Activation of the Diguanylate Cyclase PleD by Phosphorylation-mediated Dimerization
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Caulobacter crescentus
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The aim of the present study was to investigate possible mechanisms that could be involved in the deactivation of the assembled, catalytically active NADPH oxidase of phagocytic cells and thereby lead to termination of O(2)(.-) production. Our major findings are the following: (1) Addition of GDP to the active oxidase is able to reduce O(2)(.-) production both in the fully purified and in a semi-recombinant cell-free activation system. (2) p67(phox) inhibits GTP hydrolysis on Rac whereas p47(phox) has no effect on Rac GTPase activity. (3) Soluble regulatory proteins (GTPase activating protein, guanine nucleotide dissociation inhibitor, and the Rac-binding domain of the target protein p21-activated kinase) inhibit activation of the NADPH oxidase but have no effect on electron transfer via the assembled enzyme complex. (4) Membrane-associated GTPase activating proteins (GAPs) have access also to the assembled, catalytically active oxidase. Taken together, we propose that the GTP-bound active form of Rac is required for sustained enzyme activity and that membrane-localized GAPs have a role in the deactivation of NADPH oxidase. PMID: 12186557
Small GTPase
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Rac GTP-Binding Proteins
Autolysis (biology)
PAK1
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Caulobacter crescentus
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Metabolic ATP is converted rapidly to IMP during platelet secretion. The conversion is rate limited by deamination of AMP to IMP. AMP deaminase has been purified to homogeneity from human platelet concentrates by phosphocellulose chromatography. Kinetic studies showed sigmoidal behavior as a function of AMP with K0.5 values of 3.5 and 4.0 mM in 0.1 M NaCl and 0.1 M KC1, respectively, at pH 6.5. Activation by saturating ATP converted the velocity versus substrate plot to hyperbolic with a Km of 1.2 mM in either salt. Addition of increasing concentrations of GTP in the presence of NaCl led to activation followed by weak competitive inhibition whereas GTP in the presence.of KC1 gave strong competitive inhibition with no apparent activation. Consequently, enzyme activity measured at 100 μM AMP in the presence of 10 μM GTP was 22 times greater in NaCl than in KC1. Studies on the effects of pH revealed pH optima around 6.5 in NaCl and 6.7 in KC1. Activation by ATP or GTP shifted the optima to lower pH values whereas inhibition by GTP shifted the optima to higher pH values. All of these results may be described by a model that does not invoke subunit-subunit interactions to explain cooperative behavior but requires obligatory binding of AMP to a distinct activator site on the same subunit before AMP can bind to the catalytic site. ATP and GTP bind to the same activator site as AMP and GTP can also bind competitively to the catalytic site. Changes in activity produced by NaCl or KC1 or pH are due to changes in the affinity of the two sites for the various nucleotides in media of different ionic composition and pH. Regulation of AMP deaminase in the intact platelet is thus envisaged as being the result of changes in ionic composition and pH that may accompany platelet stimulation rather than changes in the concentration of effector nucleotides.
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In crude extracts of epidermal papillomas induced by an initiation-promotion protocol, ornithine decarboxylase (OrnDCase) activity was increased by the addition of GTP to the enzyme assay. No effect of GTP on the phorbol ester-induced enzyme isolated from normal epidermis was observed. Kinetic analyses indicated that the major effect of the nucleotide on the tumor-derived enzyme was to lower the apparent Km for L-ornithine. When papilloma OrnDCase was partially purified by gel-filtration chromatography, two forms of the enzyme were resolved, only one of which was found in an epidermal extract from phorbol 12-myristate 13-acetate-treated mice. The enzymatic properties of the two forms of papilloma enzyme were compared. The higher molecular weight form (peak I) was activated by GTP, while the lower molecular weight form (peak II) was not. As expected from the kinetic analyses of the crude papilloma extracts, the apparent Km of peak I enzyme for L-ornithine was very high (1.25 mM) but was much lower in the presence of GTP (0.02 mM). The two forms of papilloma OrnDCase differed in their sensitivities to heat inactivation and the ability of GTP to protect against heat inactivation. The K1/2 for activation of peak I OrnDCase by GTP was 0.1 microM. The activation process was irreversible and did not require Mg2+. When several nucleotides were tested for their ability to activate peak I OrnDCase, only GTP, dGTP, and the nonhydrolyzable derivative GTP[gamma-S] were effective, while GDP, GMP, ATP, and CTP were relatively ineffective. Our results demonstrated the existence of two forms of OrnDCase in epidermal tumor extracts, of which one can be activated by GTP and one cannot. The significance of these findings for the regulation of this enzyme in normal and tumor cells is discussed.
12-O-Tetradecanoylphorbol-13-acetate
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Guanine nucleotide exchange factors (GEFs) and their associated GTP-binding proteins (G-proteins) are key regulatory elements in the signal transduction machinery that relays information from the extracellular environment into specific intracellular responses. Among them, the MAPK cascades represent ubiquitous downstream effector pathways. We have previously described that, analogous to the Ras-dependent activation of the Erk-1/2 pathway, members of the Rho family of small G-proteins activate the JNK cascade when GTP is loaded by their corresponding GEFs. Searching for novel regulators of JNK activity we have identified Epac (exchange protein activated by cAMP) as a strong activator of JNK-1. Epac is a member of a growing family of GEFs that specifically display exchange activity on the Rap subfamily of Ras small G-proteins. We report here that while Epac activates the JNK severalfold, a constitutively active (G12V) mutant of Rap1b does not, suggesting that Rap-GTP is not sufficient to transduce Epac-dependent JNK activation. Moreover, Epac signaling to the JNKs was not blocked by inactivation of endogenous Rap, suggesting that Rap activation is not necessary for this response. Consistent with these observations, domain deletion mutant analysis shows that the catalytic GEF domain is dispensable for Epac-mediated activation of JNK. These studies identified a region overlapping the Ras exchange motif domain as critical for JNK activation. Consistent with this, an isolated Ras exchange motif domain from Epac is sufficient to activate JNK. We conclude that Epac signals to the JNK cascade through a new mechanism that does not involve its canonical catalytic action, i.e. Rap-specific GDP/GTP exchange. This represents not only a novel way to activate the JNKs but also a yet undescribed mechanism of downstream signaling by Epac. Guanine nucleotide exchange factors (GEFs) and their associated GTP-binding proteins (G-proteins) are key regulatory elements in the signal transduction machinery that relays information from the extracellular environment into specific intracellular responses. Among them, the MAPK cascades represent ubiquitous downstream effector pathways. We have previously described that, analogous to the Ras-dependent activation of the Erk-1/2 pathway, members of the Rho family of small G-proteins activate the JNK cascade when GTP is loaded by their corresponding GEFs. Searching for novel regulators of JNK activity we have identified Epac (exchange protein activated by cAMP) as a strong activator of JNK-1. Epac is a member of a growing family of GEFs that specifically display exchange activity on the Rap subfamily of Ras small G-proteins. We report here that while Epac activates the JNK severalfold, a constitutively active (G12V) mutant of Rap1b does not, suggesting that Rap-GTP is not sufficient to transduce Epac-dependent JNK activation. Moreover, Epac signaling to the JNKs was not blocked by inactivation of endogenous Rap, suggesting that Rap activation is not necessary for this response. Consistent with these observations, domain deletion mutant analysis shows that the catalytic GEF domain is dispensable for Epac-mediated activation of JNK. These studies identified a region overlapping the Ras exchange motif domain as critical for JNK activation. Consistent with this, an isolated Ras exchange motif domain from Epac is sufficient to activate JNK. We conclude that Epac signals to the JNK cascade through a new mechanism that does not involve its canonical catalytic action, i.e. Rap-specific GDP/GTP exchange. This represents not only a novel way to activate the JNKs but also a yet undescribed mechanism of downstream signaling by Epac. Epac 1The abbreviations used are: Epac, exchange protein activated by cAMP; GEF, guanine nucleotide exchange factor; GAP, GTPase-activating protein; MAPK, mitogen-activated protein kinase; SAPK, stress activated proteins kinase; JNK, c-Jun NH2-terminal kinase; ERK, extracellular regulated kinase; GST, glutathione S-transferase; DEP, dishevelled, Egl-10, and pleckstrin; REM, Ras exchange motif; CBD, cAMP binding domain; PKA, protein kinase A; MOPS, 4-morpholinepropanesulfonic acid; HA, hemagglutinin.1The abbreviations used are: Epac, exchange protein activated by cAMP; GEF, guanine nucleotide exchange factor; GAP, GTPase-activating protein; MAPK, mitogen-activated protein kinase; SAPK, stress activated proteins kinase; JNK, c-Jun NH2-terminal kinase; ERK, extracellular regulated kinase; GST, glutathione S-transferase; DEP, dishevelled, Egl-10, and pleckstrin; REM, Ras exchange motif; CBD, cAMP binding domain; PKA, protein kinase A; MOPS, 4-morpholinepropanesulfonic acid; HA, hemagglutinin. (exchange protein activated by cAMP) is a newly discovered guanine nucleotide exchange factor (GEF) that selectively activates members of the Rap family of G-proteins (1Rooij J. Zwartkruis F.J.T. Verheijen M.H.G. Cool R.H. Nijman S.M.B. Wittinghofer A. Bos J.L. Nature. 1998; 396: 474-477Crossref PubMed Scopus (1606) Google Scholar, 2Kawasaki H. Springett G.M. Mochizuki S.T. Nakaya M. Matsuda M. Housman D.E. Graybel A.M. Science. 1998; 282: 2275-2279Crossref PubMed Scopus (1166) Google Scholar, 3Rooij J. Rehmann H. van Trest M. Cool R.H. Wittinghofer A. Bos J.L. J. Biol. Chem. 2000; 275: 20829-20836Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar). Rap proteins, like all G-proteins, are biochemical transducers (4Boguski M.S. McCormick F. Nature. 1993; 366: 643-654Crossref PubMed Scopus (1755) Google Scholar, 5Kitashama H. Sugimoto Y. Matsuzaki T. Ikawa Y. Noda M. Cell. 1989; 56: 77-84Abstract Full Text PDF PubMed Scopus (760) Google Scholar) that function as allosteric regulatory elements, switching between an inactive GDP-bound and an active GTP-bound conformation (4Boguski M.S. McCormick F. Nature. 1993; 366: 643-654Crossref PubMed Scopus (1755) Google Scholar). The switch mechanism consists of activation by exchange of bound GDP for GTP, and inactivation by hydrolysis of GTP into GDP, catalyzed by GEFs and GTPase-activating proteins (GAPs), respectively. Similar to all GEFs, Epac acts catalytically on the rate-limiting step for G-protein activation, i.e. dissociation of bound GDP (4Boguski M.S. McCormick F. Nature. 1993; 366: 643-654Crossref PubMed Scopus (1755) Google Scholar). The involvement of Rap1 in signaling mechanisms is demonstrated by the variety of second messengers mediating its activation. Recently, a number of exchange factors were identified as mediators of these specific activities (6Bos J.L. de Rooij J. Reedquist K.A. Nat. Rev. 2001; 2: 369-377Crossref Scopus (512) Google Scholar). Among them, Epac was characterized as a molecule responsible for cAMP-dependent Rap1 activation (1Rooij J. Zwartkruis F.J.T. Verheijen M.H.G. Cool R.H. Nijman S.M.B. Wittinghofer A. Bos J.L. Nature. 1998; 396: 474-477Crossref PubMed Scopus (1606) Google Scholar, 2Kawasaki H. Springett G.M. Mochizuki S.T. Nakaya M. Matsuda M. Housman D.E. Graybel A.M. Science. 1998; 282: 2275-2279Crossref PubMed Scopus (1166) Google Scholar). Epac is a multidomain protein comprised of a COOH-terminal catalytic and an NH2-terminal regulatory module. The COOH-terminal module encompasses the Rap-specific GEF catalytic core and the Ras exchange motif (REM) (1Rooij J. Zwartkruis F.J.T. Verheijen M.H.G. Cool R.H. Nijman S.M.B. Wittinghofer A. Bos J.L. Nature. 1998; 396: 474-477Crossref PubMed Scopus (1606) Google Scholar). According to the structure solved for SOS GEF, the REM domain does not provide any catalytic residues but rather participates in GEF stability (7Boriack-Sjodin A.P. Margarit M.S. Bar-Sagi D. Kuriyan J. Nature. 1998; 394: 337-343Crossref PubMed Scopus (615) Google Scholar). The NH2-terminal regulatory module contains a DEP domain whose function seems to be related to membrane/cytoskeleton association, and a cAMP-binding domain (CBD) homologous to the CBD originally identified in the regulatory subunit of PKA (1Rooij J. Zwartkruis F.J.T. Verheijen M.H.G. Cool R.H. Nijman S.M.B. Wittinghofer A. Bos J.L. Nature. 1998; 396: 474-477Crossref PubMed Scopus (1606) Google Scholar). Although cAMP binding and GEF activity are energetically uncoupled processes (8Kraemer A. Rehmann H.R. Cool R.H. Theiss C. de Rooij J. Bos J.L. Wittinghofer A. J. Mol. Biol. 2001; 306: 1167-1177Crossref PubMed Scopus (58) Google Scholar), deletion of the NH2-terminal regulatory module renders a constitutively active (cAMP-independent) GEF, which is suggestive of an auto-inhibitory mechanism that is relieved upon cAMP binding (1Rooij J. Zwartkruis F.J.T. Verheijen M.H.G. Cool R.H. Nijman S.M.B. Wittinghofer A. Bos J.L. Nature. 1998; 396: 474-477Crossref PubMed Scopus (1606) Google Scholar). The mitogen-activated protein kinases (MAPKs) represent an example of fundamental biochemical processes regulated by G-proteins (9Gutkind, J. S. (2000) Science's STKE http://stke.sciencemag.org/cgi/content/full/sigtrans;2000/40/RE1.Google Scholar). Three major MAP kinase cascades have been described so far (10Pearson G. Robinson F. Beers Gibson T. Xu B. Karandikar M. Berman K. Cobb M.H. Endocr. Rev. 2001; 22: 153-183Crossref PubMed Scopus (3500) Google Scholar): the Ras-dependent MAPKs as ERK-1/2 are typically associated with cell proliferation (11Sasaoka T. Langlois W.J. Leitner J.W. Draznin B. Olefsky J.M. J. Biol. Chem. 1994; 269: 32621-32625Abstract Full Text PDF PubMed Google Scholar, 12Chang L. Karin M. Nature. 2001; 410: 37-40Crossref PubMed Scopus (4354) Google Scholar). The stress-activated proteins kinases (SAPKs) as the c-Jun kinase or JNK and the p38 kinases are triggered in response to environmental stresses (13Davis R.D. Cell. 2000; 103: 239-252Abstract Full Text Full Text PDF PubMed Scopus (3611) Google Scholar, 14Martin-Blanco E. Bioessays. 2000; 22: 637-645Crossref PubMed Scopus (172) Google Scholar), although they have also been linked to proliferative conditions depending on the cellular setting (9Gutkind, J. S. (2000) Science's STKE http://stke.sciencemag.org/cgi/content/full/sigtrans;2000/40/RE1.Google Scholar, 12Chang L. Karin M. Nature. 2001; 410: 37-40Crossref PubMed Scopus (4354) Google Scholar). The discovery of additional MAPKs as ERK-5 and others add diversity to the MAPK scenario (15Kyriakis J.M. Avruch J. Physiol. Rev. 2001; 81: 807-869Crossref PubMed Scopus (2864) Google Scholar). The signaling cascades that end in MAPKs and regulate their activity include several sequential events of phosphorylation in the cytoplasm that define phosphorylation cascades. MAPKs are activated by dual phosphorylation at two residues, a tyrosine and a threonine by kinases known as MAPKKs (16Marshall C.J. Curr. Biol. 1994; 4: 82-89Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). Particularly JNK is activated by the prototypical JNKKs, MKK4 (SEK1) and MKK7 (SEK2), which are in turn activated by upstream phosphorylation events. A large group of JNKKKs has been reported (13Davis R.D. Cell. 2000; 103: 239-252Abstract Full Text Full Text PDF PubMed Scopus (3611) Google Scholar). However, the mechanisms involved in the activation of these JNKKKs, and the link between a specific stress signal and its mediators are still ill defined. The module SOS/Ras/Raf/MEK/Erk-1–2 constitutes a typical example of a MAPK signaling pathway (10Pearson G. Robinson F. Beers Gibson T. Xu B. Karandikar M. Berman K. Cobb M.H. Endocr. Rev. 2001; 22: 153-183Crossref PubMed Scopus (3500) Google Scholar). In recent years we have contributed to the development of the dominant concept that probably all of the MAPKs are activated in an analogous fashion when we discovered that Dbl, a GEF specific for the Rho family of small G-proteins, can activate the JNK pathway (17Coso O.A. Chiariello M. Yu J.C. Termoto H. Crespo P. Xu N. Miki T. Gutkind J.S. Cell. 1995; 81: 1137-1146Abstract Full Text PDF PubMed Scopus (1561) Google Scholar) through the Rho family members Rac and Cdc42 and a series of kinases that include MLK3, MEKK, and SEK (17Coso O.A. Chiariello M. Yu J.C. Termoto H. Crespo P. Xu N. Miki T. Gutkind J.S. Cell. 1995; 81: 1137-1146Abstract Full Text PDF PubMed Scopus (1561) Google Scholar, 18Yan M. Dai T. Deak J.C. Kyriakis J.M. Zon L.I. Woodget J.R. Templeton D.J. Nature. 1994; 372: 798-800Crossref PubMed Scopus (658) Google Scholar, 19Sanchez I. Hughes R.T. Mayer B.J. Yee K. Woodget J.R. Avruch J. Kyriakis J.M. Zon L.I. Nature. 1994; 372: 794-798Crossref PubMed Scopus (916) Google Scholar, 20Teramoto H. Coso O.A. Miyata H. Igishi T. Miki T. Gutkind J.S. J. Biol. Chem. 1996; 271: 27225-27228Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar). It is well now established that the Rho-like small G-proteins Rac and Cdc42 represent a link between environmental stimuli and JNK activation (21Bar-Sagi D. Hall A. Cell. 2000; 103: 227-238Abstract Full Text Full Text PDF PubMed Scopus (700) Google Scholar). These G-proteins as well as their corresponding GEFs are also regulators of the actin cytoskeleton dynamics (22Nobes C.D. Hall A. Cell. 1995; 81: 53-62Abstract Full Text PDF PubMed Scopus (3714) Google Scholar). Expression of the small G-protein Rap1b in Swiss 3T3 fibroblasts induces an anchorage-dependent transformed phenotype (23Ribeiro-Neto F. Altschuler D.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7475-7479Crossref PubMed Scopus (123) Google Scholar), accompanied by changes in focal contacts and actin cytoskeleton. 2D. Altschuler, unpublished observations.2D. Altschuler, unpublished observations. These results prompted us to address whether Rap G-proteins, like Rac and Rho, might participate in signaling to the JNKs. Unexpectedly, we have found that Epac effectively signals to the JNK pathway, while its cognate G-protein Rap1b does not. Our studies identified a new function for the Rap-selective guanine nucleotide exchange factor Epac: JNK activation, which is independent of Rap1 activation and unrelated to its GEF activity. Moreover, this signal is specific for JNK, since other SAPKs like p38 MAPK are not activated. We can circumscribe downstream signaling to JNK to a region in Epac devoid of exchange activity on Rap, the REM domain, which is sufficient to activate JNK. This is to our knowledge not only the first report of a transduction mechanism that connects this GEF to the MAPKs acting independently of its G-protein counterpart but also describes a novel, Rap-independent, Epac function. Cell Lines and Transfections—HEK-293T cells were maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum. Transfections were performed by the calcium phosphate precipitation technique, adjusting the total amount of plasmid DNA to 3–6 μg/plate with empty vector. Additional transfections were performed using FuGENE transfection reagent (Roche Molecular Biochemicals), adjusting the total amount of DNA plasmid to 0.5–1 μg/plate as directed by the manufacturer. Kinase Assays—48 h after transfection, cells were starved with serum free media for 2 h, stimulated if necessary, washed with cold phosphate-buffered saline, and lysed at 4 °C in a buffer containing 25 mm HEPES, pH 7.5, 0.3 m NaCl, 1.5 mMMgCl2, 0.2 mm EDTA, 0.5 mm dithiothreitol, 1% Triton X-100, 0.1% SDS, 20 mm β-glycerophosphate, 1 mm sodium vanadate, and 1 mm phenylmethylsulfonyl fluoride. AU5-JNK was precipitated from the cleared lysates by incubation with the specific antibody against AU5 (MMS-135R, Covance) for 1.5 h at 4 °C. Complexes were recovered with the aid of Gamma-Bind Sepharose beads (Santa Cruz) and washed one time with phosphate-buffered saline containing 1% Nonidet P-40 and 2 mm sodium vanadate, once with 100 mm Tris, pH 7.5, 0.5 m LiCl, and three times in kinase reaction buffer (12.5 mm MOPS, pH 7.5, 12.5 mm β-glycerophosphate, 7.5 mm MgCl2, 0.5 mm EGTA, 0.5 mm sodium fluoride, and 0.5 mm sodium vanadate). The JNK activity present in the inmunoprecipitates was determined by resuspension in 30 μl of kinase reaction buffer containing 10 μCi of [γ-32P]ATP per reaction and 20 μm unlabeled ATP, using 1 μg of purified bacterially expressed, GST-ATF2 protein as substrate. After 30 min at 30 °C, reactions were terminated by addition of 10 μl of 5× Laemmli buffer. Samples were heated at 95 °C for 5 min and analyzed by SDS-gel electrophoresis on 12% acrylamide gels. Autoradiography was performed with the aid of an intensifying screen. Parallel inmunoprecipitates were processed for Western blot analysis using the same antiserum as described previously (17Coso O.A. Chiariello M. Yu J.C. Termoto H. Crespo P. Xu N. Miki T. Gutkind J.S. Cell. 1995; 81: 1137-1146Abstract Full Text PDF PubMed Scopus (1561) Google Scholar). To assay p38 activity, cells were transfected with a plasmid that expresses HA-p38, which was inmunoprecipitated from the cell lysates with a specific antibody against the HA epitope (MMS-101R, Covance) and processed as described above for JNK (20Teramoto H. Coso O.A. Miyata H. Igishi T. Miki T. Gutkind J.S. J. Biol. Chem. 1996; 271: 27225-27228Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar). DNA Constructs—pCEFLAU5-JNK was constructed transferring the JNK cDNA insert from pCDNA3HA-JNK (17Coso O.A. Chiariello M. Yu J.C. Termoto H. Crespo P. Xu N. Miki T. Gutkind J.S. Cell. 1995; 81: 1137-1146Abstract Full Text PDF PubMed Scopus (1561) Google Scholar) as a BglII-NotI fragment to the corresponding expression vector. Plasmids pCDNAonc-Dbl, pCDNA3Rac1QL, and pCEFL HA-p38 have already been described (17Coso O.A. Chiariello M. Yu J.C. Termoto H. Crespo P. Xu N. Miki T. Gutkind J.S. Cell. 1995; 81: 1137-1146Abstract Full Text PDF PubMed Scopus (1561) Google Scholar, 20Teramoto H. Coso O.A. Miyata H. Igishi T. Miki T. Gutkind J.S. J. Biol. Chem. 1996; 271: 27225-27228Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar). pCMV myc-Epac and pCMVmyc-Δ-Epac were generated transferring SalI-NotI fragments from pMT2SMHA-Epac pCMVmyc-N-Epac was cloned using a SalI-BglII fragment, which contains the DEP, CBD, and REM domains (1–1270 nucleotides). pCGNHA-Rap1bN17, pCGNHA-Rap1bG12V, and pMT2SMHA-Rap1GAP have already been described (24Altschuler D.L. Lapetina E.G. J. Biol. Chem. 1993; 268: 7527-7531Abstract Full Text PDF PubMed Google Scholar, 25Reedquist K.A. Ross E. Koop E.A. Wolthuis R.M.F. Zwartkruis F.J.T. van Kooyk Y. Salmon M. Buckley C.D. Bos J.L. J. Cell Biol. 2000; 148: 1151-1158Crossref PubMed Scopus (363) Google Scholar). The Epac REM and DEP domains were isolated by PCR amplification using oligonucleotides 5′-acGGATCCacagtgatgtctggcacc-3′ and 5′-tgGAATTCtcactgctcgctgccacccgc-3′ for REM and 5′-acGGATCCatgacccgagaccggaagtacc-3′ and 5′-gaGAATTCgtagaattgggcatctcg-3′ for DEP. The PCR products were digested with BamHI and EcoRI enzymes and ligated in the corresponding sites in a pCEFL-GST vector. pCEFL GST-REM expresses amino acids 345–410 from Epac as a GST fusion protein. The same is true for amino acids 74–140 from Epac in pCEFL-GSTDEP. Western Blots—Lysates taken from transfected cells, containing similar amounts of protein, were analyzed for protein expression levels by SDS-PAGE followed by Western blotting, with the corresponding antibodies. Complexes were visualized by enhanced chemiluminescence detection using horseradish peroxidase-conjugated secondary antibodies (Santa Cruz) and Luminol as a substrate (Sigma) as described previously (17Coso O.A. Chiariello M. Yu J.C. Termoto H. Crespo P. Xu N. Miki T. Gutkind J.S. Cell. 1995; 81: 1137-1146Abstract Full Text PDF PubMed Scopus (1561) Google Scholar). Mouse monoclonal antibodies anti-HA and anti-AU5 epitopes were purchased from Covance. Rap Activation Assay Using RalGDS-RBD—Cells transfected with a plasmid expressing HA-Rap1b were lysed with a buffer containing 200 mm NaCl, 50 mm Tris-HCl, pH 7.5, 1% Nonidet P-40, 10% glycerol, 2 mm MgCl2, 1 mm phenylmethylsulfonyl fluoride, 2 μm leupeptin, and 2 μm aprotinin. Lysis was performed at 4 °C for 10–30 min. Lysates were clarified by centrifugation at maximal speed in an Eppendorf centrifuge for 10 min at 4 °C. 10 μg of bacterially expressed RalGDS-RBD (26Franke B. Akkerman J.W. Bos J.L. EMBO J. 1997; 16: 252-259Crossref PubMed Scopus (366) Google Scholar) coupled to glutathione-Sepharose beads (Amersham Biosciences) were added to the supernatants and incubated at 4 °C for 60min with slight agitation. Beads were washed four times in the same lysis buffer. After the final wash, Laemmli sample buffer was added to the samples. Proteins were fractionated in a 15% SDS-PAGE and transferred to a nitrocellulose membrane. HA-Rap1-GTP was visualized by Western blotting with an anti-HA antibody as described previously (17Coso O.A. Chiariello M. Yu J.C. Termoto H. Crespo P. Xu N. Miki T. Gutkind J.S. Cell. 1995; 81: 1137-1146Abstract Full Text PDF PubMed Scopus (1561) Google Scholar). Epac Is a JNK Activator—Expression of the Rap1-specific GEF Epac into HEK-293T cells triggered a strong JNK response, about a 6-fold increase in its kinase activity. This Epac-mediated effect was similar in magnitude to the one produced by canonical JNK activators as Onc-Dbl and the protein synthesis inhibitor anisomycin (Fig. 1, upper panel and bars). The lower panel of Fig. 1 shows that coexpression of the GEFs did not alter the JNK protein levels, which is indicative of changes in specific JNK activity rather than changes in the amount of enzyme present. As reported previously for MAPK activation (27Karim M. Hunter T. Curr. Biol. 1995; 5: 747-757Abstract Full Text Full Text PDF PubMed Scopus (662) Google Scholar), parallel experiments showed that a green fluorescence protein (GFP)-JNK fusion protein translocates to the nucleus upon contransfection with plasmids that express Epac or Onc-Dbl (data not shown). Opposite to Epac, a Constitutively Active Mutant of Rap1b Fails to Stimulate JNK Activity—We have previously found that expression of constitutively active forms of small G-proteins of the Rho subfamily were highly effective in triggering JNK activation to levels comparable with its corresponding GEFs, Ost and Dbl (17Coso O.A. Chiariello M. Yu J.C. Termoto H. Crespo P. Xu N. Miki T. Gutkind J.S. Cell. 1995; 81: 1137-1146Abstract Full Text PDF PubMed Scopus (1561) Google Scholar). Epac is a Rap-specific GEF that turns out to be a significant JNK activator (Fig. 1). If its effects on JNK activity are conveyed through Rap activation, we reasoned that expression of a constitutively active Rap form should fully mimic the action of Epac. We found that while Rac1QL and Epac display a 5-fold induction in JNK activation (Fig. 2A), expression of a GTPase-deficient form of Rap1b (RapG12V) did not produce any changes in JNK activity, despite high levels of expression as measured in Western blots (Fig. 2, A and B). The HA-RapG12V construct is expressed on its active form when transfected into HEK-293T cells as shown by its ability to bind to the Rap binding domain of RalGDS, which binds Rap1 on its active GTP-bound conformation (not shown). These results demonstrate that in contrast to Epac, Rap1b-G12V does not activate JNK, suggesting that the enzymatic activity described for Epac (GTP loading unto Rap) is not sufficient to transduce the effects of Epac on JNK activity. Rap Activity Is Not Necessary for Epac-mediated JNK Activation—Results from the two preceding sections suggest two potential alternatives for Epac signaling. One of them would be mediated by Rap activation, as has been previously demonstrated, while the other one would be Rap-independent. The first scenario should be mimicked by expression of RapG12V; meanwhile, the second one should not be altered by the presence of Rap inhibitory molecules. We observed no JNK activation by RapG12V; therefore, to assess the second possibility, endogenous Rap1 was inactivated, and the effects of Epac on JNK were tested. Rap1 inactivation was attained by expression of two inhibitory molecules: dominant negative RapN17 and RapGAP, which inactivate Rap by different mechanisms. As shown in Fig. 3A, we found that RapGAP that inactivates active Rap-GTP by driving the equilibrium toward its inactive Rap-GDP form (25Reedquist K.A. Ross E. Koop E.A. Wolthuis R.M.F. Zwartkruis F.J.T. van Kooyk Y. Salmon M. Buckley C.D. Bos J.L. J. Cell Biol. 2000; 148: 1151-1158Crossref PubMed Scopus (363) Google Scholar) did not modify Epac-mediated JNK activity despite these proteins being expressed at high levels (Fig. 3B) and able to inhibit Epac-mediated Rap1 activation (see below). Like the so-called dominant negative RasN17 mutant (28Powers S. O'Neill K. Wigler M. Mol. Cell. Biol. 1989; 9: 390-395Crossref PubMed Scopus (131) Google Scholar), RapN17 inhibits activation of endogenous Rap by binding and sequestering GEFs, upstream activators of Rap1. As expected, expression of RapN17 partially inhibited Epac activation of JNK activity (Fig. 3A). This seemingly contradictory result could be explained assuming that RapN17 is actually an inhibitor of the upstream GEF regulator Epac, and not of the small G-protein itself, acting presumably through titration of "free" Epac molecules, thereby preventing Epac actions including JNK activation. Since RapG12V does not mimic the action of Epac on JNK, and expression of a downstream negative regulator of Rap1 (RapGAP) has no effect on Epac-stimulated JNK activity, we conclude that Rap1 activity should not be necessary for Epac-mediated JNK activation. Stimulation of JNK by Epac Is Independent of Its cAMP-dependent GEF Activity—The general organization of Epac is schematized in Fig. 4A. The COOH-terminal domain contains the catalytic exchange core (GEF), responsible for the GDP/GTP exchange on Rap. The NH2-terminal regulatory domain negatively modulates this catalytic activity. Binding of cAMP to the CBD site in this NH2-terminal domain relieves the negative constrain, leading to GEF-dependent Rap activation (1Rooij J. Zwartkruis F.J.T. Verheijen M.H.G. Cool R.H. Nijman S.M.B. Wittinghofer A. Bos J.L. Nature. 1998; 396: 474-477Crossref PubMed Scopus (1606) Google Scholar). Accordingly, deletion of the NH2-terminal domain, including the CBD, renders a constitutively active GEF mutant (Δ-Epac), which maximally activates Rap1 even in the absence of agonist stimulation (cAMP) (1Rooij J. Zwartkruis F.J.T. Verheijen M.H.G. Cool R.H. Nijman S.M.B. Wittinghofer A. Bos J.L. Nature. 1998; 396: 474-477Crossref PubMed Scopus (1606) Google Scholar). We reasoned that if Rap exchange activity is involved in JNK signaling, maximum JNK activation should be observed when Δ-Epac dose responses are compared with full-length Epacs, working in the absence of cAMP stimulation. In other words, when measuring JNK activity the dose-response curve to Epac should present a higher EC50 relative to that of Δ-Epac. In addition, cAMP-signaling agents should potentiate Epac′, but not Δ-Epac′, dose-response curves. As shown in Fig. 4B, Epac and Δ-Epac stimulation of JNK activity showed identical profiles in the absence of cAMP stimulation. A 15-min treatment with forskolin, a cAMP-elevating agent, did not increase JNK activity induced by either Epac versions (not shown). We verified that Δ-Epac is functional as a Rap1GEF, as shown by GST-RalGDS-RBD pull-down assays (Fig. 4C). Moreover, coexpression of RapGAP reduced the amount of pulled HA-Rap to values below basal, thus confirming that the observed Δ-Epac-induced increase in HA-Rap represents active GTP-loaded Rap. In addition, RapGAP coexpression did not affect JNK activation by Δ-Epac (data not shown). We conclude that Epac stimulation of JNK is independent of cAMP-mediated Epac exchange activity on Rap1b. Epac-mediated JNK Activation Is Dissociated from the GEF Activity of Epac—Although the results described above strongly suggest that the nucleotide exchange on Rap1 Epac is not involved in Epac-mediated JNK activation, we directly tested this premise experimentally. We asked whether the presence of the GEF domain was necessary for Epac-mediated JNK activation. A construct containing the NH2-terminal regulatory domain (N-Epac), but devoid of the catalytic GEF domain, was sufficient to activate JNK at levels comparable with both Epac and Δ-Epac (Fig. 5A, compare with Figs. 3A and 4B). As expected, Δ-Epac stimulated the loading of GTP onto Rap1, while N-Epac did not (Fig. 5B). Most importantly, while the JNK stimulating activity of full-length Epac was partially sensitive to RapN17 (Fig. 3A), the JNK response to N-Epac was completely resistant to both Rap inhibitory molecules RapN17 and RapGAP (Fig. 5A). These results demonstrate unequivocally that Epac activation of JNK is independent of its guanine nucleotide exchange function. Finding a New Effector Domain in Epac; a Role for the REM Domain in JNK Activation—What is the minimal domain of Epac responsible for downstream signaling into JNK? The three Epac versions used in this study (Epac, N-Epac, and Δ-Epac) activate JNK comparably with known JNK activators such as Dbl or anisomycin. That N-Epac fully mimics the effect of full-length Epac on JNK suggests that the JNK activation domain of Epac resides outside the GEF domain in a region common to the three mutants. That Δ-Epac is as a potent JNK activator as N-Epac suggests that the minimal Epac domain responsible for downstream signaling into JNK is a region encompassing the REM domain (amino acids 345–410), which is common to these three constructs (Fig. 4A). To assess this possibility we decided to isolate the REM domain and challenge its ability to activate JNK; another Epac region that contains the DEP domain was also isolated as a control, and both were expressed as GST fusion proteins. Fig. 6 shows that while being expressed to the same levels (Fig. 6A), GST-DEP did not induce changes in JNK activity but GST-REM exerted an effect comparable with N-Epac (Fig. 6B). We understand that the REM domain of Epac is responsible for this JNK activatio
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The aim of the present study was to investigate possible mechanisms that could be involved in the deactivation of the assembled, catalytically active NADPH oxidase of phagocytic cells and thereby lead to termination of O(2)(.-) production. Our major findings are the following: (1) Addition of GDP to the active oxidase is able to reduce O(2)(.-) production both in the fully purified and in a semi-recombinant cell-free activation system. (2) p67(phox) inhibits GTP hydrolysis on Rac whereas p47(phox) has no effect on Rac GTPase activity. (3) Soluble regulatory proteins (GTPase activating protein, guanine nucleotide dissociation inhibitor, and the Rac-binding domain of the target protein p21-activated kinase) inhibit activation of the NADPH oxidase but have no effect on electron transfer via the assembled enzyme complex. (4) Membrane-associated GTPase activating proteins (GAPs) have access also to the assembled, catalytically active oxidase. Taken together, we propose that the GTP-bound active form of Rac is required for sustained enzyme activity and that membrane-localized GAPs have a role in the deactivation of NADPH oxidase.
Small GTPase
Rac GTP-Binding Proteins
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