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Aurora kinase A-mediated phosphorylation triggers structural alteration of Rab1A to enhance ER complexity during mitosis

Nature Structural & Molecular Biology volume 31, pages 219–231 (2024)Cite this article

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Abstract

Morphological rearrangement of the endoplasmic reticulum (ER) is critical for metazoan mitosis. Yet, how the ER is remodeled by the mitotic signaling remains unclear. Here, we report that mitotic Aurora kinase A (AURKA) employs a small GTPase, Rab1A, to direct ER remodeling. During mitosis, AURKA phosphorylates Rab1A at Thr75. Structural analysis demonstrates that Thr75 phosphorylation renders Rab1A in a constantly active state by preventing interaction with GDP-dissociation inhibitor (GDI). Activated Rab1A is retained on the ER and induces the oligomerization of ER-shaping protein RTNs and REEPs, eventually triggering an increase of ER complexity. In various models, from Caenorhabditis elegans and Drosophila to mammals, inhibition of Rab1AThr75 phosphorylation by genetic modifications disrupts ER remodeling. Thus, our study reveals an evolutionarily conserved mechanism explaining how mitotic kinase controls ER remodeling and uncovers a critical function of Rab GTPases in metaphase.

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Fig. 1: Rab1A is phosphorylated by mitotic kinase during mitosis.
Fig. 2: Rab1A phosphomimetic remodels switch II conformation and disfavors GDI binding.
Fig. 3: p-Rab1AThr75 accumulates on the ER during mitosis.
Fig. 4: ER accumulation of p-Rab1AThr75 enhances mitotic ER complexity.
Fig. 5: Rab1A binds to ER-shaping proteins, promoting the curvature-stabilizing oligomerization that is required for increased mitotic ER complexity.
Fig. 6: Rab1A promotes curvature-stabilizing oligomerization in a phosphorylation-dependent manner.
Fig. 7: AURKA–Rab1 axis-controlled mitotic ER complexity is conserved in metazoans.

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Data availability

The crystal structure of GDP-bound Rab1A-T75D is available in the Protein Data Bank under accession code PDB 7EQ2. SILAC–MS data shown in Extended Data Figure 1f are available with the manuscript. Source data are provided with this paper.

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Acknowledgements

We would like to thank H. Cheng (Peking University) and T. Tang (Chinese Academy of Sciences) for discussions, H. Li and Q. Zhang (Sun Yat-sen University) for their assistance with TEM characterization, and S. Chen, Z. Li, and S. Wu (Sun Yat-sen University Cancer Center) for technical support. We thank SYSU Instrumental Analysis and Research Center for performing MS. This work was supported by the National Key R&D Program of China (2019YFA0110300, 2017YFA0505600-04 and 2022YFA1104002 to Q.L., 2018YFA0508300 to S.G.), the National Natural Science Foundation of China (81820108024, 81630005 and 82341020 to Q.L., 82173098 to S.G., 82173367 and 81773166 to Z.W., 81972594 to M.Y., 32100589 to W.Z.), the Innovative Research Team in University of Ministry of Education of China (IRT-17R15 to Q.L.), the Natural Science Foundation of Guangdong (2016A030311038 and 2017A030313608 to Q.L., 2022A1515010915 and 2017A020215098 to Z.W., 2018A0303130299 and 2020A1515010608 to M.Y.), the Fundamental Research Funds for the Central Universities (19ykpy187 to M.Y.)., the Science and Technology Planning Project of Guangzhou (201804020044 to Q.L.), and the Cancer Innovative Research Program of Sun Yat-sen University Cancer Center (CIRP-SYSUCC-0019 to Q.L.).

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Author notes

  1. These authors contributed equally: Wei Zhang, Zijian Zhang, Yun Xiang, Dong-Dong Gu, Jinna Chen.

Authors and Affiliations

  1. State Key Laboratory of Oncology in South China, Guangdong Provincial Clinical Research Center for Cancer, Sun Yat-sen University Cancer Center, Guangzhou, P.R. China

    Wei Zhang, Zijian Zhang, Dong-Dong Gu, Jinna Chen, Bin He, Min Yan, Zifeng Wang, Yu-Lu Cao, Bing Deng, Deshun Zeng, Jie Lei, Junxiao Zhuo, Xinxing Lei, Song Gao & Quentin Liu

  2. Department of Clinical Immunology, The Third Affiliated Hospital, Sun Yat-sen University, Guangzhou, China

    Wei Zhang

  3. Institute of Cancer Stem Cell, Dalian Medical University, Dalian, China

    Zijian Zhang, Jie Xu, Bilian Jin & Quentin Liu

  4. National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China

    Yun Xiang, Yong Liu, Tao Jiang, Chong Liu, Dong Li & Junjie Hu

  5. University of Chinese Academy of Sciences, Beijing, China

    Yun Xiang, Yifan Chen, Yong Liu, Tao Jiang, Chong Liu, Dong Li, Yidong Shen & Junjie Hu

  6. State Key Laboratory of Cell Biology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai, China

    Yifan Chen & Yidong Shen

  7. MOE Key Laboratory of Laser Life Science and College of Biophotonics, South China Normal University, Guangzhou, China

    Shixian Zhai & Tongsheng Chen

  8. Department of Hematology, The Third Affiliated Hospital, Sun Yat-sen University, Guangzhou, China

    Zijie Long

  9. Institute of Hematology, Sun Yat-sen University, Guangzhou, China

    Zijie Long & Quentin Liu

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  1. Wei Zhang
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Contributions

W.Z. and Z.Z. developed the experimental protocol; designed, performed, and analyzed the experiments; and wrote the manuscript. Y.X., D.-D.G., and J.C. designed, performed, and analyzed the experiments. Y.S. and Y.C. designed and performed the C. elegans embryo mitosis experiment. S.Z. and T.C. designed and performed the E-FRET experiment. J.X. contributed to development of the experimental protocols. B.D., D.Z., J.L., J.Z., X.L., Y.C. Y.-L.C., T.J., and C.L. contributed to the experiments. B.H., Z.L., M.Y., Z.W., B.J., and D.L. revised the experimental data and contributed to the discussion. S.G. and J.H. conceptualized, designed, and analyzed the experiments and wrote the manuscript. Q.L. directed the project, conceptualized and designed the experiments, interpreted the results, and wrote the manuscript.

Corresponding authors

Correspondence to Junjie Hu, Song Gao or Quentin Liu.

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Nature Structural & Molecular Biology thanks Richard Bayliss, Blake Riggs and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Katarzyna Ciazynska and Carolina Perdigoto, in collaboration with the Nature Structural & Molecular Biology team. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Rab1A is phosphorylated and activated during mitosis.

a, The possible phosphorylation sites of Rab1A during mitosis. b, The conserved AURKA recognition motif of Rab1A. c, Cell lysates from serum-starved (G1), double thymidine blocked (S), or nocodazole-arrested (M) MDA-MB-231 cells with or without VX680 (100 nM) treatment were analyzed by immunoblotting. d, Cell lysates were prepared at the indicated times after release from arrest with nocodazole and analyzed by immunoblotting. e, Schematic depiction of the experimental workflow of SILAC-MS analysis. Protein lysates from stable WT Flag-Rab1A or Flag-Rab1A-T75A-expressing MDA-MB-231 cells grown in ‘heavy’ and ‘light’ SILAC medium were used in anti-Flag immunoprecipitation and LC-MS/MS analyses. f, SILAC H/L ratios. The differential binding preferences (log2, y-axis) of all 63 proteins identified and quantified by LC-MS/MS (x-axis) are shown. g, Anti-Flag immunoprecipitates were prepared from MDA-MB-231 cells that stably expressed wild-type or mutant Flag-Rab1A. Immunoprecipitates and cell lysates were analyzed by immunoblotting. h, Anti-Flag immunoprecipitates were prepared from MDA-MB-231 cells that stably expressed wild-type or mutant Flag-Rab1B, Flag-Rab10, or Flag-Rab18. Immunoprecipitates and cell lysates were analyzed by immunoblotting. i, Cell lysates from MDA-MB-231 cells expressing Flag-tagged wild-type or mutant Rab1A were incubated with GTP-agarose beads in the presence or absence of GTP and the bound proteins analyzed by immunoblotting. Data shown in c, d, g, h, i are representative of two independent experiments.

Source data

Extended Data Fig. 2 Structural analysis of Rab1AThr75 phosphorylation.

a, Structural overlay of the two molecules (chains) in the asymmetric unit of the Rab1A-T75D crystal. Switch II region is specified. Note the consistent configuration of T75D in the two chains. b, Local interactions of the switch II loop (coloured magenta) in the crystal packing of Rab1A-T75D. The two crystallographic Rab1A-T75D dimers are coloured cyan and grey, respectively. NC, non-crystallographic. c, Comparison of the nucleotide binding pockets of WT Rab1A (PDB code 2FOL) and Rab1A-T75D. Note that R72 (yellow) blocks the exit of the pocket in Rab1A-T75D. d, Structural comparison between Rab1A-T75D-•GDP and Rab8A-Q67L-pT72•GTP:RILPL2 complex (PDB code 6RIR). Note the different conformations of switch II in Rab1A and Rab8A. e, Complex structures of Rab1A•GDP•AlF3 with Shigella VirA (left, 4FMB), E. Coli EspG (middle, 4FMD), and Legionella LepB (right, 4JVS). Note the different distances between Rab1A Thr75 and these GAPs.

Extended Data Fig. 3 Phosphorylation promotes Rab10 and Rab18, but not Rab1B, accumulating on the ER.

a, MDA-MB-231 cells co-expressing ER-RFP and GFP-tagged wild-type or mutant Rab1B were analyzed by confocal fluorescent microscopy. Scale bars = 20 μm. b, Colocalization of ER-RFP and GFP-tagged wild-type or mutant Rab1B was quantified in multiple cells, and evaluated by Pearson’s correlation coefficient. WT, n = 22; T72A, n = 19; T72D, n = 27. c, MDA-MB-231 cells co-expressing ER-RFP and GFP-tagged wild-type or mutant Rab10 were analyzed by confocal fluorescent microscopy. Scale bars = 20 μm. d, Colocalization of ER-RFP and GFP-tagged wild-type or mutant Rab10 was quantified in multiple cells, and evaluated by Pearson’s correlation coefficient. WT, n = 15; T73A, n = 15; T73D, n = 21. e, MDA-MB-231 cells co-expressing ER-RFP and GFP-tagged wild-type or mutant Rab18 were analyzed by confocal fluorescent microscopy. Scale bars = 20 μm. f, Colocalization of ER-RFP and GFP-tagged wild-type or mutant Rab18 was quantified in multiple cells, and evaluated by Pearson’s correlation coefficient. WT, n = 16; T72A, n = 17; T72D, n = 16. g, Whole cell lysate and ER extract from MDA-MB-231 cells were analyzed by immunoblotting. Data shown in g are representative of two independent experiments. Data from a-f were examined over three independent experiments. Data from b, d, f were analyzed using one-way Welch’s ANOVA test (P = 0.2830 in b, P < 0.0001 in d and f), and pairwise comparisons were performed using Dunnett’s T3 multiple comparisons test. Data are presented as mean ± s.e.m.

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Extended Data Fig. 4 Nocodazole, but not STLC, disrupts mitotic ER morphology.

a, Schematic of living cell imaging of ER-GFP-labeled mitotic cells using three-dimensional structured illumination microscopy (3D-SIM). The length of ER elements was calculated using ImageJ software. b, Left, representative 3D-SIM images of the ER in ER-GFP-labeled MDA-MB-231 cells treated with DMSO, nocodazole, or STLC. Red boxed regions were enlarged and subjected to 3D reconstruction. Scale bars = 5 μm. Right, the length of ER elements from multiple cells in the optical SIM sections. DMSO, n = 8; Nocodazole, n = 8; STLC, n = 7. Data from b were examined over three independent experiments, and analyzed using one-way Welch’s ANOVA test (P < 0.0001), pairwise comparisons were performed using Games-Howell’s multiple comparisons test. Data are presented as mean ± s.e.m.

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Extended Data Fig. 5 ER undergoes a wave of remodeling along with mitotic progression.

a, Schematic illustrating the FLIP experiments in ER-GFP-labeled mitotic cells. The repeatedly bleached region (Bleach region, circle) and fluorescence test region (Test region, square) are represented by circles and rectangles, respectively. b, c, FLIP experiments in mitotic COS-7 cells expressing ER-GFP. b, Representative images recorded by time-lapse microscopy at each time point. Scale bars = 5 μm. c, Left, average fluorescence intensity of ER-GFP from multiple cells at each time point in the test region. Right, quantification of fluorescence intensity for ER-GFP at t = 20 s derived from non-linear fitted curves. PROM, prometaphase (n = 6); META, metaphase (n = 5); ANA, anaphase (n = 5). d, Left, representative 3D-SIM images of the ER in ER-GFP-labeled mitotic COS-7 cells. Red boxed regions were enlarged and subjected to 3D reconstruction. Scale bars = 5 μm. Right, the length of ER elements from multiple cells in the optical SIM sections. PROM, n = 8; META, n = 8; ANA, n = 8. Data from b-d were examined over three independent experiments. Data from c, d were analyzed using one-way Welch’s ANOVA test (P = 0.1132 in c, P < 0.0001 in d), pairwise comparisons in c were performed using Dunnett’s T3 multiple comparisons test, pairwise comparisons in d were performed using Games-Howell’s multiple comparisons test. Data are presented as mean ± s.e.m.

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Extended Data Fig. 6 AURKA-Rab1 axis enhances ER complexity during mitosis.

a, Cell lysates from shcontrol or shAURKA MDA-MB-231 cells were analyzed by immunoblotting. b, c, Doxycycline-induced shAURKA MDA-MB-231 cells were labeled with ER-GFP and used in FLIP experiments. b, Representative images recorded by time-lapse microscopy at each time point. Scale bars = 5 μm. c, Left, average fluorescence intensity for ER-GFP from multiple cells at each time point in the test region. Right, quantification of fluorescence intensity for ER-GFP at t = 20 s derived from non-linear fitted curves. shCtrl, n = 8; shAURKA, n = 7. d, Left, doxycycline-induced shAURKA MDA-MB-231 cells were labeled with ER-GFP and analyzed by 3D-SIM. Red boxed regions were enlarged and subjected to 3D reconstruction. Scale bars = 5 μm. Right, the length of ER elements from multiple cells in the optical SIM sections. shCtrl, n = 8; shAURKA, n = 8. e, Left, doxycycline-induced shAURKA MDA-MB-231 cells were visualized by thin-section electron microscopy. Scale bars = 2 μm. Right, the length of ER elements was quantified. shCtrl, n = 5; shAURKA, n = 5. f, Cell lysates from shcontrol or shRab1A MDA-MB-231 cells were analyzed by immunoblotting. g, Left, representative 3D-SIM images of the ER in ER-GFP-labeled MDA-MB-231 cells expressing control or Rab1A shRNA. Red boxed regions were enlarged and subjected to 3D reconstruction. Scale bars = 5 μm. Right, the length of ER elements from multiple cells in the optical SIM sections. shCtrl, n = 8; shRab1A#1, n = 7; shRab1A#2, n = 8. h, Left, endogenous Rab1A-knockdown MDA-MB-231 cells overexpressing Flag-tagged wild-type or mutant Rab1A were visualized by thin-section electron microscopy. Scale bars = 2 μm. Right, the length of ER elements was quantified. shRab1A+Flag-Rab1AWT, n = 5; shRab1A+Flag-Rab1A-T75A, n = 5; shRab1A+Flag-Rab1A-T75D, n = 5. Data shown in a, f are representative of three independent experiments. Data from b, c, d, e, g, h were examined over three independent experiments. Data from g, h were analyzed using one-way Welch’s ANOVA test (P < 0.0001 in g and h), and pairwise comparisons were performed using Games-Howell’s multiple comparisons test. Data from c, d, e were analyzed using two-tailed unpaired Student’s t-test. Data are presented as mean ± s.e.m.

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Extended Data Fig. 7 p-Rab1AThr75 has a subtle effect on ER morphology in interphase cells.

a, Left, endogenous Rab1A-knockdown MDA-MB-231 cells co-expressing mCherry-tagged wild-type or mutant Rab1A and ER-GFP were localized by N-SIM. Scale bars = 5 μm. Right, quantification of ER three-way junctions in MDA-MB-231 cells with wild-type or mutant Rab1A overexpression. WT, n = 21; T75A, n = 21; T75D, n = 21. b, Endogenous Rab1A-knockdown MDA-MB-231 cells co-expressing mCherry-tagged wild-type or mutant Rab1A and ER-GFP were treated with nocodazole and analyzed by 3D-SIM. Left, representative images of high- or low-complexity ER. Red box regions were enlarged and subjected to 3D reconstruction. Scale bars = 5 μm. Right, percentage of cells co-expressing mCherry-tagged wild-type or mutant Rab1A and ER-GFP with high-complexity ER. WT, n = 286; T75A, n = 309; T75D, n = 229. Data from a, b were examined over three independent experiments. Data from a were analyzed using one-way Welch’s ANOVA test (P = 0.5245), and pairwise comparisons were performed using Dunnett’s T3 multiple comparisons test. Data are presented as mean ± s.e.m.

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Extended Data Fig. 8 Rab1A binds to ER-shaping proteins.

a-d, GST-Flag or GST-Flag-Rab1A immobilized to glutathione Sepharose 4B was used for the binding assays with the HepG2 (a), HEK293 (b, c) or Hela (d) cell lysates and the samples analyzed by immunoblotting. e, GST-Flag or GST-Flag-Rab1A immobilized to glutathione Sepharose 4B was used for the binding assays in COS-7 cell lysates and analyzed by immunoblotting. f, GST-Flag-tagged Rab1A immobilized to glutathione Sepharose 4B was used for the binding assays in COS-7 cell lysates overexpressing HA-tagged Reep1 or HA-tagged Reep5 and the samples analyzed by immunoblotting. g, Anti-GFP immunoprecipitates were prepared from MDA-MB-231 cells expressing GFP-tagged RTN4B and analyzed by immunoblotting. h, HA-tagged Reep1 bound to HA magnetic beads was used for binding assays with the COS-7 cell lysates and the samples analyzed by immunoblotting. i, j, Anti-mCherry immunoprecipitates were prepared from MDA-MB-231 cells expressing mCherry-tagged ATL1 (i) or ATL3 (j) and analyzed by immunoblotting. k, HA-tagged Reep5 bound to HA magnetic beads was used for binding assays with the COS-7 cell lysates, the samples were analyzed by immunoblotting. l, N-terminal cytosolic domains of ATL1, ATL2 and ATL3 (cyt-ATL) with HA tags were purified and incubated with GST-Flag-Rab1A in vitro for binding assays and analyzed by immunoblotting. m, HA-ATL3 was bound to anti-HA magnetic beads and then incubated with purified GST-Flag-Rab1A, the samples were analyzed by immunoblotting. n, The cytosolic loop connecting two transmembrane hairpins (TMHs) is relatively conserved in REEPs. Left, Sequence alignment of REEPs. Right, Membrane topology of REEPs. o, Binding of Rab1A to RTN peptides. Nogo-40: RIYKGVIQAIQKSDEGHPFRAYLESEVAISEELVQKYSNS (purple dots), RTN4P-1: RIYKGVIQAIQKSDEGH (pink dots), RTN4P-2: PFRAYLESEVAISEELVQKYSNS (green dots), RTN4P-3: ALGHVNCTIKELRR (deep blue dots). The protein-bound peptide fractions (bound/total) were calculated from the MST signals at each peptide concentration. p, Left, cell lysates from wild-type or ATL DKO COS-7 cells were treated with the indicated concentrations of ethylene glycol bis (EGS) and analyzed by immunoblotting with anti-Reep5 antibody. *, monomer; **, dimer; ***, trimer. Right, Reep5 dimers were quantified from three independent experiments. Data shown in a-f, h-m, p are representative of three independent experiments, and in g are from two independent experiments.

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Extended Data Fig. 9 Rab1A has a subtle effect on ATL functions.

a, Rab1A has a subtle effect on ATL1, ATL2 and ATL3 GTPase activity. b, c, Left, Representative traces. Right, the gradient was fractionated into five 50 μL fractions and the reconstitution efficiency of dmATL and membrane-anchored efficiency of dmRab1 analyzed by SDS-PAGE. Data shown in b, c are representative of three independent experiments.

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Extended Data Fig. 10 Phosphorylation at Thr75 promotes Rab1A interacting with ER-shaping proteins.

a, Left, anti-mCherry immunoprecipitates were prepared from MDA-MB-231 cells expressing mCherry-tagged wild-type or mutant Rab1A and analyzed by immunoblotting. Right, co-immunoprecipitated RTN4B was quantified. b, Left, anti-GFP immunoprecipitates were prepared from MDA-MB-231 cells expressing GFP-tagged wild-type or mutant Rab1A and analyzed by immunoblotting. Right, co-immunoprecipitated ATL3 was quantified. c, HA-ATL3 was bound to anti-HA magnetic beads and then incubated with purified GST-Flag-Rab1A (wild-type or mutant). The samples were analyzed by immunoblotting. d, GST-tagged wild-type or mutant Rab1A immobilized to glutathione Sepharose 4B was used for the binding assay with the COS-7 cell lysates and the samples analyzed by immunoblotting. e, mCherry-ATL3 was co-expressed with Flag-tagged WT Rab1A, Rab1A-T75A, or Rab1A-T75D in MDA-MB-231 cells. Anti-mCherry immunoprecipitation was performed and analyzed by immunoblotting. f, Left, cell lysates from COS-7 cells overexpressing RTN4B alone or co-expressing RTN4B and Rab1A (wild-type or mutant) were treated with the indicated concentrations of EGS and analyzed by immunoblotting. *, monomer; **, dimer. Right, RTN4B dimers were quantified. g, Left, cell lysates from COS-7 cells expressing wild-type or mutant Rab1A were treated with the indicated concentrations of EGS and analyzed by immunoblotting with anti-Reep5 antibody. *, monomer; **, dimer; ***, trimer. Right, Reep5 dimers were quantified. h, Left, cell lysates from COS-7 cells overexpressing Reep5 alone or co-expressing Reep5 and Rab1A (wild-type or mutant) were treated with the indicated concentrations of EGS and analyzed by immunoblotting. *, monomer; **, dimer; ***, trimer. Right, Reep5 dimers were quantified. Data shown in a-h are representative of three independent experiments. Data from a, b were analyzed using two-tailed unpaired Student’s t-test. Data are presented as mean ± s.e.m.

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Zhang, W., Zhang, Z., Xiang, Y. et al. Aurora kinase A-mediated phosphorylation triggers structural alteration of Rab1A to enhance ER complexity during mitosis. Nat Struct Mol Biol 31, 219–231 (2024). https://doi.org/10.1038/s41594-023-01165-7

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