Oncogene (2009) 28, 3332–3344 & 2009 Macmillan Publishers Limited All rights reserved 0950-9232/09 $32.00 www.nature.com/onc ORIGINAL ARTICLE Heat shock protein 27 is involved in SUMO-2/3 modification of heat shock factor 1 and thereby modulates the transcription factor activity M Brunet Simioni1,2, A De Thonel1,2, A Hammann1,2, AL Joly1,2, G Bossis3,4,5, E Fourmaux1, A Bouchot1, J Landry6, M Piechaczyk3,4,5 and C Garrido1,2,7 1INSERM U866, Dijon, France; 2Faculty of Medicine and Pharmacy, University of Burgundy, Dijon, Burgundy, France; 3Institut de Ge´ne´tique Mole´culaire UMR 5535 CNRS, Montpellier cedex 5, France; 4Universite´ Montpellier 2, Montpellier cedex 5, France; 5Universite´ Montpellier 1, Montpellier cedex 2, France; 6Centre de Recherche en Cance´rologie et De´partement de Me´decine, Universite´ Laval, Quebec City, Que´bec, Canada and 7CHU Dijon BP1542, Dijon, France Heat shock protein 27 (HSP27) accumulates in stressed otherwise lethal conditions. This stress response is cells and helps them to survive adverse conditions. We have universal and is very well conserved through evolution. already shown that HSP27 has a function in the Two of the most stress-inducible HSPs are HSP70 and ubiquitination process that is modulated by its oligomeriza- HSP27. Although HSP70 is an ATP-dependent chaper- tion/phosphorylation status. Here, we show that HSP27 is one induced early after stress and is involved in the also involved in protein sumoylation, a ubiquitination- correct folding of proteins, HSP27 is a late inducible related process. HSP27 increases the number of cell HSP whose main chaperone activity is to inhibit protein proteins modified by small ubiquitin-like modifier aggregation in an ATP-independent manner (Garrido (SUMO)-2/3 but this effect shows some selectivity as it et al., 2006). HSP27 has the ability to form oligomers of neither affects all proteins nor concerns SUMO-1. More- up to 1000 kDa. HSP27 oligomerization is a highly over, no such alteration in SUMO-2/3 conjugation is dynamic process modulated by the phosphorylation of achievable by another HSP, such as HSP70. Heat shock the protein. Human HSP27 can be phosphorylated on factor 1 (HSF1), a transcription factor responsible for HSP three serines (S15, S78 and S82) and the phosphoryla- expression, is one of the targets of HSP27. In stressed cells, tion provokes a shift toward small oligomers (Lambert HSP27 enters the nucleus and, in the form of large et al., 1999; Rogalla et al., 1999). The different activities oligomers, binds to HSF1 and induces its modification by of HSP27 seem to be modulated by the oligomerization SUMO-2/3 on lysine 298. HSP27-induced HSF1 modifica- pattern of the chaperone. We have shown, in vitro and tion by SUMO-2/3 takes place downstream of the in vivo, that large non-phosphorylated oligomers of transcription factor phosphorylation on S303 and S307 HSP27 are responsible for the caspase-dependent anti- and does not affect its DNA-binding ability. In contrast, this apoptotic effect of this chaperone (Bruey et al., 2000). modification blocks HSF1 transactivation capacity. These Other groups have shown that large oligomers are data show that HSP27 exerts a feedback inhibition of needed for HSP27 antioxidant activity (Rogalla et al., HSF1 transactivation and enlighten the strictly regulated 1999). Large non-phosphorylated HSP27 oligomers interplay between HSPs and HSF1. As we also show that bind to actin and the two proteins dissociate HSP27 binds to the SUMO-E2-conjugating enzyme, Ubc9, upon HSP27 phosphorylation (During et al., 2007). our study raises the possibility that HSP27 may act as a In contrast, it is proven that phosphorylated HSP27 SUMO-E3 ligase specific for SUMO-2/3. directly interacts with Daxx (Charette et al., 2000). Oncogene (2009) 28, 3332–3344; doi:10.1038/onc.2009.188; Moreover, small oligomers of HSP27 are also the form published online 13 July 2009 of the protein, which displays affinity for ubiquitin chains and accelerates the degradation of certain Keywords: HSP27; SUMOylation; HSF1; transcriptional proteins under stress conditions (Parcellier et al., activity; stress; cancer 2006). These results suggest that oligomerization/phos- phorylation of the protein alters HSP27 conformation and, hence, determines its capacity to interact with its Introduction different partners. Expression of HSP genes is regulated by the family of Stress or heat shock proteins (HSPs) are induced by the heat shock transcription factors, which bind to the different stresses and help the cells to cope with these heat shock element (HSE) in the promoter region of heat shock genes and, thereby stimulate their transcrip- tion. Three members have been identified in mammals, Correspondence: Dr C Garrido, Faculty of Medicine, INSERM U866, of which heat shock factor 1 (HSF1) is the major stress- 7 boulevard Jeanne d’Arc, 21000 Dijon, Burgundy, France. E-mail: [email protected] responsive family member as no other HSF is able to Received 24 January 2009; revised 17 May 2009; accepted 29 May 2009; functionally substitute for HSF1 or to rescue the published online 13 July 2009 heat shock response in HSF1-deficient cells or mice HSP27 facilitates HSF1 sumoylation M Brunet Simioni et al 3333 (McMillan et al., 1998; Xiao et al., 1999). In response to (Gill, 2003; Hay, 2005). Many transcription factors stressful stimuli such as elevated temperatures, oxidants, such as HSF1 have been shown to be sumoylated and heavy metals, and bacterial or viral infections, HSF1 is this modification, in most cases, impairs the transactiva- activated by trimerization and hyperphosphorylation tion capacity of the substrate (Geiss-Friedlander and (Pirkkala et al., 2001; Holmberg et al., 2002). As a Melchior, 2007). result, HSF1 acquires the ability to bind to HSE and to Heat shock factor 1 has been shown to undergo stress- activate transcription of heat shock genes, which results inducible SUMO-2/3 modification on a conserved lysine, in accumulation of HSPs such as HSP27 and HSP70 K298 (Hietakangas et al., 2003). Further, it has been with, as a final outcome, cell protection (Pirkkala et al., shown that HSF1 has to be previously phosphorylated 2001; Didelot et al., 2006; Garrido et al., 2006; Schmitt on serine 303 for its subsequent sumoylation (Hietakan- et al., 2007; Anckar and Sistonen, 2007a, b). Once the gas et al., 2003). Considering the need of regulated stress conditions are over, HSP gene transcription must chaperone levels in maintaining cellular homoeostasis, immediately stop. HSF1 activity is therefore tightly repression of HSF1 activity by interaction with HSPs is regulated, in particular, through various types of post- essential. The objective of this paper was to study translational modifications. Thus, HSF1 can be phos- whether the main inducible heat shock proteins, HSP70 phorylated at different serine residues (Holmberg et al., or HSP27, have a role in the overall sumoylation process 2001, 2002) and be ubiquitinated (Lee et al., 2008). More and, more particularly, whether they could regulate recently, it has been shown that HSF1 can also be HSF1 sumoylation. We found that HSP27, in the form sumoylated (Hong et al., 2001; Hietakangas et al., 2003, of large oligomers, induced HSF1 SUMO-2/3 modifica- 2006; Anckar et al., 2006). tion and blocked its transactivation capacity. By doing Small ubiquitin-like modifier (SUMO) proteins are so, HSP27 allows a fast and precise modulation of HSP about 100 amino acids in size with a three-dimensional gene transcription, including that of its own gene. structure reminiscent to that of ubiquitin. However, SUMO and ubiquitin share o20% amino acid sequence identity and are different in their overall surface charge Results and Discussion distribution (Geiss-Friedlander and Melchior, 2007). Like ubiquitination, sumoylation results in the forma- HSP27 favours HSF1 modification by SUMO-2/3 tion of an isopeptide bond between the glycine residue To explore whether the two main stress-inducible HSPs, of the modifier protein and the e-amino group of a lysine HSP27 and HSP70, play a role in the sumoylation residue in the acceptor protein. SUMO conjugation process, we assessed their effect on HSF1 modification utilizes a multistep enzymatic pathway, in which by SUMO-2/3. To this aim, we transiently transfected proteolytically processed SUMO initially forms a HeLa cells with a vector for either Myc-tagged wild-type thioester bond with Sae1/2, the E1 SUMO-activating HSF1 (HSF1-wt) or a non-sumoylable HSF1 mutant in enzyme (Johnson, 2004). The SUMO moiety is subse- which K298 has been replaced by arginine (HSF1- quently transferred to Ubc9, the single E2 SUMO- K298R), in the presence or in the absence of vectors for conjugating enzyme, which usually binds directly the haemagglutinin (HA)-tagged HSP27 (HSP27) or HSP70 target protein at the level of sumoylated sites (Geiss- (HSP70) (Figure 1a). SUMO-2/3 modification being Friedlander and Melchior, 2007). E2-substrate interac- inducible by stress (Saitoh and Hinchey, 2000), there- tion may be facilitated by SUMO-E3 factors. The E3 fore, the absence of detection of HSF1 modified by factors increase sumoylation efficiency in a substrate- SUMO-2/3 was not surprising under our experimental specific manner, either by accelerating the transfer of conditions. The only exception for HSF1-SUMO-2/3 SUMO from Ubc9 to the substrate or merely by detection was in cells overexpressing HSP27 and HSF1- providing a scaffold (Guo et al., 2005; Geiss-Friedlander wt, as shown in Figure 1a, left panel, and confirmed on and Melchior, 2007). The SUMO family consists of four the right panel by reversing the immunoprecipitation/ members, SUMO-1, -2, -3 and -4, but SUMO-4 has only immunodetection. As expected, this SUMO-2/3 modi- been detected at the RNA level (Guo et al., 2005; Geiss- fication of HSF1 induced by HSP27 disappeared in cells Friedlander and Melchior, 2007).