nature cell biology volume 11 | number 1 | JAnuArY 2009 11

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Ran out of the nucleus for apoptosisAndrew Wilde and Yixian Zheng

induction of apoptosis causes active dissipation of the RanGTP gradient across an intact nuclear envelope by immobilizing RCC1, the guanine nucleotide exchange factor for RanGTPase, on chromatin. The subsequent reduction in nuclear trafficking prevents the movement of nF-κB into the nucleus, thereby allowing apoptosis to proceed.

Apoptosis is a highly regulated cell death pro-gram, essential for the development of multicel-lular organisms. Cells can receive specific signals to undergo or suppress apoptosis. These signals can be extrinsic through the binding of ligands to cell surface receptors, or intrinsic through sensing stresses such as DNA damage. However, the signalling thresholds in the cell that may tip the balance towards life or death are not fully understood. One common regulatory theme of apoptosis involves the redistribution of factors from one cellular compartment to another, in particular, the redistribution of factors between the cytosol, mitochondria and nucleus. For example, in some cells, movement of the tumour suppressor p53 from the nucleus to the mito-chondria can trigger apoptosis1, whereas block-ing transport of NF-κB from the cytoplasm to the nucleus allows apoptosis resistance2. Indeed changes in nuclear trafficking have long been suggested to have a role in apoptosis3.

An early characteristic of cells undergo-ing apoptosis is the appearance of condensed chromosomes, a phenotype that can also be induced by depletion of RCC1, the nucleotide exchange factor for the GTPase Ran4. The GTPase Ran is essential for nuclear trafficking, as it controls the assembly of nuclear trans-port receptor/cargo complexes in a manner dependent on its guanine nucleotide-bound state. Formation of RanGTP is catalysed by RCC1 in the nucleus, whereas RanGTP hydrolysis occurs in the cytoplasm, thus cre-ating a gradient of RanGTP across the nuclear membrane that drives the directed transport of cargo across the membrane.

To determine whether the onset of apop-tosis could be linked to alterations in the RanGTP gradient, Wong et al.5 (page 36 of this issue) treated cells with the topoisomerase II inhibitor VP16 (etoposide), which causes

DNA damage and induces apoptosis. VP16 treatment leads to redistribution of Ran from the nucleus to the cytosol and a reduction in RanGTP levels. Mislocalization of Ran was also seen in tsBN2 cells, at the non-permissive temperature that induces RCC1 degradation in these cells. These data suggest that during apoptosis the dissipation of the RanGTP gra-dient across the nuclear membrane could be due to the inhibition of RCC1.

As RCC1 protein levels did not change dur-ing this early phase of apoptosis, one possible model was that RCC1 activity was inhibited. Previously, Li and co-workers had shown that RCC1 activity was coupled to its dynamic interaction with chromosomes, an interaction that can be mediated in part through binding to histones H2A and H2B6,7. Using fluores-cence recovery after photobleaching (FRAP) and fluorescence loss in photobleacing (FLIP), Wong et al. found that RCC1–GFP bound more tightly to chromosomes on stimulation of apoptosis, by treating cells with etoposide. The key question then is how the signal to initi-ate apoptosis is translated into tighter binding of RCC1 to chromosomes and the presumed loss in RCC1 activity?

Previous studies have shown that phospho-rylation of Ser 14 of histone H2B (H2BS14) regulates chromosome condensation in apop-tosis8. Therefore, Wong et al. asked whether this phosphorylation event could regulate the interaction between RCC1 and the core histones on the chromatin. Upon expression of histone H2B containing S14D (H2BS14D), a phosphomimetic mutation, GFP–RCC1 was more tightly bound to chromosomes when compared with cells expressing wild-type or H2BS14A, the unphosphorylatable mutant. Furthermore, in cells expressing H2BS14D, Ran redistributed to the cytoplasm and there was less RanGTP, compared with control cells, suggesting that phosphorylation of histone H2B caused immobilization and inhibition of RCC1 during apoptosis. Various histone modifications have been linked to transcrip-tional regulation. However, the study of Wong

et al. provides evidence that histone modifica-tion can be interpreted in a non-transcriptional fashion by RCC1 to regulate RanGTP produc-tion during apoptosis.

To further determine the function of H2B phosphorylation in signalling, RCC1 immobi-lization and inhibition, Wong et al. examined the role of the Mst1 kinase, which is known to phosphorylate H2BS14 during apoptosis8. In unperturbed cells, Mst1 is cytosolic but dur-ing apoptosis, a carboxy-terminal domain of Mst1 that contains a nuclear export signal is cleaved off by caspases 3 and 9, resulting in a nuclear form of Mst1 which can phosphorylate H2BS14. The authors found that on expression of a fragment of Mst1 that corresponds to the caspase cleaved form, GFP–RCC1 became more tightly bound to chromosomes with the corre-sponding redistribution of Ran into the cyto-plasm and the general reduction in RanGTP levels. These phenotypes were reminiscent of those seen after induction of apoptosis by DNA damage. Furthermore, reducing Mst1 protein levels by siRNA suppressed the Ran and RCC1 phenotypes normally induced by DNA damage at the onset of apoptosis. Therefore, activation of apoptosis creates a phospho-histone code on the chromatin, which is read by RCC1 to dampen the production of RanGTP.

An important question is whether and how RanGTP dissipation could facilitate apopto-sis. The disruption of the RanGTP gradient could be a by-product of inducing apoptosis or have an active role in promoting cell death. To address this question, Wong et al. examined the localization of NF-κB, a transcription fac-tor that normally localizes to the cytoplasm in a repressed form but can be stimulated by TNFα to enter the nucleus where it suppresses apoptosis2. Wong et al. found that when apop-tosis was induced using the same conditions that collapsed the RanGTP gradient across the nuclear membrane, NF-κB failed to relocalize to the nucleus upon TNFα treatment. However, following addition of TNFα in the absence of Mst1, NF-κB relocalized to the nucleus after treatment with DNA damaging agents. This

Andrew Wilde is in the Department of Molecular Genetics, University of Toronto, M5S 1A8 Toronto, ON, Canada. Yixian Zheng is in the Department of Embryology, Carnegie Institution for Science and Howard Hughes Medical Institute, Baltimore, Maryland 21218, USA.e-mail: andrew.wilde@utoronto.ca

© 2009 Macmillan Publishers Limited. All rights reserved.

12 nature cell biology volume 11 | number 1 | JAnuArY 2009

n e w s a n d v i e w s

entry of NF-κB into the nucleus rescued cells from apoptosis. Taken together these data sug-gest that during the onset of apoptosis, collapse of the RanGTP gradient as a result of histone phosphorylation could tip the balance in the cell in favour of continuing down the apoptotic pathway by preventing the activation of an anti-apoptotic pathway (Fig. 1).

It is tempting to speculate that these find-ings represent the tip of the iceberg and that during the early stages of apoptosis, dissipa-tion of the RanGTP gradient across the nuclear membrane affects the subcellular localization of multiple factors involved in promoting apoptosis. Indeed, a recent study found that overexpression of Ran suppressed paclitaxel-induced apoptosis, in part by preventing the redistribution of the pro-apoptotic factor Bax to mitochondria9. Therefore, dissipation of the RanGTP gradient need not necessarily be restricted to preventing relocalization of fac-tors to the nucleus, but could also be involved in redistribution of nuclear proteins into the cytosol. Indeed, several factors have been

found to move from the nucleus to the cytosol to regulate apoptosis. In the case of p53, this leads to activation of apoptosis by initiating permeabilization of the outer mitochondrial membrane, which is independent of the tran-scriptional activity of p53 (ref. 1). Prohibitin also leaves the nucleus upon induction of apoptosis, a relocation that may contribute to its pro-apoptotic activity10. Furthermore, the release of SIR-2.1, the Caenorhabditis elegans homologue of the human histone deacetylase SIRT1, from nuclei is also linked to a pro-apop-totic pathway11, raising the possibility that there could be further modifications to the histone code during apoptosis.

The change in RanGTP level may also affect apoptosis through the nuclear export factor Cas/Cse1, which on binding to RanGTP in the nucleus, exports cargo to the cytoplasm. The mammalian Cas/Cse1 was initially found to increase the susceptibility of cells to apop-totic stimuli12. Cas/Cse1 has recently been shown to enhance expression of a subset of p53 target genes known to promote apoptosis13.

As RanGTP binds to Cas/Cse1, reduction of RanGTP in the cell could affect the ability of Cas/Cse1 to regulate the expression of pro-apoptosis genes.

Interestingly, the change in nuclear RanGTP levels also regulates microtubule reorgani-zation during apoptosis. A study by Moss et al.14 found, in agreement with Wong et al. that RanGTP moves to the cytoplasm during apop-tosis. Concomitantly, TPX2, a protein involved in spindle assembly in mitosis, is released into the cytoplasm to reorganize microtubules.

Given the broad effect of RanGTP levels on apoptosis, the Ran system could be a poten-tial target for developing therapeutic reagents that either promote or suppress cell death. A recent study suggests that this maybe a practical approach. When Ran expression was suppressed specifically in neuroblastoma cells by siRNA in mice, both tumour growth and apoptosis were reduced15.

The findings by Wong et al. suggest that an important part of promoting apoptosis is the active disruption of nuclear trafficking before the wholesale breakdown of the nuclear enve-lope and mixing of the cytosolic and nuclear compartments. These findings lay the basis for a deeper understanding of how spatial and temporal signals are organized within apoptotic cells. It will be interesting to see the full extent to which dissipation of the RanGTP gradient drives the cell towards its ultimate destruction.

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527 (2004).8. Cheung, W. L. et al. Cell 113, 507–517 (2003).9. Woo, I. S. et al. Apoptosis 13, 1223–1231 (2008).10. Rastogi, S., Joshi, B., Fusaro, G. & Chellappan, S. J.

Biol. Chem. 281, 2952–2959 (2006).11. Greiss, S., Hall, J., Ahmed, S. & Gartner, A. Genes Dev.

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(2008).

RCC1

RanGDP RanGTP

Mst1

Apoptosis

H2BMst1 H2B-p

NF-κB

Cytoplasm Nucleus

Chromatin

Figure 1 Diagram showing how apoptotic stimuli target Ran and NF-κB. Apoptotic stimuli activate caspases (data not shown), which cleave the nuclear export signal of the Mst1 kinase, allowing it to localize to the nucleus to phosphorylate histone H2B. Phosphorylated H2B inhibits the chromatin-coupled production of RanGTP in the nucleus. The resulting dissipation of nuclear RanGTP in turn blocks entry of NF-κB into the nucleus and prevents the initiation of the anti-apoptotic program.

© 2009 Macmillan Publishers Limited. All rights reserved.