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Subcellular Localization and Nucleocytoplasmic Transport of the Chromosomal Passenger Proteins Before Nuclear Envelope Breakdown

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Subcellular Localization and Nucleocytoplasmic Transport of the Chromosomal Passenger Proteins Before Nuclear Envelope Breakdown Oncogene (2006) 25, 4867–4879 & 2006 Nature Publishing Group All rights reserved 0950-9232/06 $30.00 www.nature.com/onc ORIGINAL ARTICLE Subcellular localization and nucleocytoplasmic transport of the chromosomal passenger proteins before nuclear envelope breakdown JA Rodriguez1, SMA Lens2, SW Span1, G Vader2, RH Medema2, FAE Kruyt1 and G Giaccone1 1Department of Medical Oncology, VU University Medical Center, Amsterdam, The Netherlands and 2Division of Molecular Biology, Netherlands Cancer Institute, Amsterdam, The Netherlands As mitosis progresses, the chromosomal passenger pro- other during mitosis forming one or more protein teins (CPPs) Survivin, Aurora B, INCENP and Borealin complexes that play important roles in early and late dynamically colocalize to mitotic structures. Chromoso- mitoticevents. Aurora B is a member of a conserved mal passenger proteins are already expressed during G2, family of serine/threonine protein kinases originally whereas the nuclear envelope is only disassembled at the identified as a mammalian homologue of Drosophila and end of prophase. However, the mechanisms that modulate yeast proteins required for chromosome segregation their nucleocytoplasmic localization before nuclear envel- (Bischoff et al., 1998; Terada et al., 1998). Aurora B ope breakdown (NEB) are poorly characterized. Using phosphorylates a variety of substrate proteins and epitope-tagged proteins, we show that Aurora B, like regulates many aspects of cell division, from chromo- Survivin, undergoes CRM1-mediated nucleocytoplasmic some condensation to cytokinesis (reviewed by Andrews shuttling, although both proteins lack identifiable ‘classi- et al., 2003). INCENP is a large protein that has been cal’ nuclear transport signals. On the other hand, proposed to act as a scaffold for the binding of the other INCENP resides more stably in the nucleus and contains passenger proteins (Bolton et al., 2002). Aurora B multiple nuclear localization signals. Finally, Borealin was phosphorylates INCENP, which, in turn, activates the detected in the nucleolus and the cytoplasm, but its kinase activity of Aurora B in a positive feedback loop cytoplasmic localization is not directly regulated by (Bishop and Schumacher, 2002; Honda et al., 2003). CRM1. Coexpression experiments indicate that the Survivin is a member of the inhibitor of apoptosis nuclear localization of Aurora B, but not of Survivin, is protein family that plays a dual role as regulator of both modulated by INCENP and that Survivin prevents the apoptosis and cell division (reviewed by Altieri, 2003a). nucleolar accumulation of Borealin. Our data reveal that, Like INCENP, Survivin has been reported to constitute in contrast to their closely related localization during both a substrate and an activator for Aurora B (Chen mitosis, the nucleocytoplasmic localization of the CPPs et al., 2003; Wheatley et al., 2004). Borealin, which was before NEB is largely unrelated. Furthermore, the specific independently isolated as DasraB, is the most recently effect of INCENP and Survivin on the localization of identified and the least characterized subunit of the Aurora B and Borealin, respectively, suggests that chromosomal passenger complex (Gassman et al., 2004; different complexes of CPPs may exist not only during Sampath et al., 2004). Borealin is phosphorylated by mitosis, as recently reported, but also before NEB. Aurora B in vitro but, unlike Survivin and INCENP, Oncogene (2006) 25, 4867–4879. doi:10.1038/sj.onc.1209499; does not appear to increase the activity of the kinase published online 20 March 2006 (Gassman et al., 2004). Several lines of evidence implicate at least two of the CPPs, Survivin and Aurora Keywords: Survivin; Borealin; INCENP; Aurora B; B, in the development and progression of human nuclear envelope breakdown; nuclear transport tumors, and these proteins hold potential as targets for novel anticancer therapies (reviewed by Altieri, 2003b; Giet et al., 2005). The hallmark of CPPs is their dynamicpattern of subcellular localization during mitosis: they associate Introduction with the centromeric region of chromosomes from prophase to metaphase, relocate to the spindle midzone The chromosomal passenger proteins (CPPs) Survivin, and equatorial cortex during anaphase and finally Aurora B, INCENP and Borealin interact with each accumulate in the midbodies during cytokinesis (Adams et al., 2001a). Although their levels are maximal during Correspondence: Dr JA Rodriguez, Department of Medical Oncology, mitosis, the CPPs are already expressed before the VU University Medical Center, Academic Hospital Vrije Universiteit, disassembly of the nuclear envelope, or nuclear envelope KRIGO BR232, Postbus 7057, 1007 MB Amsterdam, The Nether- breakdown (NEB), takes place at the transition between lands. E-mail: [email protected] prophase and prometaphase (reviewed by Burke and Received 8 September 2005; revised 13 January 2006; accepted 9 Ellenberg, 2002). Before NEB, the transport of proteins February 2006; published online 20 March 2006 between the nucleus and cytoplasm occurs exclusively Localization of chromosomal passenger proteins before NEB JA Rodriguez et al 4868 through the nuclear pore complex, and is usually an tion, we generated mammalian expression plasmids active process, mediated by soluble import/export encoding full-length human Aurora B, INCENP receptors that bind to nuclear localization signals and Borealin proteins with small VSV-epitope tags (NLSs) and nuclear export signals (NESs) in the cargo fused at their amino-terminal end, and their localization protein, respectively (Mattaj and Englmeier, 1998). in transiently transfected MCF-7 cells was analysed Dynamic nucleocytoplasmic transport constitutes an by immunofluorescence using antitag antibodies important mechanism that regulates the subcellular (Figure 1a). As a marker of nuclear envelope integrity, localization and function of several cancer-associated green fluorescent protein (GFP)-lamin A was co- proteins (Fabbro and Henderson, 2003). transfected with the CPPs. Consistent with our previous In contrast to the detailed knowledge on the findings, Flag-Survivin was predominantly detected in mitotic localization of CPPs, the mechanisms that the cytoplasm in cells where the nuclear envelope contribute to regulate their subcellular distribution in remained intact. VSV-Aurora B was detected in the the presence of an intact nuclear envelope are poorly nucleus and cytoplasm. Variable levels of the protein in characterized. In this respect, we have previously each compartment were observed in different cells, shown that Survivin behaves as a nuclear shuttling ranging from predominantly nuclear (N>C) to mostly protein before NEB, and is actively excluded from the cytoplasmic (C>N). Similar results were obtained using nucleus by a mechanism mediated by the nuclear Aurora B-HA, which bears a carboxy-terminal HA tag export receptor CRM1 (Rodriguez et al., 2002). In the (not shown). On the other hand, VSV-INCENP showed present study, we show that Aurora B also accesses, a clear nuclear localization in most transfected cells. directly or indirectly, the CRM1-dependent pathway Finally, VSV-Borealin was detected in the nucleus, with of nuclear export. In contrast, CRM1 does not appear a prominent nucleolar accumulation, and in the to play a direct role in the nucleocytoplasmic distribu- cytoplasm. Importantly, the three VSV-tagged CPPs tion of INCENP or Borealin. Furthermore, we readily localized to the centromeres during metaphase have functionally tested several potential nuclear and the midbody during telophase/cytokinesis as ex- transport signals in Aurora B and INCENP, and pected (Figure 1a), thus confirming that the small tags identified three independent NLSs in INCENP. Inter- do not grossly interfere with the subcellular targeting of estingly, our results suggest that, despite their closely these proteins. We determined the localization of each of related localization during mitosis, the subcellular the CPPs in at least 200 transfected cells. As illustrated localization of the CPPs before NEB is partially in Figure 1b, Survivin and INCENP were consistently unrelated. In this respect, coexpression experiments localized in the cytoplasm and nucleus, respectively, showed that INCENP induces the nuclear accumulation whereas Aurora B and Borealin showed a more of Aurora B, but not of Survivin. On the other hand, heterogeneous distribution in the population of trans- Survivin, through its carboxy-terminal domain, prevents fected cells. the targeting of Borealin to the nucleolus. Such specific These results indicate that despite their close related effect of INCENP and Survivin on the localization of targeting during mitosis, the CPPs show different Aurora B and Borealin, respectively, suggests that patterns of subcellular localization before NEB, suggest- different complexes of these CPPs may exist before ing that the distribution of these proteins in the presence NEB, in line with recent evidence indicating that of an intact nuclear envelope may be, at least partially, chromosomal passenger complexes of different compo- regulated in an independent manner. sition may exist later during mitosis (Gassman et al., 2004). Our findings, in summary, begin to elucidate the Aurora B undergoes CRM1-mediated nuclear export molecular mechanisms that modulate the subcellular The nucleocytoplasmic distribution of Aurora B fused distribution of the CPPs in the presence of an intact to a MYC tag, similar in size to the VSV and HA nuclear envelope,
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