The viral SV40 T antigen cooperates with dj2 to enhance hsc70 chaperone function Athanasia Salma, Apostolos Tsiapos and Ioannis Lazaridis Laboratory of General Biology, Medical School, University of Ioannina, Greece The heat shock protein 70 (hsp70) family of molecu- lar chaperones consists of stress inducible (e.g. hsp70) and constitutively expressed (e.g. heat shock cognate 70 kDa; hsc70) members, which actively participate in a number of vital cellular functions such as protein folding, translocation, degradation and the acquisition of cell thermotolerance [1–4]. In order to perform their functions, the hsp70s cooperate with a wide range of divergent proteins collectively known as co-chaperones. Among them, the DnaJ family mem- bers seem to be the major and critical partners in regulating at least the folding activity of the hsp70 chaperone machine [5]. DnaJ proteins exert their function by binding to hsc70 and stimulating its ATPase activity. ATP hydro- lysis converts hsc70 from an open to a closed state with low exchange rates, therefore promoting a cycle of substrate binding and release [6]. In mammals, more than 20 DnaJ homologues have been reported and classified into three groups on the basis of their domain characteristics [7]. All members of the DnaJ family contain the J domain, which is a 70-amino acid sequence structured in four helices with a loop between helices II and III containing the highly conserved tri- peptide HPD, which is necessary for binding to hsc70 [8]. The type I DnaJs, in addition to the J domain, contain a Gly ⁄ Phe-rich region followed by a stretch of cysteine repeats. Type II DnaJs lack the cysteine repeats and type III DnaJs lack both the G ⁄ F region and the cysteine repeats. Type I and Type II proteins seem to have similar functions and they bind non- native substrates, in contrast to type III proteins which may not bind denatured polypeptides and thus proba- bly function as ‘specialized’ molecular chaperones [9]. dj2 is the best characterized type I mammalian DnaJ and it has been defined as the mammalian homologue of the bacterial DnaJ and the yeast Ydj1 [10–12]. It is mainly cytosolic and acts as a cochaperone to hsc70 assisting the folding of denatured proteins and facilitat- ing the protein import into the mitochondria [13–15]. As a critical member of the hsc70 chaperone machine, dj2 was found, when overexpressed, to suppress aggregate Keywords DnaJ; hsc70; molecular chaperone; protein folding; T antigen Correspondence I. Lazaridis, Department of Biology, Faculty of Medicine, University of Ioannina, 453 32 Ioannina, Greece Fax: +302 6510 97863 Tel: +302 6510 97752 E-mail: ilazarid@cc.uoi.gr (Received 2 April 2007, revised 9 July 2007, accepted 30 July 2007) doi:10.1111/j.1742-4658.2007.06019.x Simian virus 40 large T antigen is a J-domain-containing protein with mul- tiple functions. Among its numerous activities, T antigen can bind heat shock cognate 70 (hsc70) but the biological significance of this interaction has not been fully understood. Here, we show that T antigen can act as an hsc70 co-chaperone enhancing the protein-folding ability of the hsc70 chap- erone machine. We also show that T antigen exerts its function in colla- boration with the mammalian homologue of DnaJ. Moreover, we show that the participation of T antigen in the hsc70 chaperone machine has cell-type-specific characteristics. Abbreviations dj2, mammalian homologue of DnaJ; GST, glutathione S-transferase; hsc70, heat shock cognate 70 kDa; hsp, heat shock protein; mutTAg, mutant TAg; SV40, simian virus 40; TAg, SV40 large T antigen; wtTAg, wild-type TAg. FEBS Journal 274 (2007) 5021–5027 ª 2007 The Authors Journal compilation ª 2007 FEBS 5021 formation of androgen receptor and huntingtin caused by expanded polyglutamine tracts [16,17]. Simian virus 40 (SV40) large T antigen (TAg) is a multifunctional oncoprotein which is able to induce transformation in multiple cell types [18]. It has been shown that T antigen can act as a molecular chaperone because it contains a functional J domain [19]. It has also been shown that the J domain is essential for T antigen to exert its functions related to transforma- tion, enhancement of cell division, release of Rb ⁄ E2F complexes and viral DNA replication [20–23]. T antigen was found to bind hsc70 in an ATP-dependent manner that requires the ATPase activity to be provided by hsc70 [24]. Although the biological significance of the above binding is not yet clear, the prevailing view states that upregulation of the hsc70 chaperone machine is required for several viral functions [25]. In this report, we show that T antigen binds to hsc70 both directly and indirectly via dj2. We also show that the above binding is ATP independent and contributes significantly to the folding activity of the hsc70 chaperone machine. Finally, we show that the formation of the heterotrimeric complex T antigen– dj2–hsc70 is cell-type-specific, because it is observed in cells nonpermissive for SV40 viral lytic infection and not in cells permissive to lytic infection. Results In order to investigate the interactions of T antigen with the hsc70 chaperone machine, we purified hsc70, dj2 and T antigen wild-type and mutant proteins as described in Experimental procedures. Our efforts to express the full-length forms of wild-type and mutant T antigen fused to glutathione S-transferase were not successful, despite the fact that we tested extensively both the bacterial and the insect cell expression systems. We believe that the difficulty in purifying full- length GST-tagged T antigen forms was due to the large size of the resulting proteins. Therefore, we decided to purify truncated forms of T antigen which included the J domain, the Rb-binding domain and the DNA-binding domain, but lacked the ATPase domain of the C-terminus. Given that the ATPase domain of the T antigen has been shown to be dispensable for hsc70 binding [24], we reasoned that the truncated T-antigen proteins would mimic the function of their full-length counterparts. T antigen binds directly to hsc70 Upon performing GST pull-down experiments, we observed that the wild-type TAg (wtTAg) bound to hsc70 in an ATP-dependent manner with a stoichiome- try of 1 : 1, as reported previously [24]. We also con- firmed the inability of the mutTAg protein to bind hsc70, even in the presence of ATP (data not shown). It is interesting to note that hsp70 exhibited the same characteristics as hsc70, in that it was found to bind wtTAg in an ATP-dependent fashion, but with signifi- cantly reduced affinity (data not shown). T antigen and dj2 cooperate in binding to hsc70 Having established the assay conditions, we tested whether the presence of a typical hsc70 co-chaperone, namely dj2, interfered or altered the TAg ⁄ hsc70-bind- ing characteristics. The first finding was that dj2 bound TAg in an ATP-independent manner (Fig. 1A, lanes 3 and 4). Although functional dimerization of DnaJs has been reported [31–33], our results clearly indicate that binding between type I and type III DnaJs is possible in an ATP-independent manner. Moreover, it was shown that unlike hsc70, dj2 bound mutTAg indicating AB Fig. 1. dj2 participation in the TAg ⁄ hsc70 complex. Pull-down assays were performed using GST-tagged wtTAg and mutTAg as described in Experimental procedures. Lanes 1, 2 and 8 represents purified pro- teins. (A) GST–wtTAg was incubated with the indicated proteins in the presence or absence of ATP. Bound proteins were detected by SDS ⁄ PAGE, stained with Coo- massie Brilliant Blue (upper), followed by western blotting (IB). (B) The same experi- ment as in (A) was performed using GST– mutTAg instead of GST–wtTAg. Enhancement of hsc70 chaperone function A. Salma et al. 5022 FEBS Journal 274 (2007) 5021–5027 ª 2007 The Authors Journal compilation ª 2007 FEBS once again that dj2 and T antigen, although both J-domain-containing proteins have clearly distinct hsc70-binding properties (Fig. 1B, lanes 3 and 4). The above data also show that dj2 and hsc70 have different binding properties to wtTAg and mutTAg. Most importantly, we observed that the presence of dj2 dis- rupted the stoichiometric balance of hsc70 ⁄ wtTAg complex reducing the amount of bound hsc70 and allowing hsc70 to bind wtTAg even in the absence of ATP (Fig. 1A, lanes 5 and 6). Similar results were obtained when we used mutTAg instead of wtTAg in our pull-down assays. More specifically, we observed that in the presence of dj2, hsc70 bound mutTAg in an ATP-independent fashion (Fig. 1B, lanes 5 and 6). These findings led us to hypothesize that dj2 binds T antigen in an ATP independent manner and the resulting heterodimer complexes with hsc70 via dj2. To verify this hypothesis we proceeded in a pull- down experimental scheme in which wtTAg was sequentially incubated with hsc70 and dj2. As shown in Fig. 2, formation of the dj2 ⁄ TAg complex prior to hsc70 addition resulted in the recruitment of increased amounts of hsc70 to the glutathione Sepharose-bound GST–wtTAg (compare lanes 1 and 2). Densitometry analysis revealed that there is a 2–2.5-fold increase in the amount of hsc70 collected in the Tag ⁄ dj2 complex. In contrast, addition of dj2 to the already formed hsc70 ⁄ TAg complex decreased the amount of bound hsc70 by 50% (compare lanes 1 and 3). Once again the absence of ATP in the binding reactions did not influ- ence hsc70 recruitment to the dj2 ⁄ TAg complex, confirming our conclusion that the participation of the T antigen in this complex is mainly mediated by dj2. The reduced amounts of hsc70 bound to TAg prior to dj2 addition (lanes 1 and 5) are probably due to the fact that the binding affinity of dj2 to hsc70 is stron- ger. The possibility of an antagonistic effect between dj2 and TAg for hsc70 binding cannot be excluded, but the finding that formation of the dj2 ⁄ TAg complex recruits more hsc70 (compare lanes 1, 3 and 5 with lanes 2, 4 and 6) indicates that the order of inter- actions is important for the final composition of the complex. T antigen enhances the folding activity of the hsc70 chaperone machine Because one of the main functions of the dj2 ⁄ hsc70 chaperone complex is the folding of denatured or par- tially folded proteins [29] we investigated the involve- ment of T antigen in the above process. Therefore, we performed luciferase-refolding experiments and observed that indeed T antigen facilitated hsc70 and dj2 in recovering of activity for denatured luciferase (Fig. 3). We also found that T antigen can act as an hsc70 co-chaperone, albeit with much reduced activity when compared with dj2. However, we observed that the presence of wtTAg significantly enhanced the fold- ing activity of the dj2 ⁄ hsc70 chaperone machine. This was verified by the finding that the maximal refolding of luciferase achieved by the hsc70 ⁄ dj2 dimer, was fur- ther increased with the addition of wtTAg. Impor- tantly, the mutTAg had no effect on the function of hsc70 alone or hsc70 with dj2 suggesting that the effect of wtTAg is dependent on its co-chaperone activity. Fig. 2. dj2 recruits increased amounts of hsc70 to the heterotrimer- ic complex. Pull-down experiments were performed using GST– wtTAg and in the assay mixture hsc70, dj2 and ATP were added sequentially as indicated. The composition of the complexes formed was analysed by SDS ⁄ PAGE followed by Coomassie Bril- liant Blue staining. Fig. 3. wtTAg enhances the folding activity of the hsc70 ⁄ dj2 chap- erone pair. Refolding assays were performed as described and luciferase activity was monitored at several time points ranging from 0 to 60 min. This diagram depicts values taken after 60 min incubation with the indicated proteins and expressed as percentage of the native luciferase activity. A. Salma et al. Enhancement of hsc70 chaperone function FEBS Journal 274 (2007) 5021–5027 ª 2007 The Authors Journal compilation ª 2007 FEBS 5023 The ATP independence of the heterotrimeric TAg–dj2–hsc70 complex formation is cell type specific The in vitro binding data raised the question of how T antigen, dj2 and hsc70 may interact in the cellular context. In order to investigate these interactions we chose two transformed cell lines, namely Cos7 and SVTT1, which constitutively express T antigen but dif- fer in that the Cos7 cells are permissive for SV40 lytic infection, whereas the SVTT1 are not. Cell extracts were immunoprecipitated with an anti-TAg coupled to protein G beads (as described in Experimental proce- dures) and the immunoprecipitates were analysed for the presence of TAg, dj2 and hsc70. In accordance with previous findings [30], we detected the formation of the hsc70 ⁄ TAg complex only in nonpermissive cells (Fig. 4B, +ATP). The amount of hsc70 bound to TAg was quantitated as 50% of the input. We also detected the presence of dj2 in the immunoprecipitates of both cell types in the presence of ATP and our quantification measurements showed that 15–20% of the input was retained in the complex. However, to our surprise, we found that in the absence of an ATP regeneration sys- tem, both dj2 and hsc70 were not able to associate with T antigen in the extracts of non permissive cells (Fig. 4B, –ATP). In contrast under the same conditions the association of T antigen with dj2 persisted in per- missive cells (Fig. 4A, ±ATP) in accordance with our in vitro findings that this binding is ATP independent. Collectively, the above results led us to conclude that the interactions between T antigen, dj2 and hsc70 are cell-type dependent. Discussion Although the ability of T antigen to bind hsc70 was extensively studied, the exact mechanism by which this binding contributes to the T-antigen-mediated cellular transformation remains elusive [34,35]. However, a model has been suggested according to which the J-domain-dependent association of T antigen with hsc70 stimulates chaperone activity, which in turn releases E2F from its pRb complex allowing the ‘free’ E2F to transactivate the genes required for cell prolif- eration [20,22,36]. Our results, without disputing the above model, indicate that T antigen might not exert its hsc70-related functions alone but in combination with dj2, a classical hsc70 co-chaperone. This sugges- tion is based on our findings that, in vitro, T antigen associates with dj2 in an ATP-independent manner and this binding persists even in the presence of hsc70 (Fig. 1A). As a result, T antigen is recruited to the hsc70 chaperone complex via dj2. Therefore, the pres- ence of T antigen allows hsc70 to utilize yet another J-domain-containing protein and enhance its chaperon- ing function. It is interesting to note that even a mutated form of T antigen unable to bind hsc70 can be recruited to the complex via dj2 (Fig. 1B). Our interpretation of the above data is that besides the formation of the expected dj2 ⁄ hsc70 (dimer : monomer) complexes, the presence of T anti- gen leads to the formation of novel heterotrimeric complex comprising of monomeric forms of TAg ⁄ dj2 ⁄ hsc70. The formation of these complexes does not exclude the possibility of direct binding between T antigen and hsc70. On the contrary, given the abun- dance of hsc70, one can envisage the presence of all three types of complex in the cellular context. This interpretation is supported by the finding that associa- tion of T antigen with dj2 prior to the addition of hsc70 leads to the recruitment of increased amounts of hsc70 to the complex. In contrast, addition of dj2 to the already formed TAg ⁄ hsc70 dimer does not increase the levels of hsc70 present in the complex, indicating that probably dj2 associates with hsc70 in a separate BC A Fig. 4. Cell-type-specific interaction of T antigen with hsc70. Immunoprecipitations of Cos7 cell extracts (A), SVTT1 extracts (B), and NIH 3T3 transfected with mutTAg extracts, as a control (C), were performed using an anti-(T antigen) serum column. Bound proteins were detected by western blotting. The presence or absence of ATP regeneration system in the lysates is indicated. FL, analysis of the flow through mate- rial; ctrl, the control IgG-protein G beads; Inp, the whole cellular lysate (10% of total). Enhancement of hsc70 chaperone function A. Salma et al. 5024 FEBS Journal 274 (2007) 5021–5027 ª 2007 The Authors Journal compilation ª 2007 FEBS complex which does not include T antigen. Interest- ingly, the increased amounts of hsc70 required by the TAg ⁄ dj2 heterodimer do not seem to be affected by the presence of ATP, indicating once more that the ATP-independent dj2 binding to hsc70 is the driving force for the creation of the heterotrimeric complex (Fig. 2). Therefore, based on our results, we believe that T antigen can bind hsc70 in two distinctly differ- ent ways. First, it can bind directly in an ATP-depen- dent fashion, and second, it can bind indirectly via dj2 in an ATP-independent manner. The functional significance of our findings was investigated by monitoring the chaperone-folding activity of the detected complexes. Indeed, we found that the presence of T antigen enhanced the ability of the hsc70 ⁄ dj2 chaperone pair to refold denatured lucif- erase. We believe that this increased folding activity was due to the function of the newly formed heterotrimeric complex (TAg ⁄ dj2 ⁄ hsc70). In contrast, the presence of the mutated form of T antigen was not able to change the folding activity of the hsc70 ⁄ dj2 chaperones. In other words, despite the fact that the trimeric complex mutTAg ⁄ dj2 ⁄ hsc70 can be clearly formed (Fig. 1B), mutTAg is unable to further enhance luciferase folding, probably due to its inability to stimulate the hsc70 ATPase activity. This last assumption is supported by the finding that although wtTAg can act as a weak hsc70 cochaperone, mutTAg seem to be unable to positively contribute to the enhancement of the hsc70 folding activity. Collectively, we concluded that T antigen facilitates the hsc70 chaperone machine by enhancing its folding function. In order to detect the existence of the described complexes in vivo, we performed immunoprecipitations with an anti-(T antigen) serum in cellular extracts of Cos7 and SVTT1 cells. As shown in Fig. 4, our approach was quite effective in that our antibody col- umn depleted most of the T antigen from the cell lysates. As expected, based on our previous data [30], formation of the TAg ⁄ hsc70 complex was observed only in nonpermissive cells, reinforcing our suggestion that this binding has cell-type-specific characteristics. Moreover, we detected the presence of dj2 in both the TAg ⁄ hsc70 complex (nonpermissive cells) and in the T antigen immunoprecipitate (permissive cells). How- ever, to our surprise, we observed that in the absence of ATP the dj2 and hsc70 did not associate with TAg in SVTT1 cells, in contrast to the Cos7 immuno- precipitates where the binding of T antigen to dj2 persisted (Fig. 4). Given that the dj2 ⁄ TAg binding is ATP independent, as shown above, we suspect that the activation of an additional, yet unidentified, cellular factor is responsible for the cell type specific dissociation of the heterotrimeric complex in the absence of ATP or conversely enabling dj2 to bind TAg in Cos7 cells. Overall, our data using cellular extracts clearly suggest the existence of a cell type specific organization of the hsc70 chaperone machine, which is probably related to the T antigen J-domain- mediated viral functions. Experimental procedures Cell lines The Cos7 and SVTT1 (NIH 3T3 cells expressing wtTAg) cell lines established from monkey or mouse embryos, respectively, were maintained in minimal essential medium supplemented with 10% fetal bovine serum at 37 °Cina humidified 5% CO2 atmosphere. Sf9 insect cells were grown in Sf900II medium (Gibco, Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum at 27 °C and used for the production of baculoviruses. Cloning, expression and purification of recombinant proteins The pQE-32-Mydj2 construct [13] was used to overexpress Mydj2–6·His in Escherichia coli SG13009. Briefly, over- night cultures of E. coli carrying the pQE-32-Mydj2 plasmid were diluted 10-fold and cultured for 1 h. After isopropyl thio-b-d-galactoside induction (1 mm) for 2 h at 37 °C, cells were collected by brief centrifugation at 6000 g using an Avanti J-25 centrifuge with JA-14 rotor (Beckman Coulter, Fullerton, CA) and cell lysates were prepared by sonication. The recombinant protein was purified using a Ni ⁄ nitrilotriacetic acid column and imidazole elution (50 ± 250 mm) as described by the manufacturers (Qiagen, Valencia, CA). The SV40 large T antigen was subcloned from pSG5-T (a generous gift from J. A. DeCaprio, Harvard Medical School, MA) into the BamH1 site of pGEX-4T-1 (AMRAD) to create the pGEX-4T-1–wtTAg construct. The resulting plasmid was digested with EcoR1 and HindII, treated with Klenow and religated to create the pGEX-4T-1–wtTAg282 construct which was used to express the first 282 amino acid residues of the wild-type T antigen. The mutated form of T antigen was subcloned from pSG5-T-H42Q (a gener- ous gift from J. A. DeCaprio) to pGEM and subsequently inserted to the EcoR1 and Sal1 sites of pGEX-4T-1. The resulting plasmid was digested with Not1 and HindIII, blunt ended with Klenow and religated to create the pGEX-4T-1–mutTAg268, which was used to express the first 268 amino acid residues of T antigen with a single amino acid substitution (H42Q). The described constructs, named GST–wtTAg and GST–mutTAg, respectively, were expressed in BL21(DE3) bacterial cells and the corresponding A. Salma et al. Enhancement of hsc70 chaperone function FEBS Journal 274 (2007) 5021–5027 ª 2007 The Authors Journal compilation ª 2007 FEBS 5025 proteins were purified from lysates according to standard procedures [26]. Baculovirus expressed His6·-tagged hsc70 and hsp70 were prepared according to Bac-To-Bac baculovirus expres- sion system procedure (Invitrogen). Pull-down assays GST fusion proteins (3 lg; wtTAg, mutTAg) or GST alone, were incubated for 30 min at room temperature with 30 lL of glutathione–agarose beads and ‘blocked’ in 1% fish gela- tin in assay buffer (150 mm NaCl, 20 mm Tris ⁄ HCl pH 7.5, 2mm MgCl 2 , 250 mm sucrose, 1% Triton X-100, 0.1 mm EGTA, 1 mm phenylmethanesulfonyl fluoride, 1 mm DTT). After washing three times with the same buffer, the beads were combined with equal amounts of purified His–hsc70 and ⁄ or His–dj2 and further incubated for 1 h at room tem- perature in the presence or absence of ATP regeneration buffer (1 mm ATP, 30 mm creatine phosphate, 0.25 mg phosphokinase per mL, 2 mm MgCl 2 ,1mm dithiothreitol). The beads were washed six times with assay buffer, before eluting the bound proteins with hot Laemmli buffer [27]. Western blots were performed using the following anti- bodies: T antigen-specific mAb PAb419, which have been produced by hybridoma cells. Hsc70-specific antibody (SPA-815, StressGen), Hsp70-specific antibody (SPA-810, StressGen) and Dj2-specific antibody (HDJ-2 ⁄ DNAJ Ab-1, NeoMarkers). Quantitation of the pulled down proteins was performed using the quantity one densitometry software provided by Bio-Rad Inc (Hercules, CA). Immunoprecipitation Whole-cell extracts were prepared from COS and SVTT1 cells in lysis buffer containing 150 mm NaCl, 20 mm Tris ⁄ HCl pH 7.5, 2 mm MgCl 2 , 250 mm sucrose, 1%NP40, 0.1 mm EGTA, 1 mm phenylmethanesulfonyl fluoride, 1mm dithiothreitol and 1 lgÆmL )1 each of protease inhibi- tors aprotinin, leupeptin and pepstatin. The extracts were centrifuged at 13 000 g for 15 min using a Sigma 1-14 microcentrifuge with 12094 rotor (Sigma-Aldrich, Munich, Germany), precleared with 20 lL of IgG–protein G (3 lg) for 30 min at room temperature and then incubated with 20 lL of anti-TAg–protein G beads (40 lg of anti-TAg serum) or with 20 lL IgG–protein G beads (40 lg of anti- [mouse IgG] serum) overnight at 4 °C, in the presence or absence of ATP regeneration buffer. The antibodies were covalently coupled to the beads using DMP (Pierce, Rock- ford, IL) as a cross-linker. After incubation, the beads were washed three times with lysis buffer. Proteins present in the immune complexes were eluted with lysis buffer supple- mented with 1 m NaCl. The eluates were trichloroacetic acid precipitated and the pellets were resuspended in 30 lL Laemmli buffer for subsequent analysis by SDS ⁄ PAGE and western blotting. Luciferase assays Luciferase activity was measured as described [28] with minor modifications. Briefly, 1 mg luciferase (Promega, Madison, WI) was chemically denatured by diluting 3-fold in buffer containing guanidinium-HCl (6 m)25mm Hepes (pH 7.6), 50 mm KC1, 5 mm MgCl 2 and 1 mm dithiothreitol for 40 min at room temperature. Refolding reactions were performed in 125 lL volumes by diluting denatured luciferase (1 lL) into refolding buffer contain- ing 25 mm Hepes (pH 7.6), 50 mm KC1, 5 m m MgCl 2 , purified chaperones, and ATP regeneration buffer. The samples were incubated at 30 °C and at the indicated times, 1 lL of each reaction was diluted into 60 lLof luciferase assay mixture (Promega). Activity was measured using a luminometer (Junior LB9509 EG & G Berthold, Bad Wildbad, Germany). Native luciferase activity was measured after first diluting the stock solution threefold into refolding buffer and then diluting 125-fold into the same buffer. All activities were calculated as a percent of the native luciferase activity and all data points are the average of at least two replicates. Acknowledgements We thank M. Cheetham and H.H. Kampinga for critically reading the manuscript. We also thank Dr P. Kouklis for helpful advice. This work was sup- ported by grants from the EU (QLRT-1999, #30720) and the Greek Ministry of Education (Hrakleitos). References 1 Hendrick JP & Hartl FU (1993) Molecular chaperone functions of heat shock proteins. Annu Rev Biochem 62, 349–384. 2 Georgopoulos C & Welch WJ (1993) Role of the major heat shock proteins as molecular chaperones. Annu Rev Cell Biol 9, 601–634. 3 Hartl FU (1996) Molecular chaperones in cellular pro- tein folding. Nature 381, 571–579. 4 Patterson C & Cyr D (2002) Welcome to the machine. Circulation 106, 2741–2746. 5 Takayama S, Xie Z & Reed JC (1999) An evolutionarily conserved family of hsp70 ⁄ hsc70 molecular chaperone regulators. J Biol Chem 274, 781–786. 6 Young JC, Agashe VR, Siegers K & Hartl FU (2004) Pathways of chaperone-mediated protein folding in the cytosol. Nat Rev Mol Cell Biol 5, 781–791. 7 Cheetham ME & Caplan AJ (1998) Structure, function and evolution of DnaJ: conservation and adaptation of chaperone function. Cell Stress Chaperones 3, 28–36. 8 Tsai J & Douglas MG (1996) A conserved HPD sequence of the J-domain is necessary for Ydj1 Enhancement of hsc70 chaperone function A. Salma et al. 5026 FEBS Journal 274 (2007) 5021–5027 ª 2007 The Authors Journal compilation ª 2007 FEBS stimulation of hsp70 ATPase activity at a site distinct from substrate binding. J Biol Chem 271, 9347–9354. 9 Kelley WL (1998) The J domain family and the recruit- ment of chaperone power. Trends Biochem Sci 23, 222–227. 10 Qiu X-B, Shao Y-M, Miao S & Wang L (2006) The diversity of the DnaJ ⁄ hsp40 family, the crucial partners for hsp70 chaperones. Cell Mol Life Sci 63, 2560–2570. 11 Chellaiah A, Davis A & Mohanakumar T (1993) Clon- ing of a unique human homologue of the Escherichia coli DNAJ heat shock protein. Biochim Biophys Acta 1174, 11–13. 12 Oh S, Iwahori A & Kato S (1993) Human cDNA encoding DnaJ protein homologue. Biochim Biophys Acta 1174, 114–116. 13 Bozidis P, Lazaridis I, Pagoulatos G & Angelidis CE (2002) Mydj2 as a potent partner of hsc70 in mamma- lian cells. Eur J Biochem 269, 1553–1560. 14 Terada K & Mori M (2000) Human DnaJ homologs dj2 and dj3 and bag-1 are positive cochaperones of hsc70. J Biol Chem 275, 24728–24734. 15 Terada K, Kanazawa M, Bukau B & Mori M (1997) The human DnaJ homologue dj2 facilitates mitochon- drial protein import and luciferase refolding. J Cell Biol 139, 1089–1095. 16 Kobayashi Y, Kume A, Li M, Doyu M, Hata M, Oht- suka K & Sobue G (2000) Chaperones hsp70 and hsp40 suppress aggregate formation and apoptosis in cultured neuronal cells expressing truncated androgen receptor protein with expanded polyglutamine tract. J Biol Chem 275, 8772–8778. 17 Wyttenbach A, Carmichael J, Swartz J, Furlong RA, Nazain Y, Rankin J & Rubinsztein DC (2000) Effects of heat shock, heat shock protein 4 (HDJ-2) and protea- some inhibition on protein aggregation in cellular mod- els of Huntington’s disease. Proc Natl Acad Sci USA 97, 2898–2903. 18 Saenz-Robles MT, Sullivan CS & Pipas JM (2001) Transforming functions of simian virus 40. Oncogene 20, 7899–7907. 19 Kelley WL & Georgopoulos C (1997) The T ⁄ t common exon of simian virus 40, JC and BK polyomavirus T antigens can functionally replace the J-domain of the Escherichia coli DnaJ molecular chaperone. Proc Natl Acad Sci USA 94, 3679–3684. 20 Srinivasan A, McClellan AJ, Vartikar J, Marks I, Cantalupo P, Li Y, Whyte P, Rundell K, Brodsky JL & Pipas JM (1997) The amino-terminal transforming region of simian virus 40 large T and small t antigens functions as a J domain. Mol Cell Biol 17, 4761–4773. 21 Studbal HJ, Zalvide J, Campbell KS, Schweitzer C, Roberts TM & DeCaprio JA (1997) Inactivation of pRB-related proteins p130 and p107 mediated by the J domain of simian virus 40 large T antigen. Mol Cell Biol 17, 4979–4990. 22 Sullivan CS, Cantalupo SP & Pipas JM (2000) The molecular chaperone activity of simian virus 40 large T antigen is required to disrupt Rb-E2F family complexes by an ATP-dependent mechanism. Mol Cell Biol 20, 6233–6243. 23 Peden KW & Pipas JM (1992) Simian virus 40 mutants with amino-acid substitutions near the amino terminus of large T antigen. Virus Genes 6, 107–118. 24 Sullivan CS, Gilbert SP & Pipas JM (2001) ATP-depen- dent simian virus 40 T-antigen–hsc70 complex forma- tion. J Virol 75, 1601–1610. 25 Sullivan CS & Pipas JM (2001) The virus–chaperone connection. Virology 287, 1–8. 26 Sambrook J, Fritsch FF & Maniatis TM (1989) Molecu- lar Cloning. A Laboratory Manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 27 Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685. 28 Levy EJ, McCarty J, Bukau B & Chirico WJ (1995) Conserved ATPase and luciferase refolding activities between bacteria and yeast hsp70 chaperones and mod- ulators. FEBS Lett 368, 435–440. 29 Kelley WL (1999) Molecular chaperones: how J domains turn on hsp70s. Curr Biol 9, R305–R308. 30 Sainis I, Angelidis C, Pagoulatos GN & Lazaridis I (2000) Hsc70 interactions with SV40 viral proteins differ between permissive and non permissive mammalian cells. Cell Stress Chaperones 5, 132–138. 31 Langer T, Lu C, Echols H, Flanagan J, Hayer MK & Hartl FU (1992) Successive action of DnaK, DnaJ and GroEL along the pathway of chaperone-mediated pro- tein folding. Nature 356, 683–689. 32 Li J, Qian X & Sha B (2003) The crystal structure of the yeast hsp40 Ydj1 complexed with its peptide sub- strate. Structure 11, 1475–1483. 33 Nemethova M, Smutny M & Wintersberger E (2004) Transactivation of E2F-regulated genes by polyomavi- rus large T antigen. Evidence for a two-step mechanism. Mol Cell Biol 24, 10986–10994. 34 Brodsky JL & Pipas JM (1998) Polyomavirus T anti- gens: molecular chaperones for multiprotein complexes. J Virol 72, 5329–5334. 35 De Caprio JA (1999) The role of the J domain of SV40 large T in cellular transformation. Biologicals 27, 23–28. 36 Zalvide J, Stubdal H & DeCaprio JA (1998) The J domain of simian virus 40 large T antigen is required to functionally inactivate pRb family proteins. Mol Cell Biol 18, 1408–1415. A. Salma et al. Enhancement of hsc70 chaperone function FEBS Journal 274 (2007) 5021–5027 ª 2007 The Authors Journal compilation ª 2007 FEBS 5027 . wtTAg. Impor- tantly, the mutTAg had no effect on the function of hsc70 alone or hsc70 with dj2 suggesting that the effect of wtTAg is dependent on its. associate with TAg in SVTT1 cells, in contrast to the Cos7 immuno- precipitates where the binding of T antigen to dj2 persisted (Fig. 4). Given that the dj2