**4. Getting into the nucleus**

The molecular weight cut off for passive import across the nuclear membrane is approximately 45kD, thus at 16.5 kD survivin is sufficiently small to access the nucleus by passive diffusion even as a homodimer, or as a monomer tagged with a large fluorescent moiety such as green fluorescent protein, GFP (27kD). Whether an active component contributes to its import is unclear, but is supported by the observation that transit from the cytoplasm to the nucleus is abrogated when cells are chilled (Rodriguez et al., 2002), and would have the potential for its regulation, as suggested above for CK2, which could be exploited under challenging or stressful conditions. As survivin itself has no recognisable nuclear localisation signal (NLS) of its own, to facilitate nuclear entry survivin would presumably require a chaperone. While its mitotic partners, borealin, inner centromeric protein (INCENP) and aurora-B all have one or more NLS, co-expression analysis demonstrated that none of them can coerce survivin into the nucleus, instead, when coexpressed with borealin, borealin no longer localises to the nucleoli but is forced to relocate to the cytoplasm (Rodriguez et al., 2006). This is somewhat surprising given that borealin interacts with survivin at its homodimerisation interface and could potentially mask part of the central NES (Figure 2B), nevertheless, it points to the subtleties lying within the structure

Nuclear Survivin: Cellular Consequences and Therapeutic Implications 337

noted in breast cancer cells, T-lymphocytes, and haemopoietic cells, reviewed in (Fukuda and Pelus, 2006). Thus one manifestation of nuclear residence is accelerated entry into the

A second consequence of nuclear residence of survivin is the loss of its cytoprotective activity. Converging data from analysis of NES mutants (Colnaghi et al., 2006), NLS fusions (Connell et al., 2008a), LMB treatment (Knauer et al., 2007), GSK3β-activated cells (Li et al., 2008), and the response of cells in which nuclear survivin naturally occurs (Temme et al., 2007), have clearly demonstrated that nuclear survivin can no longer inhibit cell death, invoked by either the intrinsic or extrinsic apoptotic pathways. An apparent paradox here is that while cytoplasmic localisation of survivin is a requirement for it to protect cells from irradiation (IR), irradiation itself induces translocation of survivin to the nucleus (Chakravarti et al., 2004). In our hands, IR induces nuclear foci of survivin, which form as early as 60 minutes post-IR and can persist for many hours (Figure 1B), although a more dynamic response has been noted by others (Capalbo et al., 2010). This paradox, however, may be explained in part by data from Chan et al., (2010) who recently reported that during early stages of cell death, the CRM1/exportin pathway collapses. Although in their case retention of survivin in the nucleus as a result of compromised export, resulted in its rapid Ub-proteasome dependent rapid clearance (Chan et al., 2010), it is formally possible that failed export contributes to an initial sequestration of survivin in the nucleus, which may be followed by an altered rate of turnover, and eventually focus formation. Another plausible explanation for these data may be that the cells are arresting in G2 in response to DNA damage and that this is a time when survivin normally enters the nucleus in preparation for mitosis. Whatever the means by which these foci form have been shown to colocalise with the DNA damage marker, gamma-H2AX, and the DNA repair proteins, DNA-PK and Ku70 (Capalbo et al., 2010). Moreover, survivin translocation may be effected by the downstream chk1 and chk2 effector kinases, which respond to the PIK-family of kinases, ATR and ATM respectively, both pivotal upstream kinases in the DNA damage-repair pathways, and DNA-PK knock out cells accumulate nuclear survivin after UV irradiation (Asumen et al., 2010). Consistent with this, we identified DNA-PK and Ku70 as potential partners by Mass Spec analysis of in survivin co-immunoprecipitating proteins post-X-irradiation (Connell and Wheatley, unpublished), and similar data was published by (Capalbo et al., 2010). Whether survivin then aids in DNA repair per se remains to be determined, but early work from (Chakravarti et al., 2004) reported fewer comet tails post-IR in glioblastoma cell lines expressing survivin compared with control cells, indicating either less damage sustained or

Finally, could expression of survivin in the nucleus interfere with the cell's transcriptional programme? Although survivin has a zinc finger in its BIR domain, this fold is distinct from zinc rich domains found in many TFs, and accordingly survivin itself cannot bind directly to DNA(Klein et al., 2006). However, it has been suggested that survivin suppresses the cytokine activated transcription factor, STAT3, and that this association may be regulated acetylation of survivin at lysine, K129, in the carboxyl alpha helix (Wang et al., 2010). Although the consequences of this interaction are not clear at present, it is nevertheless intriguing that this could be one reason to eliminate survivin from the nucleus. In this regard it is important to note that significant new insight in the mitosis field has revealed that survivin interacts directly with the NH2 tail of histone H3, which protrudes from the

cell cycle.

more efficient repair of DNA strand breaks.

of this tiny protein. As mentioned above redistribution of survivin into the nucleus is promoted by activation of GSK3B, and although it is not yet known whether survivin is a substrate of this kinase, GSK3B can bind survivin directly and has a bipartite NLS (Li et al., 2008).
