Proc. Nati. Acad. Sci. USA Vol. 91, pp. 2587-2591, March 1994 Biochemistry RanGAP1 induces GTPase activity of nuclear Ras-related Ran (GTPase-activating protein/RCCl/TC4/G2 checkpoint) F. RALF BISCHOFF*t, CHRISTIAN KLEBEt, JURGEN KRETSCHMER*, ALFRED WITrINGHOFERt, AND HERWIG PONSTINGL* *Division for Molecular Biology of Mitosis, German Cancer Research Center, D-69120 Heidelberg, Federal Republic of Germany; and *Abteilung Strukturelle Biologie, Max-Planck-Institut ffr Molekulare Physiologie, D-44139 Dortmund, Federal Republic of Germany Communicated by Hans Neurath, December 3, 1993 ABSTRACT The nuclear Ras-related protein Ran binds DMAE-650/M (Merck; Superformance, 26 x 115 mm) in 20 guanine nucleotide and is involved in cell cycle regulation. mM Bis-Tris-propane HCl, pH 7.0/1 mM DTT with a linear Models of the signal pathway predict Ran to be active as gradient of NaCl from 0.05 M to 1 M at a flow rate of 5 Ran GTP at the initiation of S phase upon activation by the ml/min. Fractions containing RanGAP were pooled and nucleotide exchange factor RCC1 and to be inactivated for the immediately applied to a hydroxylapatite column (Merck; onset of mitosis by hydrolysis of bound GTP. Here a nuclear Superformance, 10 x 150 mm) in 20 mM potassium phos- homodimeric 65-kDa protein, RanGAPl, is described, which phate, pH 7.0/1 mM DTT, with a linear gradient from 20 mM we believe to be the immediate antagonist of RCC1. It was to 1 M phosphate at a flow rate of 2 ml/min. To fractions purified from HeLa cell lysates and induces GTPase activity of containing RanGAP, ammonium sulfate in 20 mM Bis-Tris- Ran, but not Ras, by more than 3 orders of magnitude. The propane HCl (pH 7.0) was added to 35% saturation within 20 Ran mutant Q69L, modeled after RasQ61L, which is unable to min. The precipitate was removed by centrifugation for 10 hydrolyze bound GTP, is insensitive to RanGAP1. min at 6000 x g, and the supernatant was loaded on a hydrophobic column (Merck; Fractogel EMD AFTA 650/S, Ras-related small guanine nucleotide-binding proteins par- Superformance, 10 x 20 mm). Chromatography was started ticipate in various intracellular signal pathways. As a rule, the with 35% saturated ammonium sulfate in 20 mM Bis-Tris- GTP-bound form represents the active state ofa signal. Their propane HCl, pH 7.0/1 mM DTT at a flow rate of 1 ml/min, with and a linear gradient of35 to 0%o ammonium sulfate saturation latent GTPase activity is induced upon interaction was applied in 20 mM Bis-Tris-propane HCl, pH 7.0/1 mM GTPase-activating proteins (GAPs). The GDP-bound form is DTT/100 mM NaCl. The eluate containing RanGAP was inactive in signaling, and nucleotide exchange factors cata- diluted 5-fold with 20 mM Tris HCl (pH 7.5) and separated on lyze reactivation by exchanging GDP for GTP. Both nucle- a Mono Q (HR 5/5; Pharmacia) column at a flow rate of 1 otides are otherwise firmly bound in the presence of Mg2+. ml/min in the same buffer containing 200 mM NaCl, with a Whereas most members of the Ras superfamily are associ- linear gradient to 1 M NaCI. RanGAP1 was also purified from ated with membranes, Ran (1) [or TC4, which was identified a 400 mM NaCl extract of the 70,000 x g pellet from HeLa by homology screening in a human teratocarcinoma cDNA cell lysate, using the same protocol. library (2)] is a soluble nuclear protein. In the GTP-bound Purification of Bacterially Expressed Ran. Ran/TC4 was form it is believed to be involved in inhibiting the onset of expressed in Escherichia coli BL21(DE3) from PET3d vector mitosis. Cells expressing mutated Ran/TC4 incapable of (11). Bacteria were grown to an OD of 0.4 in a 2-liter culture, hydrolyzing GTP are arrested in G2 (3). In contrast, inacti- and expression of the protein was induced by 0.5 mM vation of its nucleotide exchange factor RCC1 results in isopropyl 3-D-thiogalactoside. After 3 h at 37°C cells were premature chromosome condensation, activation ofthe cdc2 harvested by centrifugation for 5 min at 5000 x g and kinase, and onset of mitosis in S-phase cells (4), presumably resuspended on ice in 8 ml of30 mM Tris HCl, pH 7.5/40 mM by depletion of active Ran-GTP. Additional effects of inac- NaCl/1 mM DTT, containing lysozyme at 1 mg/ml and tivating or removing RCC1 have been described in various protease inhibitors. After 20 min on ice, cells were sonicated systems: Exit from mitosis is inhibited (5), G1 cells do not 15 times for 10 sec (Branson Sonifier B15, microtip, 50%o enter S phase (4), synthesis of double-stranded DNA is not cycle, output control 4) followed by a 30-min centrifugation initiated (6), and mRNA transcripts are processed incom- at 30,000 x g. Ran was precipitated from the supernatant at pletely (7-9). To identify further members of this signal 35-55% ammonium sulfate saturation and centrifuged at pathway, we have used [y32P]GTP'Ran to monitor the iso- 70,000 x g for 20 min. The pellet was dissolved in 25 mM lation of a protein (RanGAP1) inducing its GTPase activity. Mes, pH 6.5/50 mM NaCl/1 mM 2-mercaptoethanol/10% glycerol and purified by gel filtration on a Sephacryl S-200 (26 MATERIALS AND METHODS x 1000 mm; Pharmacia) column by using the same buffer at a flow rate of 1 ml/min. The fraction containing Ran was Purification of RanGAP. Fifty milliliters of packed unsyn- incubated in 10 mM EDTA with 1 mM GTP for 4 h on ice. chronized HeLa cells was thawed in 150 ml oflysis buffer [20 Final purification was achieved on Fractogel EMD SO3 650 mM Bis-Tris-propane HCl, pH 7.0/1 mM EDTA/1 mM di- (S) (Merck; 10 x 50 mm) in the same buffer with a step elution thiothreitol (DTT)/protease inhibitors (10)] and swollen on of 500 mM NaCl at a flow rate of 2 ml/min. ice for 20 min. They were lysed by 10 strokes with an S-type The Q69L substitution was introduced into Ran by site- Dounce homogenizer and centrifuged at 70,000 x g for 60 directed mutagenesis in M13mp (Amersham), and the mutant min. The pellet was extracted with 100 mM NaCl in lysis protein was expressed and purified by the method described buffer and recentrifuged at 70,000 x g for 30 min. Superna- tants were pooled and chromatographed on Fractogel EMD Abbreviations: DTT, dithiothreitol; GAP, GTPase-activating pro- tein; DMAE, dimethylaminoethyl. The publication costs ofthis article were defrayed in part by page charge tTo whom reprint requests should be addressed at: Division for payment. This article must therefore be hereby marked "advertisement" Molecular Biology of Mitosis, German Cancer Research Center, Im in accordance with 18 U.S.C. §1734 solely to indicate this fact. Neuenheimer Feld 280, D-69120 Heidelberg, F.R.G. 2587 Downloaded by guest on September 25, 2021 2588 Biochemistry: Bischoff et al. Proc. Natl. Acad Sci. USA 91 (1994) by Klebe et al. (11) for recombinant wild-type Ran/TC4. gradient (75/30%o). After centrifugation for 10 min at 1200 x g, RasGAP and the catalytic domain ofNF1 were expressed and the resulting nuclei and the mitotic chromatin at the 30%6/75% purified as described by Gideon etal. (12) and by Wiesmniller interphase were resuspended in lysis buffer containing 100mM and Wittinghofer (13). NaCa and sonicated for 10 sec as described for bactena. Ihe RanGAP Assays. Twenty-five micromolar RanGTP or suspension was centrifuged for 10 min at 5000 x g to separate Ras-GTP (14) was incubated for 30 min on ice with 13 ,uM the soluble material (nucleoplasm in case ofthymidine arrested ['-t32P]GTP (15 Ci/mmol; 1 Ci = 37 GBq) in the presence of cells) from the insoluble material. 20 mM EDTA in a final volume of 150 A4. The buffer was changed to 20 mM Hepes, pH 7.4/5 mM MgCl2/0.05% hydrolyzed gelatin (incubation buffer) on a NAP-5 column RESULTS (Pharmacia). Comparison of Ran from HeLa Cells and Recombinant TC4 To identify RanGAP activity, dilutions of fractions in a as Substrates for Monitoring RanGAP Purificon. Sequence total volume of 10 ml were added to 40 pA of 60 nM Rank-le alignments of all known members of the Ran/TC4 family and 32P]GTP (in incubation buffer). The GTPase reaction was of most ras-related proteins showed a conserved arginine stopped after shaking for 6 min at 250C by addition of 1 ml of residue at the position corresponding to Arg-129 in the charcoal suspension [7% (wt/vol) charcoal/l0o (vol/vol) Ran/TC4 sequence. After reexamining our previous data (1), ethanol/0.1 M HCl/10 mM KH2PO4 (15)]. After centrifuga- we could confirm Arg-129. Now all amino acid sequences tion at 10,000 x g, release of P2P]phosphate was determined obtained from HeLa Ran peptides fit the corrected reading in 700 ILI ofthe supernatant in a Packard 1500 Tri-Carb liquid frame of TC4 (16). Furthermore, Ran from HeLa and the scintillation counter. RanGAP activities are expressed as the human TC4 gene product expressed in E. coli show identical percentage of Ran-bound [yt32P]GTP hydrolyzed within 6 mobilities in PAGE, and RCCl-catalyzed GDP exchange (11) min, corrected for buffer control. Enrichment of RanGAP as well as RanGAP-stimulated GTP hydrolysis activity was calculated by determining the amount of the (data not fraction leading to 50% hydrolysis of Ran-bound [('y32P]GTP shown) yielded similar results.