RanGAP1*SUMO1 is phosphorylated at the onset of mitosis and remains associated with RanBP2 upon NPC disassembly - PubMed (original) (raw)

RanGAP1*SUMO1 is phosphorylated at the onset of mitosis and remains associated with RanBP2 upon NPC disassembly

Sowmya Swaminathan et al. J Cell Biol. 2004.

Abstract

The RanGTPase activating protein RanGAP1 has essential functions in both nucleocytoplasmic transport and mitosis. In interphase, a significant fraction of vertebrate SUMO1-modified RanGAP1 forms a stable complex with the nucleoporin RanBP2/Nup358 at nuclear pore complexes. RanBP2 not only acts in the RanGTPase cycle but also is a SUMO1 E3 ligase. Here, we show that RanGAP1 is phosphorylated on residues T409, S428, and S442. Phosphorylation occurs before nuclear envelope breakdown and is maintained throughout mitosis. Nocodazole arrest leads to quantitative phosphorylation. The M-phase kinase cyclin B/Cdk1 phosphorylates RanGAP1 efficiently in vitro, and T409 phosphorylation correlates with nuclear accumulation of cyclin B1 in vivo. We find that phosphorylated RanGAP1 remains associated with RanBP2/Nup358 and the SUMO E2-conjugating enzyme Ubc9 in mitosis, hence mitotic phosphorylation may have functional consequences for the RanGTPase cycle and/or for RanBP2-dependent sumoylation.

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Figures

Figure 1.

Figure 1.

Cell cycle–dependent phosphorylation of RanGAP1. (A) HeLa cells arrested in S-phase with thymidine were released from the S-phase block, harvested at the indicated times, and analyzed by immunoblotting with antibodies to RanGAP1, cyclin A, and cyclin B. For optimal resolution of RanGAP1 species a, b, and c, 6% SDS-PAGE was used for the top panel. Note that all time points were run on the same gel. (B) Mitotic index was determined by visually scoring fixed cells stained with Hoechst for the presence of condensed chromosomes. About 200 cells were scored at each time point. (C) Cell cycle analysis: DNA content of each sample was determined by propidium iodine staining and analysis by flow cytometry.

Figure 2.

Figure 2.

Mitotic RanGAP1 is phosphorylated at residues T409, S428, and S442 in vivo. (A) RanGAP1 species required for mass spectrometry (indicated as bands a, b, and c) were enriched by immunoprecipitation from HeLa cell extracts. A fraction of the precipitates was subjected to immunoblotting with α-RanGAP1 antibodies. The remainder was separated on SDS-PAGE (not depicted), and Coomassie-stained bands a, b, and c were excised from lanes 2, 3, and 1, respectively. cyc, cycling cells; nocodazole, nocodazole-arrested cells; dig, digitonin lysis; hyp, hypotonic swelling extracts. (B) MALDI-TOF mass spectra of a tryptic digest of RanGAP1 species enriched as described in A. Top, band c; middle, band a; bottom, band b. A peptide (amino acids 407–445 in human RanGAP1) containing up to three phosphates was identified. A selected mass range containing different phosphorylation stages (0, 1, 2, and 3 Ph) of this peptide is depicted. (C) Scheme showing the localization of two phosphorylation sites through fragmentation of the doubly phosphorylated peptide (amino acids 414–445) of RanGAP1 using an electrospray ionization ion trap mass spectrometer. The number of phosphorylation sites that were observed on each fragment is added as an arabic number to the arrows. (D) RanGAP1 immunoprecipitated with α RanGAP1 antibodies from cycling and nocodazole-arrested HeLa cells was analyzed by immunoblotting with the indicated phosphospecific antibodies. (E) All three phosphorylation sites of human RanGAP1 are conserved in mouse, areas surrounding S442 and S428 are also conserved in Xenopus laevis RanGAP1.

Figure 3.

Figure 3.

RanGAP1 phosphorylation takes place before nuclear envelope breakdown. (A) HeLa cells fixed and permeabilized with 4% PFA and 0.2% Triton X-100 were subjected to indirect immunofluorescence with α p-T409 antibody. (B) HeLa cells were fixed with 2% PFA, permeabilized with 0.2% Triton X-100, and stained with primary antibodies as indicated. (C) At least three distinct phosphorylated RanGAP1 species exist during mitosis. (lanes 2–7) α RanGAP1 immunoprecipitates from cells harvested at indicated times after release from thymidine (same cells as in Fig. 1). (lanes 1, 8, and 9) Total extracts from cycling (cyc) and nocodazole (noc) extracts served as controls. Immunoblotting was done with the indicated phosphospecific antibodies.

Figure 4.

Figure 4.

RanGAP1 phosphorylation by Cdks. (A) Phosphorylation of RanGAP1 in HeLa cell extracts is inhibited by p27. Recombinant sumoylated RanGAP1 was incubated with nocodazole extracts in the presence of ATP and analyzed by immunoblotting with α RanGAP1. Phosphorylation results in a mobility shift (arrows). Where indicated, extracts were preincubated with p27 before RanGAP1*SUMO1 addition. (B) In vitro phosphorylation of recombinant RanGAP1 with cyclin A/Cdk2 or cyclin B/Cdk1. After incubation, samples were analyzed by SDS-PAGE and autoradiography. p27 was included to demonstrate specificity of the kinase preparations. Mouse RanGAP1 mutants: T-D, RanGAP1-T411D; S-D, RanGAP1-S444D; DD, RanGAP1-T411D, S444D. (C) In vivo phosphorylation of human RanGAP1 T409 correlates with levels and localization of cyclin B. HeLa cells fixed with 2% PFA and permeabilized with 0.2% Triton X-100 were stained with α p-T409 antibodies and α cyclin B.

Figure 5.

Figure 5.

RanGAP1*SUMO1 phosphoforms a and b remain associated with RanBP2 and Ubc9 in nocodazole-arrested HeLa cells. (A) α RanGAP1 and α RanBP2 immunoprecipitates from cycling and nocodazole-arrested cells (RIPA extracts) were analyzed for the presence of RanGAP1 (top) and Ubc9 (bottom) by immunoblotting. IgGs 1 and 2 were from the respective preimmune sera and served as specificity controls. (B) Model: the RanGAP1*SUMO1–RanBP2 complex serves a dual function as an activator of Ran-GTP hydrolysis and as a SUMO E3 ligase. Either process may be affected by mitotic phosphorylation of RanGAP1.

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