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Cell Biology: Ran, Mitosis and the Cancer Connection

2006; Elsevier BV; Volume: 16; Issue: 12 Linguagem: Inglês

10.1016/j.cub.2006.05.032

1879-0445

Helen S. Sanderson, Paul R. Clarke,

Nuclear Structure and Function

The small GTPase Ran has been shown to regulate HURP, a protein that interacts with several mitotic spindle assembly factors. This discovery sheds new light on the role of Ran in the fidelity of mitosis and in cancer. The small GTPase Ran has been shown to regulate HURP, a protein that interacts with several mitotic spindle assembly factors. This discovery sheds new light on the role of Ran in the fidelity of mitosis and in cancer. Cell division requires that one copy of each chromosome is faithfully segregated to each of the daughter cells during mitosis, a remarkable feat that is achieved by the mitotic spindle, a bipolar array of microtubules focussed at each pole by a centrosome. One of the most important features of a successful mitosis is the proper biorientation of all chromosomes on the spindle. This is accomplished through the attachment of microtubules to the kinetochore region of each chromosome, and the stabilization and bundling of microtubules to form kinetochore fibres (k-fibres). Biorientation and congression of a chromosome to the middle of the spindle is relatively slow, but once achieved, may serve as a highway for monoorientated chromosomes, that is those attached to microtubules from only one pole of the spindle, enabling them to hitchhike to the centre of the cell where the density of searching spindle microtubules is high [1Wollman R. Cytrynbaum E.N. Jones J.T. Meyer T. Scholey J.M. Mogilner A. Efficient chromosome capture requires a bias in the 'search-and-capture' process during mitotic-spindle assembly.Curr. Biol. 2005; 15: 828-832Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 2Kapoor T.M. Lampson M.A. Hergert P. Cameron L. Cimini D. Salmon E.D. McEwen B.F. Khodjakov A. Chromosomes can congress to the metaphase plate before biorientation.Science. 2006; 311: 388-391Crossref PubMed Scopus (320) Google Scholar]. Until all chromosomes are successfully attached to microtubules in a biorientated manner, with each kinetochore from the duplicated sister chromatids attached to microtubules from opposite poles of the spindle, the spindle checkpoint prevents separation of the chromatids and premature cell division. When the fidelity of this process is compromised, abnormal numbers of chromosomes can be distributed to the daughter cells (aneuploidy), which is often associated with cancer. Embedded at the regulatory heart of these processes is the small GTPase Ran. The active GTP-bound configuration of Ran is generated by the chromatin-associated guanine nucleotide exchange factor RCC1 and promotes the release of spindle assembly factors from inhibitory complexes with importins [3Clarke P.R. Zhang C. Ran GTPase: a master regulator of nuclear structure and function during the eukaryotic cell division cycle?.Trends Cell Biol. 2001; 11: 366-371Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar]. The generation of Ran-GTP at chromosomes is proposed to guide spindle assembly by providing a positional gradient signal, which has been visualised using fluorescent reporters in Xenopus egg extracts and mammalian cells, most recently by Caudron et al.[4Caudron M. Bunt G. Bastiaens P. Karsenti E. Spatial coordination of spindle assembly by chromosome-mediated signaling gradients.Science. 2005; 309: 1373-1376Crossref PubMed Scopus (241) Google Scholar] and Kalab et al.[5Kalab P. Pralle A. Isacoff E.Y. Heald R. Weis K. Analysis of a RanGTP-regulated gradient in mitotic somatic cells.Nature. 2006; 440: 697-701Crossref PubMed Scopus (288) Google Scholar]. Of the growing portfolio of factors which fall under the aegis of mitotic regulation by Ran-GTP and importins, perhaps the most prominent identified to date is TPX2, which when released from importin-α triggers activation of the Aurora A protein kinase [6Gruss O.J. Vernos I. The mechanism of spindle assembly: functions of Ran and its target TPX2.J. Cell Biol. 2004; 166: 949-955Crossref PubMed Scopus (182) Google Scholar]. During mitosis, activated Aurora A phosphorylates a broad array of proteins, including the tumour suppressors Lats2 and BRCA1, and the close association of Aurora A with the development of human cancers tallies with its central role in centrosome functioning and mitotic progression [7Meraldi P. Honda R. Nigg E.A. Aurora kinases link chromosome segregation and cell division to cancer susceptibility.Curr. Opin. Genet. Dev. 2004; 14: 29-36Crossref PubMed Scopus (286) Google Scholar]. Two papers published recently in Current Biology[8Koffa M.D. Casanova C.M. Santarella R. Kocher T. Wilm M. Mattaj I.W. HURP (Hepatocarcinoma-Upregulated) is part of a Ran-dependent complex involved in spindle.Curr. Biol. 2006; 16: 743-754Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 9Silljé H.H.W. Nagel S. Körner R. Nigg E.A. HURP is a Ran-importin beta regulated protein that stabilizes kinetochore microtubules in the vicinity of chromosomes.Curr. Biol. 2006; 16: 731-742Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar] show that another oncoprotein, hepatocarcinoma-upregulated protein (HURP), is under the direct regulatory control of Ran during mitosis (Figure 1). HURP was first identified as a potential oncogene that is aberrantly expressed in human hepatocellular carcinoma [10Tsou A.P. Yang C.W. Huang C.Y. Yu R.C. Lee Y.C. Chang C.W. Chen B.R. Chung Y.F. Fann M.J. Chi C.W. et al.Identification of a novel cell cycle regulated gene, HURP, overexpressed in human hepatocellular carcinoma.Oncogene. 2003; 22: 298-307Crossref PubMed Scopus (113) Google Scholar]. The expression profile of HURP correlates with that of Aurora A, being periodically expressed during the cell cycle and peaking at G2/M. HURP is a substrate for the kinase, suggesting that they may be coordinately regulated through stabilisation of HURP by Aurora A [11Yu C.T. Hsu J.M. Lee Y.C. Tsou A.P. Chou C.K. Huang C.Y. Phosphorylation and stabilization of HURP by Aurora-A: implication of HURP as a transforming target of Aurora-A.Mol. Cell Biol. 2005; 25: 5789-5800Crossref PubMed Scopus (99) Google Scholar]. In the new studies, Koffa et al.[8Koffa M.D. Casanova C.M. Santarella R. Kocher T. Wilm M. Mattaj I.W. HURP (Hepatocarcinoma-Upregulated) is part of a Ran-dependent complex involved in spindle.Curr. Biol. 2006; 16: 743-754Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar] employed Xenopus extracts to identify novel mitotic targets of Ran that promote bipolar spindle formation and characterised a HURP-containing complex with such activity. They demonstrated that HURP is a microtubule-associated protein (MAP) in mitotic extracts [8Koffa M.D. Casanova C.M. Santarella R. Kocher T. Wilm M. Mattaj I.W. HURP (Hepatocarcinoma-Upregulated) is part of a Ran-dependent complex involved in spindle.Curr. Biol. 2006; 16: 743-754Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar]. Complementary cell analyses by Silljé et al.[9Silljé H.H.W. Nagel S. Körner R. Nigg E.A. HURP is a Ran-importin beta regulated protein that stabilizes kinetochore microtubules in the vicinity of chromosomes.Curr. Biol. 2006; 16: 731-742Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar] revealed a striking subcellular distribution, with HURP residing predominantly at k-fibres in prometaphase, where it remains until telophase when cellular levels sharply decline. This decrease is consistent with earlier studies indicating that CDK1/cyclin B phosphorylation (at distinct sites to those targeted by Aurora A) triggers ubiquitination of HURP by SCFFbx7 and targets it for proteosomal degradation [12Hsu J.M. Lee Y.C. Yu C.T. Huang C.Y. Fbx7 functions in the SCF complex regulating Cdk1-cyclin B-phosphorylated hepatoma up-regulated protein (HURP) proteolysis by a proline-rich region.J. Biol. Chem. 2004; 279: 32592-32602Crossref PubMed Scopus (88) Google Scholar]. Koffa et al.[8Koffa M.D. Casanova C.M. Santarella R. Kocher T. Wilm M. Mattaj I.W. HURP (Hepatocarcinoma-Upregulated) is part of a Ran-dependent complex involved in spindle.Curr. Biol. 2006; 16: 743-754Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar] showed that, in mitotic Xenopus extracts, HURP forms part of a complex comprising two additional MAPs, TPX2 and XMAP215, together with the plus-end directed kinesin Eg5, and Aurora A. As a unit, this complex is required to establish a bipolar spindle from aster-like structures. Aurora A kinase activity is essential for the formation and function of the complex, and it may target not only HURP, but potentially also other complex members. Given the established functions of these spindle assembly factors — TPX2 in chromosome-associated microtubule nucleation and k-fibre formation [13Tulu U.S. Fagerstrom C. Ferenz N.P. Wadsworth P. Molecular requirements for kinetochore-associated microtubule formation in mammalian cells.Curr. Biol. 2006; 16: 536-541Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar], XMAP215 in microtubule nucleation [14Popov A.V. Severin F. Karsenti E. XMAP215 is required for the microtubule-nucleating activity of centrosomes.Curr. Biol. 2002; 12: 1326-1330Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar], Eg5 in maintenance of spindle bipolarity [15Goshima G. Wollman R. Stuurman N. Scholey J.M. Vale R.D. Length control of the metaphase spindle.Curr. Biol. 2005; 15: 1979-1988Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar] — the finding that they are united in a single complex provides the potential for coordination of their independent activities, under the regulatory control of Aurora A. Aurora A, TPX2 and possibly also Eg5 have been independently verified as mitotic targets of Ran [6Gruss O.J. Vernos I. The mechanism of spindle assembly: functions of Ran and its target TPX2.J. Cell Biol. 2004; 166: 949-955Crossref PubMed Scopus (182) Google Scholar]. In Xenopus, Ran-GTP stimulates the interaction between TPX2 and Aurora A (Eg2) and activates Aurora A in a microtubule-dependent manner, whilst simultaneously preventing protein phosphatase 1-induced inactivation [6Gruss O.J. Vernos I. The mechanism of spindle assembly: functions of Ran and its target TPX2.J. Cell Biol. 2004; 166: 949-955Crossref PubMed Scopus (182) Google Scholar, 16Tsai M.Y. Wiese C. Cao K. Martin O. Donovan P. Ruderman J. Prigent C. Zheng Y. A Ran signalling pathway mediated by the mitotic kinase Aurora A in spindle assembly.Nat. Cell Biol. 2003; 5: 242-248Crossref PubMed Scopus (305) Google Scholar]. It is possible that Ran may regulate the intrinsic activities of HURP, and potentially also the formation of the larger multifunctional complex described by Koffa et al.[8Koffa M.D. Casanova C.M. Santarella R. Kocher T. Wilm M. Mattaj I.W. HURP (Hepatocarcinoma-Upregulated) is part of a Ran-dependent complex involved in spindle.Curr. Biol. 2006; 16: 743-754Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar]. It will be interesting to establish whether this complex is present in somatic cells — Aurora A and the other mammalian counterparts of Xenopus complex members have been localised mainly to the spindle poles [7Meraldi P. Honda R. Nigg E.A. Aurora kinases link chromosome segregation and cell division to cancer susceptibility.Curr. Opin. Genet. Dev. 2004; 14: 29-36Crossref PubMed Scopus (286) Google Scholar]. Silljé et al.[9Silljé H.H.W. Nagel S. Körner R. Nigg E.A. HURP is a Ran-importin beta regulated protein that stabilizes kinetochore microtubules in the vicinity of chromosomes.Curr. Biol. 2006; 16: 731-742Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar] suggest that HURP may be phosphorylated at the spindle poles by Aurora A, allowing HURP to translocate to k-fibres in a Ran-GTP-dependent manner. Knockdown of HURP by RNA interference (RNAi) leads to impaired chromosome capture and increased duration of congression in HeLa cells, resultant from loss of k-fibre integrity. Nonetheless, cells do eventually align their chromosomes and progress through mitosis. These data indicate that HURP is an essential component in k-fibre formation and/or stabilisation, a view further strengthened by in vitro analysis demonstrating that recombinant human HURP induces tubulin polymerisation, stabilises existing microtubules and induces microtubule bundling in Xenopus egg extracts [8Koffa M.D. Casanova C.M. Santarella R. Kocher T. Wilm M. Mattaj I.W. HURP (Hepatocarcinoma-Upregulated) is part of a Ran-dependent complex involved in spindle.Curr. Biol. 2006; 16: 743-754Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar]. Notably, these functions appear to be independent of microtubule nucleation, and therefore suggest that the primary roles of HURP relate to microtubule polymerisation and stabilisation. HURP can also be added to the growing list of spindle assembly factors whose activity is regulated by importins. Silljé et al.[9Silljé H.H.W. Nagel S. Körner R. Nigg E.A. HURP is a Ran-importin beta regulated protein that stabilizes kinetochore microtubules in the vicinity of chromosomes.Curr. Biol. 2006; 16: 731-742Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar] present evidence for a direct interaction between importin-β and HURP and show that this interaction inhibits the microtubule 'bundling' activity of HURP in vitro and localisation to k-fibres in cells. This suggests that localised release of HURP from importin β near chromatin in prometaphase may be the trigger for k-fibre stabilisation which persists until cytokinesis is complete. Interestingly, the distinctive localisation of HURP was similarly observed for another essential component of the mitotic apparatus, NuSAP [17Raemaekers T. Ribbeck K. Beaudouin J. Annaert W. Van Camp M. Stockmans I. Smets N. Bouillon R. Ellenberg J. Carmeliet G. NuSAP, a novel microtubule-associated protein involved in mitotic spindle organization.J. Cell Biol. 2003; 162: 1017-1029Crossref PubMed Scopus (160) Google Scholar]. This newly characterised protein possesses potent microtubule stabilisation and bundling activities in Xenopus egg extracts, distinct functions that are differentially regulated by importin-α, importin-β and importin-7 [18Ribbeck K. Groen A.C. Santarella R. Bohnsack M.T. Raemaekers T. Kocher T. Gentzel M. Gorlich D. Wilm M. Carmeliet G. et al.NuSAP, a mitotic RanGTP target that stabilizes and cross-links microtubules.Mol. Biol. Cell. 2006; 17: 2646-2660Crossref PubMed Scopus (94) Google Scholar]. Together, the papers by Silljé et al.[9Silljé H.H.W. Nagel S. Körner R. Nigg E.A. HURP is a Ran-importin beta regulated protein that stabilizes kinetochore microtubules in the vicinity of chromosomes.Curr. Biol. 2006; 16: 731-742Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar] and Koffa et al.[8Koffa M.D. Casanova C.M. Santarella R. Kocher T. Wilm M. Mattaj I.W. HURP (Hepatocarcinoma-Upregulated) is part of a Ran-dependent complex involved in spindle.Curr. Biol. 2006; 16: 743-754Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar] provide important new insights into the role of HURP in the control of microtubule organisation and the coordination of this function within a larger complex that is essential for the formation of the bipolar spindle. The overexpression of HURP and Aurora A in cancers suggests that misregulation of this complex plays a role in carcinogenesis. It remains to be fully elucidated what the functional contributions of the individual components are, how they may cooperate, and precisely how the complex is regulated by Ran-GTP and importins. Understanding the regulation of mitotic spindle function by Ran may lead to important new insights into the development of cancer.