welch H.C. Coadwell W.J. Ellson C.D. Ferguson G.J. Andrews S.R. Erdjument-Bromage H. Tempst P. Hawkins P.T. Stephens L.R. Cell. 2002; 108: 809-821Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar), a phospholipid messenger produced in cells via the activation of phosphatidylinositol 3-kinases (2Kerchner K.R. Clay R.L. McCleery G. Watson N. McIntire W.E. Myung C.S. Garrison J.C. J. Biol. Chem. 2004; 279: 44554-44562Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). 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 (1Welch H.C. Coadwell W.J. Ellson C.D. Ferguson G.J. Andrews S.R. Erdjument-Bromage H. Tempst P. Hawkins P.T. Stephens L.R. Cell. 2002; 108: 809-821Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar, 3Donald S. Hill K. Lecureuil C. Barnouin R. Krugmann S. John C.W. Andrews S.R. Walker S.A. Hawkins P.T. Stephens L.R. Welch H.C. FEBS Lett. 2004; 572: 172-176Crossref PubMed Scopus (82) Google Scholar, 4Rosenfeldt H. Vazquez-Prado J. Gutkind J.S. FEBS Lett. 2004; 572: 167-171Crossref PubMed Scopus (72) Google Scholar). Although there is an inositol polyphosphate 4-phosphatase (InsPx 4-phosphatase) domain contained within P-Rex1 and P-Rex2a, neither of these proteins demonstrate InsPx 4-phosphatase activity (1Welch H.C. Coadwell W.J. Ellson C.D. Ferguson G.J. Andrews S.R. Erdjument-Bromage H. Tempst P. Hawkins P.T. Stephens L.R. Cell. 2002; 108: 809-821Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar, 3Donald S. Hill K. Lecureuil C. Barnouin R. Krugmann S. John C.W. Andrews S.R. Walker S.A. Hawkins P.T. Stephens L.R. Welch H.C. FEBS Lett. 2004; 572: 172-176Crossref PubMed Scopus (82) Google Scholar) and this domain is not needed for the protein to act as a Rac-GEF (5Hill K. Krugmann S. Andrews S.R. Coadwell W.J. Finan P. Welch H.C. Hawkins P.T. Stephens L.R. J. Biol. Chem. 2005; 280: 4166-4173Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). 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 (6Schmidt A. Hall A. Genes Dev. 2002; 16: 1587-1609Crossref PubMed Scopus (984) Google Scholar, 7Zheng Y. Trends Biochem. Sci. 2001; 26: 724-732Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar, 8Cherfils J. Chardin P. Trends Biochem. Sci. 1999; 24: 306-311Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar). 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 intramolecular interactions; (c) regulation by phosphoinositol kinases; (d) activation by α and βγ subunits of GTP-binding proteins; and (e) activation by protein kinases (6Schmidt A. Hall A. Genes Dev. 2002; 16: 1587-1609Crossref PubMed Scopus (984) Google Scholar, 7Zheng Y. Trends Biochem. Sci. 2001; 26: 724-732Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar). There have only been a limited number of studies published on the activity and regulation of the three P-Rex family members. These proteins display differential tissue localization, with P-Rex1 being highly expressed in the brain and peripheral blood leukocytes, such as neutrophils (1Welch H.C. Coadwell W.J. Ellson C.D. Ferguson G.J. Andrews S.R. Erdjument-Bromage H. Tempst P. Hawkins P.T. Stephens L.R. Cell. 2002; 108: 809-821Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar). In contrast, P-Rex2a is abundant in skeletal muscle, heart, placenta, kidney, small intestine, and lung (3Donald S. Hill K. Lecureuil C. Barnouin R. Krugmann S. John C.W. Andrews S.R. Walker S.A. Hawkins P.T. Stephens L.R. Welch H.C. FEBS Lett. 2004; 572: 172-176Crossref PubMed Scopus (82) Google Scholar), and P-Rex2b seems most abundant in cardiac tissues (4Rosenfeldt H. Vazquez-Prado J. Gutkind J.S. FEBS Lett. 2004; 572: 167-171Crossref PubMed Scopus (72) Google Scholar). Interestingly, neither P-Rex2a nor P-Rex2b are present in peripheral blood leukocytes (3Donald S. Hill K. Lecureuil C. Barnouin R. Krugmann S. John C.W. Andrews S.R. Walker S.A. Hawkins P.T. Stephens L.R. Welch H.C. FEBS Lett. 2004; 572: 172-176Crossref PubMed Scopus (82) Google Scholar, 4Rosenfeldt H. Vazquez-Prado J. Gutkind J.S. FEBS Lett. 2004; 572: 167-171Crossref PubMed Scopus (72) Google Scholar). Furthermore, P-Rex1 is mainly a cytosolic protein that can be partially membrane associated in non-stimulated cells and is not substantially recruited to the membrane upon cell stimulation (1Welch H.C. Coadwell W.J. Ellson C.D. Ferguson G.J. Andrews S.R. Erdjument-Bromage H. Tempst P. Hawkins P.T. Stephens L.R. Cell. 2002; 108: 809-821Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar), suggesting that the PH domain in this protein may act as an inhibitory domain for GEF activity (1Welch H.C. Coadwell W.J. Ellson C.D. Ferguson G.J. Andrews S.R. Erdjument-Bromage H. Tempst P. Hawkins P.T. Stephens L.R. Cell. 2002; 108: 809-821Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar). The regulation of P-Rex proteins via PIP3 produced by the activation of phosphoinositol kinases, specifically by the p110γ isoform of phosphatidylinositol 3-kinase, has been reported (1Welch H.C. Coadwell W.J. Ellson C.D. Ferguson G.J. Andrews S.R. Erdjument-Bromage H. Tempst P. Hawkins P.T. Stephens L.R. Cell. 2002; 108: 809-821Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar, 4Rosenfeldt H. Vazquez-Prado J. Gutkind J.S. FEBS Lett. 2004; 572: 167-171Crossref PubMed Scopus (72) Google Scholar). Welch et al. (1Welch H.C. Coadwell W.J. Ellson C.D. Ferguson G.J. Andrews S.R. Erdjument-Bromage H. Tempst P. Hawkins P.T. Stephens L.R. Cell. 2002; 108: 809-821Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar) have demonstrated that when co-expressed in Sf9 cells, phosphatidylinositol 3-kinase, P-Rex1, and Gβ1γ2 work synergistically to produce Rac activation. Similar results have been observed for P-Rex2 (3Donald S. Hill K. Lecureuil C. Barnouin R. Krugmann S. John C.W. Andrews S.R. Walker S.A. Hawkins P.T. Stephens L.R. Welch H.C. FEBS Lett. 2004; 572: 172-176Crossref PubMed Scopus (82) Google Scholar, 4Rosenfeldt H. Vazquez-Prado J. Gutkind J.S. FEBS Lett. 2004; 572: 167-171Crossref PubMed Scopus (72) Google Scholar). A recent study by Hill et al. (5Hill K. Krugmann S. Andrews S.R. Coadwell W.J. Finan P. Welch H.C. Hawkins P.T. Stephens L.R. J. Biol. Chem. 2005; 280: 4166-4173Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar) has explored the regions of P-Rex1 that are important for its regulation by PIP3 and Gβγ subunits (5Hill K. Krugmann S. Andrews S.R. Coadwell W.J. Finan P. Welch H.C. Hawkins P.T. Stephens L.R. J. Biol. Chem. 2005; 280: 4166-4173Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Using targeted deletions, they found that P-Rex1 mutants lacking the PH domain (ΔPH) could not be stimulated by PIP3, demonstrating that the PH domain is required for PIP3-dependent activation of P-Rex1 (5Hill K. Krugmann S. Andrews S.R. Coadwell W.J. Finan P. Welch H.C. Hawkins P.T. Stephens L.R. J. Biol. Chem. 2005; 280: 4166-4173Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Moreover, the ΔPH mutant was 10 times more active than the wild-type P-Rex1, suggesting that the PH domain plays an inhibitory role in the wild-type P-Rex1 molecule (5Hill K. Krugmann S. Andrews S.R. Coadwell W.J. Finan P. Welch H.C. Hawkins P.T. Stephens L.R. J. Biol. Chem. 2005; 280: 4166-4173Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Similarly, the P-Rex1 mutant containing only the isolated DH/PH tandem of P-Rex1 was highly active compared with wild-type P-Rex1. These findings suggest that the PH domain along with the other domains in wild-type P-Rex1 are responsible for the intramolecular regulation of basal P-Rex1 activity (5Hill K. Krugmann S. Andrews S.R. Coadwell W.J. Finan P. Welch H.C. Hawkins P.T. Stephens L.R. J. Biol. Chem. 2005; 280: 4166-4173Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). In hematopoietic cells, where P-Rex1 is highly expressed, Gi-coupled receptors, such as the fMet-Leu-Phe receptor, and Gs-coupled receptors, such as the adenosine A2a receptor, have been shown to stimulate and inhibit chemotaxis, respectively (9Baggiolini M. Dewald B. Moser B. Annu. Rev. Immunol. 1997; 15: 675-705Crossref PubMed Scopus (1987) Google Scholar, 10Locati M. Murphy P.M. Annu. Rev. Med. 1999; 50: 425-440Crossref PubMed Scopus (253) Google Scholar, 11Luster A.D. N. Engl. J. Med. 1998; 338: 436-445Crossref PubMed Scopus (3259) Google Scholar, 12Mantovani A. Immunol. Today. 1999; 20: 254-257Abstract Full Text Full Text PDF PubMed Scopus (631) Google Scholar, 13Sullivan G.W. Linden J. Buster B.L. Scheld W.M. J. Infect. Dis. 1999; 180: 1550-1560Crossref PubMed Scopus (104) Google Scholar, 14Sullivan G.W. Rieger J.M. Scheld W.M. MacDonald T.L. Linden J. Br. J. Pharmacol. 2001; 132: 1017-1026Crossref PubMed Scopus (143) Google Scholar, 15Burkey T.H. Webster R.O. Biochim. Biophys. Acta. 1993; 1175: 312-318Crossref PubMed Scopus (30) Google Scholar, 16Cronstein B.N. Haines K.A. Kolasinski S. Reibman J. Blood. 1992; 80: 1052-1057Crossref PubMed Google Scholar). Because Rac activation is a key step in hematopoietic cell chemotaxis and P-Rex1 plays a major role in Rac activation, the regulation of P-Rex1 by multiple G protein α subunits (17Mayeenuddin L.H. McIntire W.E. Garrison J. J. Biol. Chem. 2006; 281: 1913-1920Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar) and βγ dimers (1Welch H.C. Coadwell W.J. Ellson C.D. Ferguson G.J. Andrews S.R. Erdjument-Bromage H. Tempst P. Hawkins P.T. Stephens L.R. Cell. 2002; 108: 809-821Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar, 17Mayeenuddin L.H. McIntire W.E. Garrison J. J. Biol. Chem. 2006; 281: 1913-1920Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar) has been studied. This work shows that P-Rex1 is not modulated by G protein α subunits but that it can be selectively activated by certain Gβγ subunits, suggesting the possibility that different receptors can release distinct Gβγ dimers (17Mayeenuddin L.H. McIntire W.E. Garrison J. J. Biol. Chem. 2006; 281: 1913-1920Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Several Rac-GEFs (Vav1–3, Ect2, and Tiam1) have been shown to be stimulated by phosphorylation (18Lopez-Lago M. Lee H. Cruz C. Movilla N. Bustelo X.R. Mol. Cell. Biol. 2000; 20: 1678-1691Crossref PubMed Scopus (142) Google Scholar, 19Fleming I.N. Elliott C.M. Buchanan F.G. Downes C.P. Exton J.H. J. Biol. Chem. 1999; 274: 12753-12758Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 20Fleming I.N. Elliott C.M. Exton J.H. FEBS Lett. 1998; 429: 229-233Crossref PubMed Scopus (38) Google Scholar, 21Fleming I.N. Elliott C.M. Collard J.G. Exton J.H. J. Biol. Chem. 1997; 272: 33105-33110Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 22Tatsumoto T. Xie X. Blumenthal R. Okamoto I. Miki T. J. Cell Biol. 1999; 147: 921-928Crossref PubMed Scopus (345) Google Scholar, 23Bustelo X.R. Mol. Cell. Biol. 2000; 20: 1461-1477Crossref PubMed Scopus (448) Google Scholar), but the regulation of P-Rex1 by protein kinases has not been studied. However, as activation of receptors such as the adenosine A2a receptor are known to raise the level of cyclic AMP and inhibit chemotaxis (13Sullivan G.W. Linden J. Buster B.L. Scheld W.M. J. Infect. Dis. 1999; 180: 1550-1560Crossref PubMed Scopus (104) Google Scholar, 14Sullivan G.W. Rieger J.M. Scheld W.M. MacDonald T.L. Linden J. Br. J. Pharmacol. 2001; 132: 1017-1026Crossref PubMed Scopus (143) Google Scholar), we investigated the possibility that phosphorylation of P-Rex1 by the cyclic AMP-dependent protein kinase (PKA) might regulate the GEF activity of P-Rex1 in cells. We found that phosphorylation of P-Rex1 by PKA caused a marked inhibition of PIP3 and Gβ1γ2-stimulated P-Rex1 activity, an effect strikingly different from the stimulatory effects reported for the phosphorylation of other Rac-GEFs (18Lopez-Lago M. Lee H. Cruz C. Movilla N. Bustelo X.R. Mol. Cell. Biol. 2000; 20: 1678-1691Crossref PubMed Scopus (142) Google Scholar, 19Fleming I.N. Elliott C.M. Buchanan F.G. Downes C.P. Exton J.H. J. Biol. Chem. 1999; 274: 12753-12758Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 20Fleming I.N. Elliott C.M. Exton J.H. FEBS Lett. 1998; 429: 229-233Crossref PubMed Scopus (38) Google Scholar, 21Fleming I.N. Elliott C.M. Collard J.G. Exton J.H. J. Biol. Chem. 1997; 272: 33105-33110Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 22Tatsumoto T. Xie X. Blumenthal R. Okamoto I. Miki T. J. Cell Biol. 1999; 147: 921-928Crossref PubMed Scopus (345) Google Scholar, 23Bustelo X.R. Mol. Cell. Biol. 2000; 20: 1461-1477Crossref PubMed Scopus (448) Google Scholar). Materials—The reagents used for Sf9 cell culture and purification of βγ dimers have been described (24Graber S.G. Lindorfer M.A. Garrison J.C. Methods Neurosci. 1996; 29: 207-226Crossref Scopus (23) Google Scholar, 25Graber S.G. Figler R.A. Garrison J.C. Methods Enzymol. 1994; 237: 212-226Crossref PubMed Scopus (30) Google Scholar). Isoproterenol, 4-[3-(cyclopentyloxy)-4-methyoxyphenyl]-2-pyrrolidinone (rolipram), amylose resin, maltose, and Triton X-100 were purchased from Sigma. Lysophosphatidic acid (LPA18:1) was purchased from Avanti Polar Lipids. Sp-cAMPS was obtained from Biomol. The Rac Activation Assay kit and the Rac monoclonal antibody were obtained from Upstate Biotechnology. PKA was obtained from Promega. PKA inhibitor peptide 5–24 was from Calbiochem. [35S]GTPγS was from PerkinElmer Life Sciences; 32P and [γ-32P]ATP were from ICN. The sources of all other reagents have been published (2Kerchner K.R. Clay R.L. McCleery G. Watson N. McIntire W.E. Myung C.S. Garrison J.C. J. Biol. Chem. 2004; 279: 44554-44562Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 17Mayeenuddin L.H. McIntire W.E. Garrison J. J. Biol. Chem. 2006; 281: 1913-1920Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Recombinant Protein Constructs—The protocols for constructing the G protein β1 and γ2 baculoviruses used in this study have been published (17Mayeenuddin L.H. McIntire W.E. Garrison J. J. Biol. Chem. 2006; 281: 1913-1920Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 26Graber S.G. Figler R.A. Kalman-Maltese V.K. Robishaw J.D. Garrison J.C. J. Biol. Chem. 1992; 267: 13123-13126Abstract Full Text PDF PubMed Google Scholar, 27McIntire W.E. MacCleery G. Garrison J.C. J. Biol. Chem. 2001; 276: 15801-15809Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). The cDNA encoding EE-tagged P-Rex1 and the recombinant baculovirus expressing EE-tagged P-Rex1 were kindly provided by Dr. Leonard R. Stephens, Cambridge University, United Kingdom (1Welch H.C. Coadwell W.J. Ellson C.D. Ferguson G.J. Andrews S.R. Erdjument-Bromage H. Tempst P. Hawkins P.T. Stephens L.R. Cell. 2002; 108: 809-821Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar). The bacterial expression plasmids encoding GST-Rac1, PAK1 p21-binding domain (PBD)-GST, and maltose-binding protein-λ phosphatase (MBP-λ-PPase) (28Satinover D.L. Leach C.A. Stukenberg P.T. Brautigan D.L. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 8625-8630Crossref PubMed Scopus (100) Google Scholar) were obtained from Dr. Ian Macara, Dr. Martin Schwartz, and Dr. Todd Stukenberg at the University of Virginia, respectively. Bacterial expression plasmids for three other Rac-GEFs, wild-type Vav2 (29Schuebel K.E. Movilla N. Rosa J.L. Bustelo X.R. EMBO J. 1998; 17: 6608-6621Crossref PubMed Scopus (223) Google Scholar), oncogenic Vav2 (29Schuebel K.E. Movilla N. Rosa J.L. Bustelo X.R. EMBO J. 1998; 17: 6608-6621Crossref PubMed Scopus (223) Google Scholar), and N-terminally trun-cated C-1199 Tiam1 (30Michiels F. Stam J.C. Hordijk P.L. van der Kammen R.A. Ruuls-Van Stalle L. Feltkamp C.A. Collard J.G. J. Cell Biol. 1997; 137: 387-398Crossref PubMed Scopus (211) Google Scholar, 31Michiels F. Habets G.G. Stam J.C. van der Kammen R.A. Collard J.G. Nature. 1995; 375: 338-340Crossref PubMed Scopus (508) Google Scholar) constructs were all provided by Dr. K. S. Ravichandran at the University of Virginia. Purification of Recombinant G βγ Dimers and EE-tagged P-Rex1 from Sf9 Cells—The protocols for the culture and infection of Sf9 cells with recombinant baculoviruses have been published (2Kerchner K.R. Clay R.L. McCleery G. Watson N. McIntire W.E. Myung C.S. Garrison J.C. J. Biol. Chem. 2004; 279: 44554-44562Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 17Mayeenuddin L.H. McIntire W.E. Garrison J. J. Biol. Chem. 2006; 281: 1913-1920Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). The methods used to purify the G protein β1γ2 dimer, to prepare the EE-antibody column, and to purify the EE-tagged P-Rex1 protein doublet from Sf9 cells have been described (17Mayeenuddin L.H. McIntire W.E. Garrison J. J. Biol. Chem. 2006; 281: 1913-1920Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Purification of Rac1 and PAK1 PBD-GST Fusion Proteins from Bacteria—The method used to purify GST-Rac1 has been described (17Mayeenuddin L.H. McIntire W.E. Garrison J. J. Biol. Chem. 2006; 281: 1913-1920Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). The human PAK1 (p21-activated kinase 1) protein contains a PBD that binds activated Cdc42 or Rac with high affinity (32Benard V. Bokoch G.M. Methods Enzymol. 2002; 345: 349-359Crossref PubMed Scopus (152) Google Scholar). The recombinant PAK1 PBD-binding domain with an N-terminal GST tag was purified using a GST resin (Amersham Biosciences). Two liters of DH5α bacterial culture expressing the GST-tagged PAK1 PBD construct were harvested and lysed using a French Press in 30 ml of lysis buffer containing 50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 5 mm MgCl2, 1 mm phenylmethylsulfonyl fluoride, 1 mm dithiothreitol (DTT) and protease inhibitors (aprotinin, leupeptin, and pepstatin (at 2 μg/ml), benzamidine at 20 μg/ml, and Pefabloc SC Plus at 100 μg/ml). One percent Triton X-100 was added to the cell lysate and the mixture incubated on ice for 30 min. The cell lysate was then centrifuged at 10,000 × g for 30 min at 4 °C. The supernatant (30 ml) was collected and incubated with 1 ml of GST resin for 2 h at 4°C with constant agitation. After the incubation, the GST-Sepharose beads were pelleted at 2000 rpm for 2 min. The beads were washed five times and pelleted at 2000 rpm for 2–3 min after each wash. The resin was washed twice with 15 ml of buffer B (50 mm Tris-HCl, pH 8.0, 150 mm NaCl, 5 mm MgCl2, 1 mm phenylmethylsulfonyl fluoride, 1 mm DTT and protease inhibitors) and 1% Triton X-100. The beads were subsequently washed three more times with 15 ml of buffer B. The beads were then aliquoted and stored in buffer B containing 50% (v/v) glycerol at –80 °C. Purification of λ-PPase—The recombinant MBP-tagged λ-PPase was purified using an amylose resin. One liter of BL21 bacterial cell culture expressing λ-PPase was harvested by centrifugation and lysed using a French press in 30 ml of MBP column buffer containing 20 mm Tris, pH 7.4, 200 mm NaCl, 1 mm EDTA, 40 units/μl DNase I, and protease inhibitors. The cell lysate was centrifuged at 10,000 × g for 30 min at 4 °C. The supernatant (30 ml) was collected and incubated with ∼3 ml of amylose resin for 2 h at 4°C with constant rocking. After the incubation, the resin was centrifuged at 600 × g for 2–3 min and the supernatant was removed. The resin was poured into a column and washed with 40 ml of MBP buffer and allowed to drain by gravity flow. The protein was eluted off the column in 2-ml fractions using MBP buffer containing 10 mm maltose. Column fractions (4 ml) containing λ-PPase were pooled and dialyzed against two changes of 2 liters of storage buffer containing 50 mm Tris, pH 7.5, 100 mm NaCl, 0.1 mm MnCl2, 0.1 mm EGTA, 2 mm phenylmethylsulfonyl fluoride, and 0.01% Brij 35. The purified protein was then dialyzed again overnight against 1 liter of storage buffer containing 50% glycerol. The dialyzed protein was resolved on an 8% SDS-PAGE to determine the purity and quantity of the λ-PPase by visualizing with Coomassie Blue. The MBP-tagged λ-PPase prepared using this protocol was highly pure and migrated as a single major band at ∼56 kDa. Approximately, 1 ml of 3 mg/ml of λ-PPase was obtained from 1 liter of BL21 bacterial culture. Preparation of λ-PPase-treated and PKA-treated P-Rex1—We used two different protocols to de-phosphorylate and phosphorylate P-Rex1. Initial experiments used the purified P-Rex1 protein doublet from Sf9 cells that was batch phosphorylated or de-phosphorylated on a small scale (10–50 μl) and then used directly for P-Rex1 activity assays. Using these small scale assays, we optimized the conditions for P-Rex1 dephosphorylation and phosphorylation (see Fig. 2). Small scale de-phosphorylation reactions using λ-PPase were carried out for 10 min at 30 °C in a buffer containing 50 mm Tris, pH 7.5, 0.1 mm Na2EDTA, 5 mm phenylmethylsulfonyl fluoride, 0.01% Brij 35 (v/v), and 2 mm MnCl2. The ability of various concentrations of the λ-PPase to de-phosphorylate P-Rex1 was tested on 80 ng/μl P-Rex1 in a 50-μl reaction volume. The reactions were terminated using either a final concentration of 2–4 mm sodium orthovanadate (33Zhuo S. Clemens J.C. Hakes D.J. Barford D. Dixon J.E. J. Biol. Chem. 1993; 268: 17754-17761Abstract Full Text PDF PubMed Google Scholar) or 25 μl of 2 × Laemmli sample buffer. Control reactions were carried out in the absence of the λ-PPase and the reactions were supplemented with the appropriate λ-PPase storage buffer. To test the P-Rex1 activity of de-phosphorylated P-Rex1, the reactions were terminated with sodium orthovanadate, placed on ice, and assayed for activity as described under “P-Rex1 Activity Assay.” Unless otherwise specified, λ-PPase-treated P-Rex1 was diluted 10-fold into the P-Rex1 activity assay to achieve a final P-Rex1 concentration of 30 nm (Fig. 1).FIGURE 1PKA can phosphorylate pure P-Rex1. P-Rex1 (500 ng) was incubated with 50 units of PKA and the reactions were carried out as described under “Experimental Procedures.” Aliquots containing about 100 ng of P-Rex1 were run on an 8% SDS-PAGE, stained with silver, dried, and the autoradiograph prepared. A, the autoradiograph; and B, the silver-stained gel of the P-Rex1 phosphorylated with PKA. C, the effect of phosphorylation on the GEF activity of P-Rex1. The Rac guanine nucleotide exchange assays to monitor P-Rex1 GEF activity were carried out on de-phosphorylated and phosphorylated P-Rex1, as described under “Experimental Procedures” in a 10-μl volume in the presence of 0.0025% CHAPS.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Small scale PKA phosphorylation reactions were carried out at 30 °C for 30 min, in a buffer containing 20 mm Hepes, pH 7.5, 200 nm microcystin, 0.1 mg/ml bovine serum albumin, 12.5 mm magnesium acetate, 1.25 mm EGTA. The total reaction volume was 10 μl and the protein to kinase ratio was 10 ng of P-Rex1:1 unit of PKA. The reactions were initiated by the addition of ATP at a final concentration of 125 μm. The reactions were terminated by the addition of the PKA inhibitor peptide (PKI residues 5–24) to a final concentration of 100 nm or by addition of 2× Laemmli sample buffer. Control reactions were carried out in the absence of the kinase, and the reactions were supplemented with the appropriate kinase storage buffers. The PKA storage buffer contained 350 mm KPO4 and 1 mm phenylmethylsulfonyl fluoride. The PKA enzyme was stored in small aliquots and used only once. To test the P-Rex1 activity of phosphorylated P-Rex1, the reactions were terminated with the above peptide inhibitor, placed on ice, and diluted 10-fold into the assay, and P-Rex1 activity was determined as above (Fig. 1). Reactions to determine the concentration of PKA needed for effective phosphorylation of P-Rex1 were carried out as above with varying concentrations of PKA in the presence of 600 ng of P-Rex1 in a volume of 10 μl for 5 min at 30 °C. Radiolabeled phosphorylation reactions were carried out as described above with the addition of [γ-32P]ATP (2.2 × 107 cpm/μl, final). Once the de-phosphorylation and phosphorylation protocols for P-Rex1 were optimized, large scale purifications of de-phosphorylated and phosphorylated P-Rex1 were undertaken using treatment of P-Rex1 on the EE-antibody column with λ-PPase and/or PKA. Purification of de-phosphorylated and phosphorylated P-Rex1 from the column allows isolation of highly pure P-Rex1 free of the λ-PPase or PKA, as these enzymes are washed away before elution of P-Rex1 from the column. This preparat" @default.
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• W2024430147 title "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" @default.
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