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Address correspondence to F. Melchior, Max-Planck Institute for Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany. Tel.: (49) 89 8578 3972. Fax: (49) 89 8578 3810. email: melchior{at}biochem.mpg.de
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Abstract |
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Key Words: RanGAP1; RanBP2/Nup358; SUMO; cyclin B/Cdk1; Ubc9
S. Swaminathan's present address is Nature Cell Biology, The Macmillan Building, 4 Crinan Street, London N19XW, UK.
Abbreviations used in this paper: MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; NPC, nuclear pore complex.
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Introduction |
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To accommodate Ran's different functions in interphase and mitosis, components of the RanGTPase cycle are likely to be cell cycle regulated. A first example is the RanGTP interacting protein RanBP1, whose levels increase from S-phase to metaphase and decline during late telophase (Guarguaglini et al., 2000). RanBP2 is hyperphosphorylated during mitosis, but the consequences of this are unknown (Favreau et al., 1996). Here, we show that RanGAP1 is subject to mitotic phosphorylation at three closely spaced residues in its COOH-terminal domain. Phosphorylation occurs at the NPC before nuclear envelope breakdown, but does not disrupt the RanGAP1*SUMO1RanBP2Ubc9 interaction. RanGAP1 phosphorylation may potentially alter RanGAP1's catalytic activity or RanBP2-mediated sumoylation in vivo.
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Results and discussion |
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Phosphorylation of RanGAP1 occurs before nuclear envelope breakdown
Next, we tested the phosphospecific antibodies in immunofluorescence. p-T409 strongly decorates cells from prophase to telophase. Fig. 3 A shows representative images for different mitotic stages. In metaphase and anaphase, strong cytoplasmic staining and some enhancement at the mitotic spindle was visible, consistent with previous reports on RanGAP1 localization in mitosis (Matunis et al., 1996; Joseph et al., 2002).
P-S428 and
P-S442 antibodies are weaker and appear to give rise to more background, but they also clearly decorate mitotic cells (Fig. 3 B). Interestingly, relative intensities of signals between prophase and telophase varied for the three antibodies, suggesting differential timing of phosphorylation or dephosphorylation. To address this issue, we immunoprecipitated RanGAP1 from cells at different time points after thymidine release and detected RanGAP1 by immunoblotting with the phosphospecific antibodies (Fig. 3 C). At 9 h after release, band a is already present, whereas little singly phosphorylated S442 (comigrating with unphosphorylated RanGAP1) and no singly phosphorylated T409 or S428 is detectable. This finding suggests that RanGAP1 is initially phosphorylated at all three sites. In contrast, dephosphorylation appears sequential, as p-T409 clearly disappears before the other two phosphorylated sites. Together, we find that RanGAP1 is phosphorylated at the onset of mitosis before nuclear envelope breakdown. Differential timing of dephosphorylation of the three sites in RanGAP1 results in at least three distinct mitotic RanGAP1 species: a triply phosphorylated species, and two species phosphorylated at both S428 and S442 or only at S442.
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Materials and methods |
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Antibodies
Goat -RanGAP1, goat
-Ubc9, rabbit
-RanBP2 (Pichler et al., 2002), and rabbit
cyclin A antibodies were described previously (Hengst and Reed, 1996). For phosphospecific antibodies, the following peptides were coupled to ovalbumin: T409, CEKSApTPSRKI; S428, CPVLSpSPPPAD; and S442, CAFPpSPEKLLR. Initial injection into goats was as an emulsion with Titermax Gold (Sigma-Aldrich), booster injections were with Freund's incomplete adjuvant. Antibodies were affinity purified on phosphopeptides coupled to EAH-Sepharose 6B (Amersham Biosciences), eluted with 0.2 M acetic acid, pH 2.7, and 0.5 M NaCl, and dialyzed against PBS. Before use, antibodies were preadsorbed against immobilized nonphosphorylated peptides. Mouse
cyclin B and rabbit
-GFP antibodies were obtained from Santa Cruz Biotechnology, Inc. Alexa 488 donkey
goat was purchased from Molecular Probes, other secondary antibodies were purchased from Jackson ImmunoResearch Laboratories.
Immunoblotting and fluorescence microscopy
Detection of antigens on nitrocellulose was performed with affinity-purified goat p-T409 (0.5 µg/ml),
p-S428 (0.4 µg/ml),
p-S442 (0.7 µg/ml), or
RanGAP1 (0.25 µg/ml) in 5% milk powder (or 0.2% gelatine) in PBS, 0.2% Tween 20 for 1 h at RT. Detection was performed by chemiluminescence. For indirect immunofluorescence, adherent HeLa cells grown on coverslips were either fixed for 10 min with 2% PFA in PBS, 1 mM MgCl2, and permeabilized for 5 min with 0.2% Triton X-100 in PBS, 1 mM MgCl2, or permeabilized and fixed for 10 min in 4% PFA, 0.2% Triton X-100, 20 mM PIPES, 1 mM MgCl2, and 10 mM EGTA (Kapoor et al., 2000). Antibodies in 2% BSA, PBS/MgCl2 were used at 0.25 µg/ml (
-RanGAP1), 1 µg/ml (
p-T409 and
p-S428), and 2 µg/ml (
p-S442), and at 1:100 (
cyclin B). Alexa 488 donkey
goat and Cy3 donkey
mouse antibodies were at 1:500. Hoechst 33342 was included with the secondary antibodies at 0.2 µg/ml. Pictures of cells mounted in ProLong Antifade (Molecular Probes) were taken with a microscope (model Axioskop II; Carl Zeiss MicroImaging, Inc.), 63x Plan Apochromat lens (aperture 1.4, oil), using a camera (model MicroMax CCD; Princeton Instruments) and Iplab software.
Immunoprecipitations
Immunoprecipitations were from SDS-lysates, digitonin cytosol, cytosol generated by hypotonic swelling, or from RIPA extracts from cycling and nocodazole-arrested HeLa cells. For SDS-lysate, HeLa suspension cells were lysed by boiling in 1% SDS and diluted 10-fold with RIPA buffer-SDS (50 mM Tris-HCl, pH 8, 150 mM NaCl, 1% NP-40, 0.5% deoxycholate, 1 mM DTT, and 1 µg/ml pepstatin, aprotinin, and leupeptin). Hypotonic swelling extracts were generated as described previously (Melchior, 1998), and buffer was changed to TB buffer with protease inhibitors and phosphatase inhibitor cocktail I (Sigma-Aldrich) using PD10 columns (Amersham Biosciences). For digitonin cytosol, HeLa cells were lysed in TB containing protease inhibitors and phosphatase inhibitor cocktail I and 0.005% digitonin (Calbiochem). For RIPA extracts, HeLa suspension cells were lysed by sonification in 4 vol RIPA buffer with protease inhibitors, phosphatase inhibitor cocktail I, and 10 mM iodoacetamide. All cell lysates were clarified at 100,000 g for 45 min before use in IP. Affinity-purified antibodies or control IgGs cross-linked at 2 mg/ml to Ultralink Immobilized Protein G Plus beads (Pierce Chemical Co.) were incubated with extracts for 90 min at 4°C. Beads were washed three times in RIPA buffer and boiled in SDS-Laemmli loading buffer.
Cell cycle analysis of RanGAP1 phosphorylation
A standard double thymidine block release protocol was used to obtain a synchronous population of suspension HeLa cells (Bonifacino et al., 1999). At indicated times, cells were harvested by centrifugation, aliquots flash frozen, and stored at -80°C. Aliquots were used for analysis by immunoblotting upon lysis in Laemmli buffer or for immunoprecipitation upon SDS-lysis. Progression through the cell cycle was monitored by FACS® analysis after cell fixation in 70% ethanol and staining with propidium iodide (Bonifacino et al., 1999). To determine the mitotic index, cells were fixed in 70% ethanol, stained using a final concentration of 4 ng/µl Hoechst 33342 (Molecular Probes), mounted with Glow mounting medium (EnerGene), and observed using a microscope (model Axioskop II; Carl Zeiss MicroImaging, Inc.).
In vitro RanGAP1 phosphorylation
Phosphorylation of 2 µg RanGAP1 with recombinant kinases was in 20 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 50 µM ATP, and 10 µCi [32P]ATP at 30°C for 30 min. Cyclin B/Cdk1 (Calbiochem) and cyclin A/Cdk2 were used at 2 U or 5 ng, respectively. Analysis was performed by SDS-PAGE and autoradiography. Mitotic extracts for RanGAP1 phosphorylation were prepared from 100 ml of nocodazole-arrested HeLa cells by freeze-thaw lysis in 1.5 ml TB buffer supplemented with phosphatase inhibitor cocktail I. 100 ng of SUMO1-modified RanGAP1 was incubated in 5 µl of extracts and 1 mM of ATP at 30°C for 2 h. Recombinant p27 at concentrations of 1 µg or 5 µg was used to pretreat mitotic cell extracts on ice for 45 min. Reactions were analyzed by immunoblotting with
RanGAP1 antibodies.
Mass spectrometry
Coomassie-stained protein bands were in-gel digested by trypsin (sequencing grade; Promega) using essentially the protocol of Shevchenko et al. (1996) and desalted using home-made miniaturized reversed-phase columns (Gobom et al., 1999). MALDI-TOF mass spectra were acquired on a Reflex III instrument (Bruker Daltonik) in positive ion reflector mode. As a matrix, 2,5 dihydroxybenzoic acid (Bruker Daltonik) was used. For peptide sequence analysis by electrospray tandem mass spectrometry, samples were filled into nano electrospray needles (Protana) and analyzed on an ion trap (model Esquire 3000+; Bruker Daltonik) mass spectrometer.
Online supplemental material
GAP assays were performed as described previously (Mahajan et al., 1997) using -[32P]Ran-GTP and immunoprecipitated or using recombinant RanGAP1. Analysis was performed by TLC. Amounts of GTP and GDP were determined using a PhosphorImager (model BAS-2500, Fuji FILM). Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200309126/DC1.
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Acknowledgments |
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This work was funded by the Bundesministerium für Bildung und Forschung (grant BioFUTURE 0311869), an Alexander von Humboldt fellowship (to S. Swaminathan), and the Max-Planck Institute for Biochemistry.
Submitted: 22 September 2003
Accepted: 18 February 2004
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