Keeping Survivin Nimble at Centromeres in Mitosis

Trần Hoàng Dũng

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Science 2 December 2005:
Vol. 310. no. 5753, pp. 1443 - 1444
DOI: 10.1126/science.1121952
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CELL BIOLOGY:
Keeping Survivin Nimble at Centromeres in Mitosis
William C. Earnshaw*
No one would expect an orchestra to perform Beethoven's ninth symphony without a conductor. It is therefore no surprise that when cells perform their most dramatic tour de force--division by mitosis--the process is carefully directed by a team of regulators. Like a conductor, these regulators activate key players in the mitotic program at precise times and locations within the cell. Some act globally. For example, activation of key cyclin-dependent kinases triggers the start of mitotic events throughout the cell, whereas activation of the ubiquitin-dependent proteolysis system by the anaphase-promoting complex/cyclosome similarly triggers the exit from mitosis. Other regulators, such as the chromosomal passenger kinase complex, link this temporal regulation with action at particular locations in the cell. They do this by moving from place to place to target critical cellular components and choreograph mitotic progression (1, 2). On page 1499 in this issue, Vong et al. (3) report the surprising discovery that the mobility of one of the essential components of the passenger complex, survivin, is regulated by its modification with the protein ubiquitin.

Survivin has attracted considerable interest--and controversy--over its brief history. Because sequence motifs in the protein resemble those of baculovirus "inhibitor of apoptosis" proteins, it was originally linked to the regulation of cell death. However, in cells or in mice lacking survivin, the phenotypes most closely correlate with defects in mitosis (4, 5). The link with mitosis was confirmed when it was realized that survivin is a member of the chromosomal passenger complex, proteins that move between chromosomes and the spindle midzone during cell division. The passenger complex also includes the aurora B protein kinase, inner centromere protein (INCENP), and borealin/Dasra-B (6, 7) (see the figure). This association of proteins corrects attachment errors between chromosomes and the mitotic spindle, regulates the quality-control checkpoint that monitors those attachments, and ensures the proper completion of cytokinesis (2).

Perhaps the most interesting aspect of chromosomal passenger behavior is the way in which the complex moves from place to place during mitosis. The complex is present and functional at centromeres (that part of the chromosome to which the mitotic spindle fibers attach) during prometaphase, when chromosomes begin to bind to microtubules. However, it relocates to the central spindle at the onset of anaphase, when chromosomes migrate to opposite poles of the cell. The complex then moves to the equatorial cortex just before cleavage furrow assembly and cytokinesis ensues.

How does this chromosomal passenger complex move from site to site? Many proteins change their behavior when they are modified by phosphorylation, but Vong et al. reveal that modification with ubiquitin has a key role regulating survivin mobility (3). They show that in the cytosol of frog eggs, where components required for cell division during early embryonic development are stockpiled, survivin is associated both with a deubiquitinating enzyme known as xFAM, and a complex of three proteins--p97, NP14, and Ufd1--that together recruit ubiquitin ligases to target proteins (8).

Ubiquitin is best known for its role in protein degradation, but it has other functions as well. Ubiquitin can be attached to a target protein as a chain, with links via the lysine 48 (K48) residue of the ubiquitin. If the polyubiquitin chain is linked to the target protein via this residue, the modification acts as a signal for degradation of the target protein. On the other hand, linkage of ubiquitin proteins to each other via lysine 63 (K63) creates a "flag" that is important for regulating the behavior of the attached protein (9). Interestingly, in mitotic cells, ubiquitin with both K43 and K68 linkages is found on survivin.


Survivin behavior at centromeres is regulated by ubiquitin. (Top left) In a prometaphase animal cell, survivin (red) is localized at centromeres, where it is highly dynamic. (Top right, enlargement) Survivin is a member of the chromosomal passenger complex, which coordinates many key events during mitosis. The Aurora B kinase regulates microtubule attachment and checkpoint function at centromeres early in mitosis. (Top right) INCENP (pink fluorescence), which colocalizes with survivin in the inner centromere, is flanked by green fluorescent dots showing the points of microtubule attachment at kinetochores. (Center) Survivin targeting to centromeres requires the attachment of ubiquitin (Ub), a modification that is stimulated by the p97-NP14-Ufd1 complex. The dynamic behavior of survivin at centromeres requires the activity of the deubiquitinating enzyme FAM. (Bottom left) Later, during anaphase, when survivin transfers to the central mitotic spindle, its behavior no longer requires p97-NP14-Ufd1 and FAM.
CREDIT: K. SUTLIFF/SCIENCE. INSET: ALASTAIR MACKAY/JOHNS HOPKINS UNIVERSITY

The protein complex of p97-NP14-Ufd1 acts as a chaperone that recruits ubiquitin ligases to target proteins. When expression of the Ufd1 subunit is reduced by RNA interference (RNAi), the amount of K63-linked polyubiquitin on survivin drops substantially, but the overall expression level of survivin does not change (3). This suggests that the K63-linked ubiquitin might be involved in regulating the behavior and function of survivin, rather than its stability. Indeed, in cells where Ufd1 expression is reduced by RNAi, survivin is unable to accumulate at centromeres, and cells have a hard time aligning their chromosomes on the spindle.
Depletion of hFAM from a human cell line by RNAi also has no effect on survivin expression, but it does cause mitotic abnormalities including misaligned chromosomes and lagging chromosomes at anaphase (3). Interestingly, although survivin depletion by RNAi causes a marked cytokinesis defect, reduction of FAM expression by RNAi does not. Thus, K63 ubiquitination of survivin is involved in regulating its function at centromeres, but not at the cleavage furrow. In keeping with this, although survivin behavior at centromeres is perturbed when FAM expression is reduced, its localization to the central mitotic spindle and midbody (the microtubule bundle that forms between the two separated daughter cell nuclei late in mitosis) appears to be unaffected.

FAM turns out to be required for survivin behavior at centromeres in a particularly interesting way. In FAM-depleted cells, the amount of survivin on the centromeres of chromosomes that are aligned on the mitotic spindle decreases, but survivin (and aurora B) on misaligned chromosomes increases, with the protein spilling over onto the chromosome arms. Studies using fluorescent probes to quantify movements of molecules inside living cells have shown that survivin localization at centromeres is dynamic (10, 11). These dynamics are substantially (up to 50-fold) quenched after FAM expression is reduced (3).

Vong et al. (3) find that when the amount of K63-linked ubiquitin on survivin is too low, the protein cannot accumulate at centromeres, and chromosome segregation is perturbed. On the other hand, if too much K63-linked ubiquitin is associated with survivin, then the protein is stuck at centromeres and cannot move around normally. This also perturbs chromosome segregation. Together, these results reveal that the balance between K63-linked ubiquitination and deubiquitination plays a key role in regulating survivin function, apparently by regulating the mobility of the protein (see the figure). Why the inhibition of survivin dynamics at centromeres, caused by overaccumulation of K63-linked ubiquitin, impairs the ability of the chromosomal passenger complex to tinker with chromosome attachments to the spindle microtubules is not known. Nor is it clear how this in turn causes defects in chromosome alignment in the mitotic spindle and segregation when cells enter anaphase. Apparently, the correct balance between K63-linked ubiquitination promoted by p97-NP14-Ufd1, and deubiquitination by FAM, ensures that survivin spends just the right amount of time at centromeres to properly regulate chromosome-spindle attachments.

In addition to illustrating a new role for ubiquitin in controlling the behavior of a key mitotic regulator during mitosis, the Vong et al. study raises interesting questions for future research. As pointed out by the authors, there are myriads of deubiquitinating enzymes and their regulators, whose functions remain to be determined. Are we on the cusp of discovering a whole new role for ubiquitin in regulating the dynamic mobility of cellular proteins in mitosis and beyond?

References and Notes

M. Carmena, W. C. Earnshaw, Nat.Rev. Mol. Cell Biol. 4, 842 (2003). [Medline]
P. Vagnarelli,W. C. Earnshaw, Chromosoma 113, 211 (2004).[Medline]
Q. P. Vong, K. Cao, H. Y. Li, P. A. Iglesias, Y. Zheng, Science 310, 1499 (2005).
E. K. Speliotes, A. Uren, D. Vaux, H. R. Horvitz, Mol. Cell 6, 211 (2000). [Medline]
A. G. Uren et al., Curr. Biol. 10, 1319 (2000). [Medline]
M. A. Bolton et al., Mol. Biol. Cell 13, 3064 (2002). [Medline]
R. Gassmann et al., J. Cell Biol. 166, 179 (2004). [Medline]
FAM (fat facet in mouse) is also known as faf (fat facet) in the fly Drosophila melanogaster and as USP9x in human. Here, I use hFAM and xFAM to refer to human and Xenopus FAM, respectively. p97 is an adenosine triphosphatase, six copies of which interact with a dimer of NP14-Udf1.
L. Sun, Z. J. Chen, Curr.Opin. Cell Biol. 16, 119 (2004). [Medline]
M. Delacour-Larose, A. Molla, D. A. Skoufias, R. L. Margolis, S. Dimitrov, Cell Cycle 3, 1418 (2004). [Medline]
V. A. Beardmore, L. J. Ahonen, G. J. Gorbsky, M. J. Kallio, J. Cell Sci. 117, 4033 (2004). [Medline]
10.1126/science.1121952
 

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