GAP1 Family Members Constitute Bifunctional Ras and Rap GTPase-activating Proteins
2006; Elsevier BV; Volume: 281; Issue: 15 Linguagem: Inglês
10.1074/jbc.m512802200
ISSN1083-351X
AutoresSabine Kupzig, Delia Deaconescu, Dalila Bouyoucef, Simon Walker, Qing Liu, Christian L. Polte, Oliver Daumke, Toshimasa Ishizaki, Peter J. Lockyer, Alfred Wittinghofer, Peter J. Cullen,
Tópico(s)Ion channel regulation and function
ResumoGAP1IP4BP is a member of the GAP1 family of Ras GTPase-activating proteins (Ras GAPs) that includes GAP1m, CAPRI, and RASAL. Composed of a central Ras GAP domain, surrounded by amino-terminal C2 domains and a carboxyl-terminal pleckstrin homology/Bruton's tyrosine kinase domain, GAP1IP4BP has previously been shown to possess an unexpected GAP activity on the Ras-related protein Rap, besides the predicted Ras GAP activity (Cullen, P. J., Hsuan, J. J., Truong, O., Letcher, A. J., Jackson, T. R., Dawson, A. P., and Irvine, R. F. (1995) Nature 376, 527–530). Here we have shown that GAP1IP4BP is indeed an efficient Ras/Rap GAP, having Kms of 213 and 42 μm and estimated kcats of 48 and 16 s–1 for Ras and Rap, respectively. For this dual activity, regions outside the Ras GAP domain are required, as the isolated domain (residues 291–569) retains a pronounced Ras GAP activity yet has very low activity toward Rap. Interestingly, mutagenesis of the Ras GAP arginine finger, and surrounding residues important in Ras binding, inhibit both Ras and Rap GAP activity of GAP1IP4BP. Although the precise details by which GAP1IP4BP can function as a Rap GAP remain to be determined, these data are consistent with Rap associating with GAP1IP4BP through the Ras-binding site within the Ras GAP domain. Finally, we have established that such dual Ras/Rap GAP activity is not restricted to GAP1IP4BP. Although GAP1m appears to constitute a specific Ras GAP, CAPRI and RASAL display dual activity. For CAPRI, its Rap GAP activity is modulated upon its Ca2+-induced association with the plasma membrane. GAP1IP4BP is a member of the GAP1 family of Ras GTPase-activating proteins (Ras GAPs) that includes GAP1m, CAPRI, and RASAL. Composed of a central Ras GAP domain, surrounded by amino-terminal C2 domains and a carboxyl-terminal pleckstrin homology/Bruton's tyrosine kinase domain, GAP1IP4BP has previously been shown to possess an unexpected GAP activity on the Ras-related protein Rap, besides the predicted Ras GAP activity (Cullen, P. J., Hsuan, J. J., Truong, O., Letcher, A. J., Jackson, T. R., Dawson, A. P., and Irvine, R. F. (1995) Nature 376, 527–530). Here we have shown that GAP1IP4BP is indeed an efficient Ras/Rap GAP, having Kms of 213 and 42 μm and estimated kcats of 48 and 16 s–1 for Ras and Rap, respectively. For this dual activity, regions outside the Ras GAP domain are required, as the isolated domain (residues 291–569) retains a pronounced Ras GAP activity yet has very low activity toward Rap. Interestingly, mutagenesis of the Ras GAP arginine finger, and surrounding residues important in Ras binding, inhibit both Ras and Rap GAP activity of GAP1IP4BP. Although the precise details by which GAP1IP4BP can function as a Rap GAP remain to be determined, these data are consistent with Rap associating with GAP1IP4BP through the Ras-binding site within the Ras GAP domain. Finally, we have established that such dual Ras/Rap GAP activity is not restricted to GAP1IP4BP. Although GAP1m appears to constitute a specific Ras GAP, CAPRI and RASAL display dual activity. For CAPRI, its Rap GAP activity is modulated upon its Ca2+-induced association with the plasma membrane. The Ras-like family of small GTPases includes, in addition to the "classic" Ras proteins H-Ras, N-Ras, and K-Ras4A and 4B, the Rap proteins Rap1A, 1B, 2A, and 2B (1Shields J.M. Pruitt K. McFall A. Shaub A. Der C.J. Trends Cell Biol. 2000; 10: 147-153Abstract Full Text Full Text PDF PubMed Scopus (690) Google Scholar, 2Hancock J.F. Nat. Rev. Mol. Cell Biol. 2003; 4: 373-384Crossref PubMed Scopus (698) Google Scholar, 3Bos J.L. Curr. Opin. Cell Biol. 2005; 17: 123-128Crossref PubMed Scopus (397) Google Scholar). These ubiquitously expressed, evolutionarily conserved proteins couple extracellular signals to various cellular responses through an ability to undergo conformational changes in response to the alternate binding of GDP and GTP. The GDP-bound "off" state and the GTP-bound "on" state recognize distinct effector proteins, thereby allowing these proteins to function as binary molecular switches (1Shields J.M. Pruitt K. McFall A. Shaub A. Der C.J. Trends Cell Biol. 2000; 10: 147-153Abstract Full Text Full Text PDF PubMed Scopus (690) Google Scholar, 2Hancock J.F. Nat. Rev. Mol. Cell Biol. 2003; 4: 373-384Crossref PubMed Scopus (698) Google Scholar, 3Bos J.L. Curr. Opin. Cell Biol. 2005; 17: 123-128Crossref PubMed Scopus (397) Google Scholar). Although Ras is the best known and best studied member of the family, Rap1A and 1B have recently attracted considerable attention (3Bos J.L. Curr. Opin. Cell Biol. 2005; 17: 123-128Crossref PubMed Scopus (397) Google Scholar, 4Stork P.J.S. Trends Biochem. Sci. 2003; 28: 267-275Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 5Caron E. J. Cell Sci. 2003; 116: 435-440Crossref PubMed Scopus (170) Google Scholar, 6Hattori M. Minato N. J. Biochem. 2003; 134: 479-484Crossref PubMed Scopus (155) Google Scholar). Rap1 was identified as a protein that could suppress the transformed phenotype of fibroblasts oncogenically transformed by one of the mutated Ras genes, K-ras (7Kitayama H. Sugimoto Y. Matsuzaki T. Ikawa Y. Noda M. Cell. 1989; 56: 77-84Abstract Full Text PDF PubMed Scopus (763) Google Scholar). This coupled with Rap1 having an effector domain virtually identical to that of Ras led to a model in which Rap1 was considered to function as an antagonist of Ras by trapping Ras effectors in an inactive complex (4Stork P.J.S. Trends Biochem. Sci. 2003; 28: 267-275Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). However, recent analysis has suggested that Rap1 may possess more complex biological functions. Active Rap1 has been implicated in several cellular processes, including superoxide formation, cyclic adenosine monophosphate (cAMP)-induced neurite outgrowth, cell proliferation, integrin-mediated cell adhesion, and secretion (3Bos J.L. Curr. Opin. Cell Biol. 2005; 17: 123-128Crossref PubMed Scopus (397) Google Scholar, 4Stork P.J.S. Trends Biochem. Sci. 2003; 28: 267-275Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 5Caron E. J. Cell Sci. 2003; 116: 435-440Crossref PubMed Scopus (170) Google Scholar, 6Hattori M. Minato N. J. Biochem. 2003; 134: 479-484Crossref PubMed Scopus (155) Google Scholar). As with other small GTPases, the extent and duration of Ras and Rap1 signaling is controlled by the interplay between guanine-nucleotide exchange factors (GEFs), 3The abbreviations used are: GEF, guanine-nucleotide exchange factor; GST, glutathione S-transferase; CHO, Chinese hamster ovary; DTE, dithioerythritol; HA, hemagglutinin; EGF, epidermal growth factor. which induce activation by stimulating the exchange of GDP for GTP, and GTPase-activating proteins (GAPs), which modulate inactivation by enhancing the intrinsic GTPase activity. A wide range of GEFs and GAPs for these GTPases have been identified, and in a few instances the mechanisms by which signals originating from activated cell surface receptors converge on these proteins have been mapped. However, for most Ras-like GEFs and GAPs we have a poor understanding of their regulation (8Donovan S. Shannon K.M. Bollag G. Biochim. Biophys. Acta. 2002; 1602: 23-45PubMed Google Scholar, 9Bernards A. Biochim. Biophys. Acta. 2003; 1603: 47-82PubMed Google Scholar, 10Bernards A. Settleman J. Trends Cell Biol. 2004; 14: 377-385Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). In mammalian cells, Ras-specific GAPs include p120GAP, NF1, GAP1 proteins, and the SynGAPs (DAB2IP, nGAP, and Syn GAP). Rap1-specific GAPs comprise Rap GAPs I and II, the SPA-1 family (SPA-1, SPAR, SPAL, E6TP1), tuberin, and DOCK4 (Ref. 11Yajnik V. Paulding C. Sordella R. McClatchey A.I. Saito M. Wahrer D.C.R. Reynolds P. Bell D.W. Lake R. van den Heuvel S. Settleman J. Haber D.A. Cell. 2003; 112: 673-684Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, reviewed in Ref. 8Donovan S. Shannon K.M. Bollag G. Biochim. Biophys. Acta. 2002; 1602: 23-45PubMed Google Scholar). Although Rap1 is a close homologue of Ras it does not possess the catalytic glutamine residue that is critical for GTP hydrolysis in all other Ras-like GTPases. The mechanism by which Ras and Rap1 GAPs enhance the GTPase activity of their respective GTPase is therefore distinct. For Ras GAPs a catalytic arginine residue, the arginine finger, is supplied by the GAP molecule into the active site of Ras, thereby stabilizing the transition state of the GTPase reaction and increasing the reaction rate by >1,000-fold (12Scheffzek K. Lautwein A. Kabsch W. Ahmadian M.R. Wittinghofer A. Nature. 1996; 384: 591-596Crossref PubMed Scopus (144) Google Scholar, 13Scheffzek K. Ahmadian M.R. Kabsch W. Wiesmuller L. Lautwein A. Schmitz F. Wittinghofer A. Science. 1997; 277: 333-338Crossref PubMed Scopus (1208) Google Scholar, 14Ahmadian M.R. Stege P. Scheffzek K. Wittinghofer A. Nat. Struct. Biol. 1997; 4: 686-689Crossref PubMed Scopus (296) Google Scholar). Rap1 GAPs are not related to other GAPs and do not employ a catalytic arginine residue (15Brinkmann T. Daumke O. Herbrand U. Kuhlmann D. Stege P. Ahmadian M.R. Wittinghofer A. J. Biol. Chem. 2002; 277: 12525-12531Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 16Chakrabarti P.P. Suveyzdis Y. Wittinghofer A. Gerwert K. J. Biol. Chem. 2004; 279: 46226-46233Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar); instead, Rap1 GAPs provide a catalytic asparagine, the asparagine thumb, to stimulate GTP hydrolysis (17Daumke O. Weyand M. Chakrabarti P.P. Vetter I.R. Wittinghofer A. Nature. 2004; 429: 197-201Crossref PubMed Scopus (116) Google Scholar). It has been proposed that the carboxamide side chain of the asparagine residue has a function similar to the glutamine residue in Ras-like GTPases in stabilizing the relative position of the nucleophilic water and γ-phosphate in the transition complex (17Daumke O. Weyand M. Chakrabarti P.P. Vetter I.R. Wittinghofer A. Nature. 2004; 429: 197-201Crossref PubMed Scopus (116) Google Scholar). The GAP1 family, which comprises GAP1m, GAP1IP4BP, CAPRI, and RASAL (18Maekawa M. Iwamatsu S.A. Morishita T. Yokota K. Imai Y. Kohsaka S. Nakamura S. Hattori S. Mol. Cell. Biol. 1994; 14: 6879-6885Crossref PubMed Scopus (119) Google Scholar, 19Cullen P.J. Hsuan J.J. Truong O. Letcher A.J. Jackson T.R. Dawson A.P. Irvine R.F. Nature. 1995; 376: 527-530Crossref PubMed Scopus (286) Google Scholar, 20Allen M. Chu S. Brill S. Stotler C. Buckler A. Gene. 1998; 218: 17-25Crossref PubMed Scopus (28) Google Scholar, 21Lockyer P.J. Wennstrom S. Kupzig S. Venkateswarlu K. Downward J. Cullen P.J. Curr. Biol. 1999; 9: 265-268Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 22Lockyer P.J. Kupzig S. Cullen P.J. Curr. Biol. 2001; 11: 981-986Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar), is characterized by a conserved domain structure comprising amino-terminal tandem C2 domains, a highly conserved central Ras GAP domain, and a carboxyl-terminal pleckstrin homology domain that is associated with a Bruton's tyrosine kinase motif (23Cullen P.J. Biochim. Biophys. Acta. 1998; 1436: 35-47Crossref PubMed Scopus (36) Google Scholar). Consistent with the presence of the Ras GAP domain all of these proteins have been shown to function as Ras GAP, although each is differentially regulated following receptor stimulation (19Cullen P.J. Hsuan J.J. Truong O. Letcher A.J. Jackson T.R. Dawson A.P. Irvine R.F. Nature. 1995; 376: 527-530Crossref PubMed Scopus (286) Google Scholar, 21Lockyer P.J. Wennstrom S. Kupzig S. Venkateswarlu K. Downward J. Cullen P.J. Curr. Biol. 1999; 9: 265-268Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 22Lockyer P.J. Kupzig S. Cullen P.J. Curr. Biol. 2001; 11: 981-986Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 24Walker S.A. Kupzig S. Bouyoucef D. Davies L.C. Tsuboi T. Bivona T. Cozier G.E. Lockyer P.J. Buckler A. Rutter G.A. Allen M.J. Philips M.R. Cullen P.J. EMBO J. 2004; 23: 1749-1760Crossref PubMed Scopus (75) Google Scholar, 25Liu Q. Walker S.A. Gao D. Taylor J.A. Dai Y-F. Arkell R.S. Bootman M.D. Roderick H.L. Cullen P.J. Lockyer P.J. J. Cell Biol. 2005; 170: 183-190Crossref PubMed Scopus (59) Google Scholar). Surprisingly, however, given that these proteins contain no detectable sequence homology with any known Rap GAP, GAP1IP4BP is also capable of enhancing the GTPase activity of Rap (19Cullen P.J. Hsuan J.J. Truong O. Letcher A.J. Jackson T.R. Dawson A.P. Irvine R.F. Nature. 1995; 376: 527-530Crossref PubMed Scopus (286) Google Scholar). This protein therefore has dual Ras and Rap GAP activity. In the current study we describe a kinetic characterization of this dual activity and have begun the process of dissecting out the mechanistic details of these activities. In addition, we address whether dual Ras/Rap GAP activity is restricted solely to GAP1IP4BP or is a function conserved within other members of the GAP1 family. Purification of GTP-binding Proteins—C-terminal-truncated Rap1B (Rap1B C′, residue 1–167) was purified from Escherichia coli strain CK600K using the ptac-expression system as described for Ras by Tucker et al. (26Tucker J. Sczakiel G. Feuerstein J. John J. Goody R.S. Wittinghofer A. EMBO J. 1986; 5: 1351-1358Crossref PubMed Scopus (226) Google Scholar). After lysis in 32 mm Tris (pH 7.5), 100 μm phenylmethylsulfonyl fluoride, 2 mm EDTA, the supernatant was applied to a Q-Sepharose column equilibrated with 32 mm Tris (pH 7.5), 10 mm MgCl2, 5 mm DTE. After washing with the same buffer, proteins were eluted using a salt gradient (0–300 mm NaCl) in 32 mm Tris (pH 7.5), 10 mm MgCl2, 5 mm DTE. Fractions containing Rap1B C′ were precipitated with ammonium sulfate (3 m final concentration). The pellet was resuspended in 64 mm Tris (pH 7.5), 10 mm MgCl2, 5 mm DTE, 200 μm GDP, 0.4 m NaCl and further purified on a Sephadex 75 gel filtration column. Fractions containing Rap1B C′ were pooled and concentrated using an Amicon concentrator (10-kDa cut-off). Truncated H-Ras-(1–166) was purified as described before (26Tucker J. Sczakiel G. Feuerstein J. John J. Goody R.S. Wittinghofer A. EMBO J. 1986; 5: 1351-1358Crossref PubMed Scopus (226) Google Scholar, 27John J. Frech M. Wittinghofer A. J. Biol. Chem. 1988; 263: 11792-11799Abstract Full Text PDF PubMed Google Scholar). Purification of GAP1IP4BP—For stability reasons, full-length wild-type GAP1IP4BP was purified as GST fusion protein GST-GAP1IP4BP, while the mutant full-length GST-GAP1IP4BP-(R371A) and the GAP domain construct GST-GAP1IP4BP-(291–569) was cleaved from GST. Freshly transformed E. coli Rosetta strain was grown in 5 liters of TB medium containing 50 μg/ml ampicillin and 50 μg/ml chloramphenicol at 37 °C. At A600 ∼0.8, expression was induced by addition of 100 μm isopropyl 1-thio-β-d-galactopyranoside overnight at 18 °C. After lysis by micro-fluidizer (Microfluidics Corp.) in 50 mm Hepes (pH 7.5), 5 mm DTE, 50 mm NaCl, 2.5 mm EDTA, 100 μm phenylmethylsulfonyl fluoride, the extract was applied to glutathione-Sepharose 4-B (Amersham Biosciences) in 50 mm Hepes (pH 7.5), 5 mm DTE, 50 mm NaCl. Following extensive washes, the protein was eluted with buffer containing 30 mm glutathione (pH 7.5). Protein was concentrated using an Amicon concentrator (30-kDa cut-off), and aliquots were snap frozen in liquid nitrogen and stored at –80 °C. Cleavage of GAP1IP4BP-(R371A) and GAP1IP4BP-(291–569) from GST was performed on the glutathione-Sepharose column with 300–600 units of thrombin (Serva) overnight at 4 °C under continuous circulation (0.5 ml/min). Proteins were eluted with 50 mm Hepes (pH 7.5), 5 mm DTE, 50 mm NaCl. GAP1IP4BP-(291–569) was further purified on a Superdex 200 gel filtration column in 50 mm Hepes (pH 7.5), 5 mm DTE, 50 mm NaCl. Proteins were concentrated using an Amicon concentrator (15 and 30-kDa cut-off), and aliquots were snap frozen in liquid nitrogen and stored at –80 °C. Full-length constructs cannot be fully purified and contain additional lower molecular mass bands, most probably derived from proteolytic degradation. Nucleotide Exchange—200 μm Rap or Ras in 25 mm Tris (pH 7.5), 100 mm NaCl, 5 mm DTE were incubated with 15 mm EDTA, 100 mm ammonium sulfate, and 10 mm GTP (stock 100 mm nucleotide in 1 m Tris, pH 7.5) for 60 min at room temperature. Exchange was terminated by the addition of 25 mm MgCl2. The separation of unbound nucleotides and EDTA from Rap-GTP and Ras-GTP was carried out by washing the proteins several times with buffer using an Amicon concentrator (10-kDa cut-off) at 4 °C. Nucleotide exchange was controlled by reversed-phase high performance liquid chromatography. Radioactive [γ-32P]GTP-bound Rap1 and Ras were prepared by incubating 1.5 mm GTP-bound protein with 20 μCi of [γ-32P]GTP (800 Ci/mmol; Amersham Biosciences) in the presence of 12 mm EDTA for 30 min on ice. The exchange reaction was stopped by the addition of 25 mm MgCl2. GAP Assay—The GAP-stimulated GTP hydrolysis of Rap1 and H-Ras was assayed by measuring Pi release using the charcoal method. Briefly, increasing amounts of radioactively labeled Rap-GTP and Ras-GTP were added to fixed concentrations of GAP1IP4BP proteins at 25 °C in standard buffer (25 mm Tris (pH 7.5), 5 mm MgCl2, 5 mm DTE, 50 mm NaCl) as described before (15Brinkmann T. Daumke O. Herbrand U. Kuhlmann D. Stege P. Ahmadian M.R. Wittinghofer A. J. Biol. Chem. 2002; 277: 12525-12531Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). To determine end points of GTP hydrolysis, all Rap-GTP and Ras-GTP were hydrolyzed by addition of a highly concentrated GAP1IP4BP solution, and a last aliquot was taken. Initial GTP hydrolysis rates were evaluated by linear regression, and Km and kcat were determined by Michaelis-Menten equation using the program Grafit (Erithacus software). GAP Assays of GAP1IP4BP Site-directed Mutants—Full-length GAP1IP4BP mutants L484A, R485Q, K517E, and K534A were isolated as GST fusion proteins, and analysis of the GAP activities of these mutants was performed under first order kinetics as described by Bottomley et al. (28Bottomley J.R. Reynolds J.S. Lockyer P.J. Cullen P.J. Biochem. Biophys. Res. Commun. 1998; 250: 143-149Crossref PubMed Scopus (26) Google Scholar). The particular GTP-binding protein was loaded with [γ-32P]GTP (3000 Ci mmol–1; Amersham Biosciences) for 5 min at 25 °C. GTPase activity was assayed at 25 °C by addition of the various GAP proteins to the loaded GTP-binding protein. After 10 min, activity was stopped by addition of 5 mm silicotungstate, 1 mm H2SO4. The liberated [32P]Pi was extracted with isobutanol/toluene (1/1 v/v), 5% (w/v) ammonium molybdate, 2 m H2SO4, and the upper phase was removed for scintillation counting. Ras Pulldown Assays—Glutathione S-transferase fusion of the Ras-GTP-binding domain from Raf-1 (GST-RBD) was purified from BL21(DE3) E. coli cells harboring the plasmid pGEX KG containing the Raf Ras-binding domain (amino acids 1–149). After induction of a bacterial culture (A600 between 0.4 and 0.6) for 3 h at 37°C with 1 mm isopropyl-1-thio-β-d-galactopyranoside, cells were lysed by sonication in phosphate-buffered saline containing 1 mm EDTA, 1% Triton X-100, 10 μg/ml of aprotinin, and 10 μg/ml of leupeptin. The lysate was clarified by centrifugation, and the resultant supernatant was stored in aliquots at –80 °C. On the required day, aliquots were thawed prior to incubation with glutathione-Sepharose (Amersham Biosciences) for 1 h at room temperature. The Sepharose beads were washed twice with phosphate-buffered saline, 1 mm EDTA, 1% (v/v) Triton X-100 before finally being suspended as a 1:1 slurry. This was used immediately in pulldown assays. Here, dishes (100 mm) of CHO-T cells (8 × 105 cells) were transiently transfected by lipofection (GeneJuice; Novagen) with 2.5 μg of H-Ras cDNA and 1 μg of vector control or vector encoding the particular GAP1 protein. The cells were serum starved for 2 h at 37 °Cin serum-free F-12 (HAM; Invitrogen) prior to the experimental procedures. On completion of the manipulation, cells were lysed in 1 ml of ice-cold extraction buffer (50 mm Hepes, pH 7.5, 100 mm NaCl, 1 mm EGTA, 5 μg/ml of benzamidine, 5 μg/ml of aprotinin, 5 μg/ml of leupeptin, 5 μg/ml of pepstatin A, 5 μg/ml of trypsin inhibitor, 0.5 mm phenylmethylsulfonyl fluoride, and 1 mm dithiothreitol containing 1% Triton X-100 and 10 mm MgCl2). Nuclear-free supernatants were incubated with GST-RBD on glutathione-Sepharose beads at 4 °C for 30 min. The beads were then collected by centrifugation and washed three times with ice-cold phosphate-buffered saline, 0.1% Triton X-100, and 10 mm MgCl2. Ras proteins were separated by SDS-PAGE and visualized by immunoblotting on nitrocellulose filters using pan-Ras antibodies (F132; Santa Cruz Biotechnology) and enhanced chemiluminescence (Amersham Biosciences). Blots were analyzed by volume integration using ImageQuant software (Amersham Biosciences) as previously described (22Lockyer P.J. Kupzig S. Cullen P.J. Curr. Biol. 2001; 11: 981-986Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 24Walker S.A. Kupzig S. Bouyoucef D. Davies L.C. Tsuboi T. Bivona T. Cozier G.E. Lockyer P.J. Buckler A. Rutter G.A. Allen M.J. Philips M.R. Cullen P.J. EMBO J. 2004; 23: 1749-1760Crossref PubMed Scopus (75) Google Scholar). Rap1 Pulldown Assays—For the analysis of active Rap, a glutathione S-transferase fusion of the Rap-GTP-binding domain from RalGDS (GST-RalGDS) was used (29Franke B. Akkerman J.W. Bos J.L. . EMBO J. 1997; 16: 252-259Crossref PubMed Scopus (367) Google Scholar). Dishes (100 mm) of CHO-T cells (8 × 105 cells) were transiently co-transfected by lipofection (GeneJuice; Novagen) with 2.5 μg of HA-tagged Rap1A cDNA and 1 μg of vector control or vector encoding the particular GAP1 protein. The cells were serum starved for 2 h at 37 °C in serum-free F-12 (HAM) medium 24 h post-transfection prior to stimulation. Cell lysis and Rap1A-GTP pulldown were carried out as described above. Immobilized Rap1A was detected using the HA probe (Y-11; Santa Cruz Biotechnology). Blots were analyzed by volume integration using ImageQuant software (Amersham Biosciences) (22Lockyer P.J. Kupzig S. Cullen P.J. Curr. Biol. 2001; 11: 981-986Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 24Walker S.A. Kupzig S. Bouyoucef D. Davies L.C. Tsuboi T. Bivona T. Cozier G.E. Lockyer P.J. Buckler A. Rutter G.A. Allen M.J. Philips M.R. Cullen P.J. EMBO J. 2004; 23: 1749-1760Crossref PubMed Scopus (75) Google Scholar). Efficient Ras and Rap GAP Activity Requires Structural Elements beyond the Ras GAP Domain of GAP1IP4BP—To extend the published characterization of the Ras and Rap GAP activity of GAP1IP4BP (19Cullen P.J. Hsuan J.J. Truong O. Letcher A.J. Jackson T.R. Dawson A.P. Irvine R.F. Nature. 1995; 376: 527-530Crossref PubMed Scopus (286) Google Scholar, 28Bottomley J.R. Reynolds J.S. Lockyer P.J. Cullen P.J. Biochem. Biophys. Res. Commun. 1998; 250: 143-149Crossref PubMed Scopus (26) Google Scholar), we initially performed a detailed kinetic analysis (Fig. 1). Here, the GAP activity was monitored by following the production of Pi using the charcoal method (15Brinkmann T. Daumke O. Herbrand U. Kuhlmann D. Stege P. Ahmadian M.R. Wittinghofer A. J. Biol. Chem. 2002; 277: 12525-12531Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). The isolated GAP domain of GAP1IP4BP, residue 291–569, corresponding to the GAP constructs GAP334 and NF1–333 from p120GAP and neurofibromin, respectively, has pronounced Ras GAP activity that, in contrast to those other Ras GAPs, cannot be saturated under the conditions used (Fig. 1, A and B). Using 100 nm GAP1IP4BP-(291–569) and 800 μm Ras-GTP, the kcat of the reaction reaches a value of 8 s–1, which is in the range observed for other Ras GAPs (30Ahmadian M.R. Hoffmann U. Goody R.S. Wittinghofer A. Biochemistry. 1997; 36: 4535-4541Crossref PubMed Scopus (108) Google Scholar). Under these conditions, no stimulation of the Rap GTPase was observed. In the presence of 20 μm GAP domain, however, Rap GAP activity was detected (Fig. 1, C and D) but reached a kcat of only 0.02 s–1 with 800 μm Rap-GTP, indicating that the apparent second order rate constant kcat/Km is at least 2 orders of magnitude lower for the Rap versus the Ras GTPase activation. Inefficient hydrolysis is at least partially due to the absence of a glutamine residue in Rap, because with the Rap-(T61Q) mutant, kcat reached 0.5 s–1, which is only 10-fold lower than that observed with Ras (Fig. 1, A and B).FIGURE 1Michaelis-Menten kinetics of the GAP domain of GAP1IP4BP. A, 100 nm GAP1IP4BP-(291–569) were incubated in standard buffer at 25 °C with increasing concentrations of H-Ras[γ-32P]GTP (•) and Rap1A[γ-32P]GTP (▪). The insert shows the reaction with mutant Rap-(T61Q) (▴) in comparison with wild-type Rap (▪). B, the data from panel A are plotted as kcat in panel B. C and D, 20 μm GAP1IP4BP-(291–569) were used to assay GTP hydrolysis with increasing concentrations of Rap1A[γ-32P]GTP. GTPase activity was monitored by measuring Pi release, and data were evaluated as described under "Materials and Methods." Data are plotted as rates (C) or rate constants (D).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Because robust Rap GAP activity has been observed before (19Cullen P.J. Hsuan J.J. Truong O. Letcher A.J. Jackson T.R. Dawson A.P. Irvine R.F. Nature. 1995; 376: 527-530Crossref PubMed Scopus (286) Google Scholar) and Ras GAP activity is apparently similar to values observed for other Ras GAPs, it became obvious that GAP1IP4BP requires additional domains for efficient Rap GAP activity. Indeed, when we used full-length GST-GAP1IP4BP enriched via GSH-Sepharose, we observed efficient Ras and Rap GAP activity (Fig. 2). Using 5 nm GAP1IP4BP we observed similar maximal rates of 0.24 μmol/s for Ras and only a 3-fold lower rate of 0.08 μmol/s for Rap (Fig. 2A). Although these rates cannot be converted to kcat values due to the degradation products present in the preparation of the full-length protein, kcat was estimated to be 48 s–1 for Ras and 16 s–1 for Rap, indicating very efficient catalysis for both GTPase reactions. Furthermore, full-length protein had a 5-fold lower Km for Rap (42 μm) versus Ras (213 μm). Thus the apparent second order rate constant kcat/Km for the GAP-mediated catalysis, estimated to be between 2.2 and 3.8 × 105 m–1 s–1, is, independent of the exact value, very similar for Ras and Rap. This indicates that the additional domain(s) not only affects the catalytic efficiency of the Rap GAP reaction but also dramatically enhances the affinity toward Rap-GTP. Thus, whereas the isolated Ras GAP domain of GAP1IP4BP is, like other Ras GAPs, sufficient to enhance the GTPase activity of Ras, other regions of the molecule such as the amino-terminal C2 domains and/or the carboxyl-terminal region including the pleckstrin homology/Bruton's tyrosine kinase domain are required for Rap GAP activity. For Ras GAPs it has been shown that the glutamine 61 residue of Ras and the arginine finger of Ras GAPs are the two crucial residues required for efficient GAP-mediated reaction (13Scheffzek K. Ahmadian M.R. Kabsch W. Wiesmuller L. Lautwein A. Schmitz F. Wittinghofer A. Science. 1997; 277: 333-338Crossref PubMed Scopus (1208) Google Scholar, 14Ahmadian M.R. Stege P. Scheffzek K. Wittinghofer A. Nat. Struct. Biol. 1997; 4: 686-689Crossref PubMed Scopus (296) Google Scholar). Rap does not have a glutamine in position 61 and Rap GAP does not act via a catalytic arginine (17Daumke O. Weyand M. Chakrabarti P.P. Vetter I.R. Wittinghofer A. Nature. 2004; 429: 197-201Crossref PubMed Scopus (116) Google Scholar), so the question arises as to how GAP1IP4BP-mediated Rap GAP activity is achieved. When we analyze the GTPase reaction using the arginine finger mutant of GAP1IP4BP, GAP1IP4BP-(R371A), the RapGAP (and RasGAP, not shown) activity was severely affected (Fig. 2A), arguing that the basic machinery for GAP activation seems to reside in the GAP domain. As with the GAP domain, catalysis is more efficient for the Rap mutant Rap-(T61Q), but the affinity is reduced because the reaction is no longer saturated with 800 μm Rap-GTP (Fig. 2B). Thus, the mechanism for GAP-mediated GTPase activity of GAP1IP4BP seems to be different from both the Ras GAP and the Rap GAP mechanisms. Further Mutations in the GAP1IP4BP GAP Domain—To extend this analysis we generated a series of site-directed mutants targeting residues that lie within the Ras-binding site of the Ras GAP domain and are known to be required for the Ras GAP activity of p120GAP and NF1. Arginine at position 485 in GAP1IP4BP is equivalent to arginine 903 and 1391 in p120GAP and NF-1, respectively, residues that by stabilizing the catalytic position of the arginine finger are vital for GTP hydrolysis on Ras (13Scheffzek K. Ahmadian M.R. Kabsch W. Wiesmuller L. Lautwein A. Schmitz F. Wittinghofer A. Science. 1997; 277: 333-338Crossref PubMed Scopus (1208) Google Scholar). The arginine finger is also stabilized by leucine 902 and 1390 in p120GAP and NF-1, respectively, equivalent to leucine 484 in GAP1IP4BP. Furthermore, lysine 949 in p120GAP resides within the variable loop that has been proposed to stabilize the α6c helix within the effector loop region of Ras-GTP. This residue is equivalent to lysine 534 of GAP1IP4BP. Finally, lysine 517 in GAP1IP4BP is equivalent to lysine 935, which has been proposed to stabilize the p120GAP/Ras-GTP complex. To examine the role of these residues in the Ras and Rap GAP activity, we introduced the following ind
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