STAT2, Caspase3 and HDAC6 as Hsp27 Client Proteins Figure 6. HeLa cells were lysed in the presence of 0.1% Triton X-100 and the 10,0006g soluble supernatants were applied to sepharose CL-6B gel filtration columns as described in Materials and methods. 29, 66, 150, 200, 443, 669 and 2000 (kDa) indicate the apparent native size of gel filtration markers. A, The presence of Hsp27, HDAC6, STAT2 and procaspase-3 in pooled fractions eluted from the columns was detected by western blot analysis. B, interactions between Hsp27 and client proteins analyzed by Co-IP experiments using a goat polyclonal anti- Hsp27 antibody. Column fractions corresponding to either procaspase-3, STAT2 or HDAC6 were pooled and used for immunoprecipitation. Immunoprecipitated proteins and input cell lysates were analyzed side by side in immunoblots probed with the indicated antibodies. A representative negative control of Co-IP was performed with a control antibody and revealed (here the anti procaspase 3). doi:10.1371/journal.pone.0029719.g006 The alteration in cytokinesis efficiency was probably the origin of the increased number of cells in G2/M phase. This may be related to the activation of cell senescence, as observed by others [27]. In HeLa cells, part of the nuclear phenotype associated with Hsp27 depletion could have resulted of a-tubulin hyper-acetylation consequently to HDAC6 degradation. In that regard it is interesting to note that HDAC6 is a major component of the cellular stress response, together with the major heat shock proteins [39], which participates in the addressing of denatured proteins to degradation [40]. In response to heat shock, large oligomers of Hsp27 store unfolded and denatured proteins, particularly when the molecular chaperone pathway is saturated [41]. In the altered protein response of cells, the first link between Hsp27 and HDAC6 is their direct interaction. Secondly, HDAC6 is able to promote hsp27 gene transcription linked to stress response [42]. Furthermore, it has been reported that HDAC6 is a powerful modifier of carcinogenesis and its stabilization by Hsp27 may contribute to oncogenic pathways activation [43]. Procaspase-3 is a major cysteine protease involved in apoptosis and differentiation pathways [44,45]. An interaction has already been described between the pro-domain of procaspase-3 and Hsp27, which modulates procaspase-3 cleavage and activation [32]. Our new finding implicates that, in HeLa cells, Hsp27 could regulate procaspase-3 half-life. We were not able to realize the same experiments in MCF-7 breast cancer cells because caspase-3 is inactivated due to a deletion mutation in exon 3 of the gene [46]. Our results are in accordance with a previous study that has already described a procaspase-3 down-regulation when Hsp27 is immuno-depleted [7]. However, the authors suggested that the down-regulation of procaspase-3 was a consequence of its cleavage in functionally caspase-3. In contrast, our results suggest that, in the absence of Hsp27, procaspase-3 is rapidly degraded through the ubiquitin/proteasome pathway. STAT2 is a central transcription factor implicated in interferon and anti-viral responses [35]. We show here that STAT2, which interacts with Hsp27 large oligomers, was degraded in Hsp27 depleted stable cells. In these cell lines, we were unable to detect a decrease in the level of STAT3, a protein that has already been demonstrated to interact with Hsp27 [21,33]. However, we found that the phosphorylation of STAT3, which reflects its transcrip- tional activation, was down-modulated in response to IL-6 and also heat shock. However, in cells transiently transfected with shRNA encoding vector, we confirmed that STAT3 was degraded and its phosphorylation down-regulated. Hence, in our cell conditions, the establishment of stable cell lines altered the degradation of STAT3 in absence of Hsp27. This reflects an adaptation mechanism as well as the tight regulation that controls STAT3 stabilization and activation [21]. It can be hypothesized that, in cancer cells, the abundance of Hsp27 may, similarly to Hsp90 [20], contribute to the stabilization of several oncogenic proteins. One example of such an activity refers to the function of Hsp27 in bortezomib treated cells where the proto-oncogene STAT3 is constitutively activated [15,21,47]. Another example, in the context of apoptosis after androgen ablation and chemotherapy, concerns the translation initiation factor 4E (eIF4E) that shows a decreased expression at the protein, but not mRNA, level in cells depleted of Hsp27 [22]. Interaction with eIF4E was confirmed but the active oligomeric size of Hsp27 is still not known. Based on the work dealing with Hsp90, a protein which, in yeast, appears to stabilize approximately 10% of the proteome [18], the number of client proteins interacting with Hsp27 complex oligomeric and phosphorylated sub-structures [4] could also be very high. Indeed, Hsp27 has been referred to act as ‘‘holdase’’ or ‘‘molecular sponge’’. Nevertheless, identification of Hsp27 client proteins is a difficult task. For example, a powerful mass-spectrometry study identified an important number of PLoS ONE | www.plosone.org 9 January 2012 | Volume 7 | Issue 1 | e29719