**3. Active export keeps survivin out of the nucleus**

In an attempt to determine the biological significance of cytoplasmic versus nuclear localisation of survivin, and prompted by the ongoing debate regarding its prognostic potential in cancer, we (Colnaghi et al., 2006), and others (Knauer et al., 2007) began to investigate and manipulate survivin distribution in controlled conditions in the lab. These data have led to the widely accepted view that survivin is predominantly cytoplasmic in interphase cells (Figure 1A), gaining access to the nucleus in G2, in readiness for mitosis. While this is consistent with the cell cycle regulation of survivin transcription, it is in fact an oversimplification. One notable exception is exemplified by Temme and coworkers (2005), who reported that in exponentially growing normal human lung fibroblasts endogenous survivin is predominantly nuclear throughout the cell cycle. Interestingly they found that survivin localisation was age dependent, with cells from an early passage exhibiting strong nuclear accumulation, while cells of late passage (>16) displaying mostly cytoplasmic survivin. In full corroboration with other reports, survivin expression was absent from confluent G0 monolayers of these cells (Temme et al., 2005). Moreover, other groups have indicated that survivin can enter the nucleus as early as S-phase (Beardmore et al., 2004; Suzuki et al., 2000). These observations highlight the fact that multiple parameters including cell type, senescence and growth conditions can impact on survivin localisation, and suggest that many pathways contribute to its whereabouts both spatially and temporally. Indeed many subcellular pools of survivin exist, including an important fraction within the mitochondria (Dohi et al., 2007; Fortugno et al., 2002), and several groups have hypothesised that these separate pools correspond to the distinct functions of this "nodal" protein, see review by Altieri, 2008.

The first evidence that survivin actively shuttles between cytoplasmic and nuclear compartments, came from the work of Rodgriguez and coworkers, who used the fungal toxin leptomycin B (LMB) to demonstrate that its cytoplasmic localisation was maintained by CRM1/exportin- mediated transport, stimulated by Ran-GTP (Rodriguez et al., 2002). Using site-directed mutagenesis we (Colnaghi et al., 2006), and others (Knauer et al., 2007), identified a leucine-rich region in the central "linker" region of survivin between the BIR (baculovirus inhibitor of apoptosis repeat) domain, and its alpha-helical C-terminus as a nuclear export sequence (NES). Although clearly positioned within the central linker domain, estimations of the precise residues involved, 89-98 and 96-106, did not fully agree. Within a classic NES hydrophobic leucines are key as they are responsible for interaction with the CRM1 (exportin) via Ran-GTP, and despite the difference between our proposed sequences, there was consensus that both encompassed L96 and L98, with the sequence

benign or malignant, this approach could yield fruitful predictions. Indeed, Gallagher, Duffy and co-workers have recently validated this approach in tumour microarrays of breast cancers, in which they have recorded not just the cytoplasmic and nuclear placement of survivin in tumours, but the ratio between their distributions. Their automated analysis clearly removes the subjectivity of the interpreter, and also permits its application to high through-put screening (Brennan et al., 2008; Rexhepaj et al., 2010). In summary, while survivin's subcellular localisation may have prognostic value across a wide spectrum of tumours, correlating its distribution, or the ratio of its distribution with disease outcome

In an attempt to determine the biological significance of cytoplasmic versus nuclear localisation of survivin, and prompted by the ongoing debate regarding its prognostic potential in cancer, we (Colnaghi et al., 2006), and others (Knauer et al., 2007) began to investigate and manipulate survivin distribution in controlled conditions in the lab. These data have led to the widely accepted view that survivin is predominantly cytoplasmic in interphase cells (Figure 1A), gaining access to the nucleus in G2, in readiness for mitosis. While this is consistent with the cell cycle regulation of survivin transcription, it is in fact an oversimplification. One notable exception is exemplified by Temme and coworkers (2005), who reported that in exponentially growing normal human lung fibroblasts endogenous survivin is predominantly nuclear throughout the cell cycle. Interestingly they found that survivin localisation was age dependent, with cells from an early passage exhibiting strong nuclear accumulation, while cells of late passage (>16) displaying mostly cytoplasmic survivin. In full corroboration with other reports, survivin expression was absent from confluent G0 monolayers of these cells (Temme et al., 2005). Moreover, other groups have indicated that survivin can enter the nucleus as early as S-phase (Beardmore et al., 2004; Suzuki et al., 2000). These observations highlight the fact that multiple parameters including cell type, senescence and growth conditions can impact on survivin localisation, and suggest that many pathways contribute to its whereabouts both spatially and temporally. Indeed many subcellular pools of survivin exist, including an important fraction within the mitochondria (Dohi et al., 2007; Fortugno et al., 2002), and several groups have hypothesised that these separate pools correspond to the distinct functions of this "nodal" protein, see

The first evidence that survivin actively shuttles between cytoplasmic and nuclear compartments, came from the work of Rodgriguez and coworkers, who used the fungal toxin leptomycin B (LMB) to demonstrate that its cytoplasmic localisation was maintained by CRM1/exportin- mediated transport, stimulated by Ran-GTP (Rodriguez et al., 2002). Using site-directed mutagenesis we (Colnaghi et al., 2006), and others (Knauer et al., 2007), identified a leucine-rich region in the central "linker" region of survivin between the BIR (baculovirus inhibitor of apoptosis repeat) domain, and its alpha-helical C-terminus as a nuclear export sequence (NES). Although clearly positioned within the central linker domain, estimations of the precise residues involved, 89-98 and 96-106, did not fully agree. Within a classic NES hydrophobic leucines are key as they are responsible for interaction with the CRM1 (exportin) via Ran-GTP, and despite the difference between our proposed sequences, there was consensus that both encompassed L96 and L98, with the sequence

within specific tumour types may be more effective.

review by Altieri, 2008.

**3. Active export keeps survivin out of the nucleus** 

proposed by (Knauer et al., 2007), more efficient at binding CRM1. Given the variance in the above studies and the proximity of the proposed NES to its homodimerisation interface (Verdecia et al., 2000), Engelsma et al., (2007) investigated this region in more detail. They proposed that residues 84-109, shown in red on the crystal structures in Figure 2, constitute a more complete sequence for the central NES. Furthermore, by mutating F101 and L102 to inhibit homodimerisation, they found that survivin was more efficiently removed from the nucleus than the wild type form, suggesting that monomeric survivin is more efficiently exported than homodimeric survivin. These authors further reported a second, unconventional NES present in the C-terminus of survivin, depicted in orange in Figure 2, and spanning the alpha-helix from residue 119 to its end (Engelsma et al., 2007; Rodriguez et al., 2002). Presumably this second NES would be operative in both monomers and dimers, and would act to further ensure survivin removal from the nucleus.

Trafficking of survivin between the nucleus and cytoplasm is also susceptible to regulation by post-translational modifications, including acetylation and phosphorylation. Survivin acetylation, which occurs at K129, and is facilitated by the acetyl transferase CREB-binding protein (CBP), has been implicated in aiding survivin transit out of the nucleus (Wang et al., 2010), presumably by affecting the activity of the C-terminal NES (Engelsma et al., 2007). Glucogen synthase kinase 3β (GSK3β) facilitates translocation of survivin into the nucleus, but whether phosphorylation of survivin by GSK3β is involved is not yet known (Li et al., 2008). Work from my lab has recently provided two lines of evidence that suggest that casein kinase 2 (CK2) phosphorylation helps to maintain survivin within the cytoplasm, as treatment with the CK2 specific inhibitor, TBB (4,5,6,7 tetrabromobenzotriazole), or mutation of threonine 48 (T48), the unique site targeted by CK2 in survivin, causes its accumulation in the nucleus (Barrett et al., 2011). This latter observation is curious because T48 lies in the BIR domain of survivin, which is from distinct from either NES, and would not affect binding to CRM1. Perhaps CK2 affects survivin import or its stability?
