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Oncogene (2012) 31, 1582–1591

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ORIGINAL ARTICLE

Molecular chaperone complexes with antagonizing activities regulate stability and activity of the tumor suppressor LKB1 H Gaude1, N Aznar1, A Delay1, A Bres1, K Buchet-Poyau1, C Caillat2, A Vigouroux2, C Rogon3, A Woods4, J-M Vanacker5, J Ho¨hfeld3, C Perret6, P Meyer2, M Billaud7 and C Forcet5 1 Centre de Recherche en Cance´rologie de Lyon (CRCL), INSERM U1052/CNRS UMR 5286, Lyon, France; 2Laboratoire d’Enzymologie et Biochimie Structurales, CNRS UPR 3082, Centre de Recherche CNRS de Gif-Sur-Yvette, Gif-Sur-Yvette, France; 3 Institute for Cell Biology and Bonner Forum Biomedizin, Rheinische Friedrich-Wilhelms-University of Bonn, Germany; 4 MRC Clinical Sciences Centre, Cellular Stress Group, Imperial College, London, UK; 5Institut de Ge´nomique Fonctionnelle de Lyon, CNRS/UCBL/ENS UMR 5242, Ecole Normale Supe´rieure de Lyon, Lyon, France; 6Institut Cochin, CNRS UMR 8104, Universite´ Paris Descartes, Paris, France and 7Institut Albert Bonniot, CRI INSERM/UJF U823, Rond-point de la Chantourne, La Tronche, France

LKB1 is a tumor suppressor that is constitutionally mutated in a cancer-prone condition, called Peutz-Jeghers syndrome, as well as somatically inactivated in a sizeable fraction of lung and cervical neoplasms. The LKB1 gene encodes a serine/threonine kinase that associates with the pseudokinase STRAD (STE-20-related pseudokinase) and the scaffolding protein MO25, the formation of this heterotrimeric complex promotes allosteric activation of LKB1. We have previously reported that the molecular chaperone heat shock protein 90 (Hsp90) binds to and stabilizes LKB1. Combining pharmacological studies and RNA interference approaches, we now provide evidence that the co-chaperone Cdc37 participates to the regulation of LKB1 stability. It is known that the Hsp90–Cdc37 complex recognizes a surface within the N-terminal catalytic lobe of client protein kinases. In agreement with this finding, we found that the chaperones Hsp90 and Cdc37 interact with an LKB1 isoform that differs in the C-terminal region, but not with a novel LKB1 variant that lacks a portion of the kinase N-terminal lobe domain. Reconstitution of the two complexes LKB1–STRAD and LKB1–Hsp90–Cdc37 with recombinant proteins revealed that the former is catalytically active whereas the latter is inactive. Furthermore, consistent with a documented repressor function of Hsp90, LKB1 kinase activity was transiently stimulated upon dissociation of Hsp90. Finally, disruption of the LKB1– Hsp90 complex favors the recruitment of both Hsp/Hsc70 and the U-box dependent E3 ubiquitin ligase CHIP (carboxyl terminus of Hsc70-interacting protein) that triggers LKB1 degradation. Taken together, our results establish that the Hsp90–Cdc37 complex controls both the stability and activity of the LKB1 kinase. This study further shows that two chaperone complexes with antagonizing

Correspondence: Dr M Billaud, Institut Albert Bonniot, CRI INSERM/UFJ U823, Rond-point de la Chantourne, 38706 La Tronche, France E-mail: [email protected] or Dr C Forcet, Institut de Ge´nomique Fonctionnelle de Lyon, CNRS/UCBL/ENS UMR 5242, Ecole Normale Supe´rieure de Lyon, Lyon, France. E-mail: [email protected] Received 21 October 2010; revised and accepted 6 July 2011; published online 22 August 2011

activities, Hsp90–Cdc37 and Hsp/Hsc70–CHIP, finely control the cellular level of LKB1 protein. Oncogene (2012) 31, 1582–1591; doi:10.1038/onc.2011.342; published online 22 August 2011 Keywords: LKB1; Hsp90; CHIP; tumor suppressor; chaperones

Introduction Molecular chaperones assist the folding of nascent polypeptides to prevent their aggregation and refold denatured proteins. The heat shock protein, Hsp90, is an abundant molecular chaperone that facilitates the folding, the maturation and/or the activity of client proteins typically involved in the control of transcription and signal transduction (reviewed in Whitesell and Lindquist, 2005; Caplan et al., 2007). Hsp90 possesses an ATPase activity and associates to essential cofactors that regulate its chaperone function. Ultimately, dissociation of the Hsp90 chaperone complex leads to the degradation of the client proteins through the ubiquitin– proteasome pathway (Whitesell and Lindquist, 2005; Caplan et al., 2007). Germline mutations of the LKB1 tumor-suppressor gene are responsible for the Peutz-Jeghers syndrome, a dominantly inherited disorder characterized by multiple hamartomatous polyps of the digestive tract and by an increased incidence of various cancer types (Hemminki et al., 1998; Jenne et al., 1998). The human LKB1 protein is a 50 kD serine/threonine kinase that forms a heterotrimeric complex with STRAD, a STE-20-related pseudokinase, and the scaffolding protein MO25 (Baas et al., 2003; Boudeau et al., 2003b; Brajenovic et al., 2004). The binding of STRAD induces a conformational modification that promotes LKB1 catalytic activity, whereas MO25 stabilizes the LKB1 activation loop in the appropriate position for kinase activity (Zeqiraj et al., 2009). LKB1 is an upstream activator of the adenosine monophosphate-activated protein kinase (AMPK), a molecular sensor that

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controls the cellular energy balance (Hawley et al., 2003; Woods et al., 2003; for review see Hardie, 2008). LKB1 signals through AMPK to downregulate several pathways known to be overactivated in malignant tumors, such as the mammalian target of rapamycin signaling cascade and fatty acid synthesis (Corradetti et al., 2004; Shaw et al., 2004). Finally, LKB1 is a key factor in the mechanisms that underlie the establishment and maintenance of cell polarity (reviewed in Hezel and Bardeesy, 2008; Jansen et al., 2009). A fine regulation of LKB1 stability is crucial because it could impact on its tumor suppressor function. In this line, we and others have previously reported that the stability of LKB1 depends on its binding to Hsp90 (Nony et al., 2003; Boudeau et al., 2003a). We subsequently decided to explore further the molecular mechanisms regulating LKB1 stabilization, and characterize the factors involved in its degradation. Here we report that the stabilization of LKB1 crucially depends on Cdc37 binding, and that its interaction with the Hsp90–Cdc37 chaperones involves the N-terminal lobe of the kinase domain. Moreover, we provide evidence that the Hsp90–Cdc37 complex is not only required for LKB1 stabilization but also functions as a repressor of LKB1 activation. Finally, we found that the chaperone Hsp/Hsc70 and the E3 ubiquitin ligase CHIP are recruited to LKB1 and promote its degradation on dissociation of LKB1 from Hsp90.

indicated that 4 h of celastrol treatment induced a 40% decrease in the level of LKB1. To confirm the role of Cdc37 in regulating the stability of LKB1, we depleted the HBL100 human mammary carcinoma cells of either Hsp90 or Cdc37 by RNA interference (RNAi). As shown in Figure 1b, transfection of the small interfering RNA (siRNA) targeting either Hsp90 or Cdc37 led to a reproducible and robust diminution of their corresponding protein levels. Interestingly, the knockdown of Cdc37 resulted in a drastic reduction of exogenous as well as endogenous LKB1 protein level (Figure 1b). The magnitude of the

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Results LKB1 stability is regulated by the molecular chaperone Cdc37 The chaperone Hsp90 interacts with and stabilizes LKB1 (Nony et al., 2003; Boudeau et al., 2003a). We and others have shown that cell treatment with geldanamycin (GA), an Hsp90-specific inhibitor, induces the proteasomemediated degradation of LKB1. It was also reported that the co-chaperone adaptator Cdc37 binds to LKB1 (Nony et al., 2003; Boudeau et al., 2003a). However, whether this interaction is involved in the control of LKB1 protein stability was not determined. To address this question, we first used the triterpene celastrol, a specific Cdc37 inhibitor that interferes with Hsp90– Cdc37 chaperoning function (Hieronymus et al., 2006; Sreeramulu et al., 2009; Zhang et al., 2009). We sought to determine the effects of celastrol on exogenous and endogenous LKB1. HeLa cells that do not express endogenous LKB1 were transfected with a plasmid encoding the human LKB1 fused to the Flag epitope tag, and then treated with 10 mM celastrol at various time points. Immunoblot analysis revealed that at 2 h after addition of celastrol, the level of LKB1 was markedly diminished. This reduction of LKB1 level was detected up to 6 h after celastrol treatment (Figure 1a). Next, we found that celastrol treatment of human kidney epithelial Bosc cells led to a net decrease of the amount of the endogenous LKB1. Quantification of the level of LKB1 normalized with the level of actin

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Figure 1 Regulation of endogenous LKB1 turnover by the chaperones Hsp90–Cdc37. (a) HeLa cells were transfected with a vector expressing Flag-LKB1, and lysed after the indicated times of treatment with 10 mM celastrol. Cell lysates were immunoblotted with the anti-Flag antibody. b-Actin was immunoblotted as a loading control (upper panels). Bosc cells were treated with 10 mM celastrol for the indicated times before lysis. Cell lysates were immunoblotted with anti-LKB1, and anti-actin antibodies (bottom panels). (b) HBL100 cells were transiently transfected with a vector expressing the Flag-LKB1 protein. Cells were also either treated with dimethyl sulfoxide or with 2 mM GA for 3 h (first two lanes), or transfected with siRNA (others lanes): the control siRNA (Ct), siRNA against Hsp90 (h1 and h2), siRNA against Cdc37 (c1 and c2) and combinations of Hsp90 and Cdc37 siRNAs (h1c1 or h2c2). Cells lysates were immunoblotted using antibodies recognizing Flag-LKB1, Hsp90, Cdc37 or actin (upper panels). HBL100 cells were treated or not with 2 mM GA for 3 h (first two lanes). In the other lanes, HBL100 were transfected with the same siRNA or combination of siRNA as above. Cells lysates extracts were immunoblotted using antibodies recognizing LKB1, Hsp90, Cdc37 or actin (bottom panels). WB, western blotting. Oncogene

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effects of the Cdc37 siRNA was similar to that achieved with the Hsp90 siRNA, or GA treatment. However, combining both Hsp90 and Cdc37 siRNAs did not result in a cumulative effect on LKB1 protein level (Figure 1b), suggesting that Cdc37 cooperates with Hsp90 to regulate LKB1 stability, as expected. Taken together, these results indicate that Cdc37 is necessary for LKB1 stabilization.

replaced by a unique 39-residue sequence (Towler et al., 2008; Denison et al., 2009). LKB1-DN is generated by the alternative use of a downstream start codon and lacks the N-terminal 124 residues of the full-length protein. The detailed characterization of both LKB1DN expression and function will be described elsewhere (CP and MB, manuscript in preparation). To analyze whether these two newly described LKB1 isoforms, LKB1-S and LKB1-DN, interact with the molecular chaperones Hsp90–Cdc37, vectors expressing these LKB1 variants were transfected in HeLa cells. As shown in Figure 2a, LKB1-S was found in association with both Hsp90 and Cdc37, whereas LKB1-DN did not bind to these chaperones. Using an anti-LKB1 antibody, we confirmed that these LKB1 isoforms were correctly expressed, although the expression level of LKB1-DN was substantially weaker than the two other isoforms (Figure 2a). This experiment was also performed in presence of sodium molybdate, an agent known to

LKB1L and LKB1S, but not LKB1-DN, are stabilized by the Hsp90/Cdc37 complex We and others have recently characterized two novel LKB1 isoforms, and we investigated whether the Hsp90–Cdc37 complex is involved in stabilizing these LKB1 isoforms. LKB1-S is generated by alternative splicing of the mRNA producing a protein in which the C-terminal 63 residues of the full-length human LKB1 (433 amino acids; here referred to as LKB1-L) are

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Figure 2 Implication of the N-terminal part of the LKB1 kinase domain in LKB1–Hsp90 interaction. (a) HeLa cells were transfected with pcDNA3 vectors expressing untagged LKB1-L, LKB1-S and LKB1-DN. LKB1 proteins were immunoprecipitated with the anti-LKB1 antibody. Cell lysates and immunoprecipitates were immunoblotted with antibodies recognizing Hsp90, Cdc37, LKB1 isoforms and actin. Asterisk (*) represent immunoglobulin G. (b) HeLa cells were transfected with vectors expressing LKB1-L, LKB1-S, LKB1-DN, and treated with 10 mM celastrol for the indicated times. Cell lysates were immunoblotted with anti-LKB1, and anti-actin antibodies. (c) HeLa cells were transiently transfected with vectors expressing the different LKB1 isoforms and treated with 100 mg/ml cycloheximide (CHX) for the indicated times. Lysates were immunoblotted with anti-LKB1 and anti-actin antibodies. (d) Signals were quantified using QuantityOne (Life Science Research, Hercules, CA, USA). LKB1 signal density was normalized to actin signal density in four independent experiments. Mean values and standard errors corresponding to each time point are indicated as percentages of the mean obtained at time 0. On the graph, dots represent mean quantification at the indicated time and curves represent the regression graphs for each isoform. Halflife times were also indicated on the abscissa axis. IP, immunoprecipitation; WB, western blotting. Oncogene

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STRADa and Hsp90 do not interact Both the Hsp90–Cdc37 complex and the STRAD– MO25 complex stabilize LKB1 (Baas et al., 2003; Nony et al., 2003; Boudeau et al., 2003a, b). To determine whether mutual interactions exist between members of these complexes, Flag-LKB1 or Flag-STRADa was expressed in Bosc cells and immunoprecipitated with the Flag antibody. As shown in Figure 3a, Hsp90 was found associated with LKB1, but not with STRADa. Immunoblotting with the anti-Flag antibody confirmed that LKB1 and STRADa proteins were correctly expressed. Next, we investigated the influence of LKB1 on the ability of STRADa to interact with Hsp90. Myc– STRADa or Flag-LKB1 or both were expressed in Bosc cells, and immunoprecipited with the anti-Myc or anti-Flag antibody. As shown in Figure 3b, LKB1 interacted with Hsp90, and the formation of this complex was not dependent on ectopic STRADa. In contrast, an interaction of STRADa with Hsp90 was detectable solely when LKB1 was coexpressed with STRADa (Figure 3b). Although Myc–STRADa

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stabilize Hsp90–client protein complexes (Stancato et al., 1997), with similar results (data not shown). Next, we examined whether celastrol treatment affects the stability of the LKB1 isoforms, HeLa cells were transfected with vectors expressing LKB1-L, LKB1-S or LKB1-DN, and then treated with 10 mM celastrol for various time points. Western blot and quantitative analysis of these results showed that LKB1-L and LKB1-S levels diminished during the time-course of celastrol treatment (Figure 2b). However, LKB1-S was more slowly degraded than LKB1-L, suggesting a more stable conformation of this isoform. As expected, LKB1-DN was insensitive to celastrol treatment (Figure 2b). As the novel variant LKB1-DN did not bind to Hsp90–Cdc37, it was expected to be less stable than the two other isoforms. To explore further this question, HeLa cells were transfected with plasmids encoding the different LKB1 isoforms and treated with the translation inhibitor cycloheximide. As shown in Figure 2c, western blot analysis revealed a similar decrease of LKB1-L and LKB1-S expression during the time-course of cycloheximide treatment. In contrast, the level of LKB1-DN was more rapidly reduced (Figure 2c). A band corresponding to a form with a molecular weight higher than LKB1-DN was detected after 1.5 h of cycloheximide treatment. This band may correspond to a post-translationally modified form of LKB1-DN or could be a crossreactivity of the antibody used, a question that was not further investigated in this study. Quantification of four independent experiments followed by regression analysis indicated that the respective half-lives of LKB1-L, LKB1-S were 1.6 h and 2.6 h, respectively, whereas the half-life of LKB1-DN was 0.7 h (Figure 2d). Taken together, these results indicate that both LKB1-L and LKB1-S isoforms, but not LKB1DN, are stabilized by the Hsp90–Cdc37 complex.

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Figure 3 Mutual interactions of LKB1 with STRADa and Hsp90. (a) HeLa cells were transfected with vectors expressing Flag-STRADa or Flag-LKB1. STRADa or LKB1 proteins were immunoprecipitated with the anti-Flag antibody. Cell lysates and immunoprecipitates were immunoblotted with antibodies recognizing Hsp90, Flag-STRADa, Flag-LKB1 and actin. (b) Bosc cells were transfected with vectors expressing Flag-LKB1 or Myc– STRADa, or co-transfected with both vectors. LKB1 or STRADa proteins were immunoprecipitated with the anti-Flag or anti-Myc antibodies. Cell lysates and immunoprecipitates were immunoblotted with antibodies recognizing Hsp90, Flag-LKB1, Myc– STRADa and actin. Asterisk (*) represent immunoglobulin G. IP, immunoprecipitation; WB, western blotting.

expression is weaker in the absence of LKB1, immunoblotting with the anti-Myc antibody demonstrated that STRADa was efficiently immunoprecipitated under both conditions (Figure 3b). Thus, these results indicate that Hsp90 interacts with LKB1 but not with STRADa. Furthermore, our data suggest a stoichiometry of two major independent complexes (as also suggested by Alessi et al., 2006), with a less abundant complex comprising LKB1–Hsp90–Cdc37 bound to STRADa. The Hsp90–Cdc37 complex functions as a repressor of LKB1 catalytic activity Previous studies have reported that Hsp90 can interfere with the kinase activities of its client proteins such as PKR, c-Src and ErbB2 (Donze´ et al., 2001; Xu et al., 2005; Koga et al., 2006; Yano et al., 2008). Oncogene

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Thus, we sought to determine whether LKB1 is active when present in the Hsp90–Cdc37 complex. To this end, we performed an in vitro autophosphorylation assay using recombinant LKB1–Hsp90–Cdc37 and LKB1– STRADa complexes purified from Escherichia coli. We also used the recombinant LKB1–STRADa–MO25a complex purified from bacculovirus-infected insect cells. As expected, LKB1 was fully active in the recombinant LKB1–STRADa–MO25a complex. Indeed, 100 ng of complex was sufficient to obtain detectable autophosphorylation of LKB1 and phosphorylation of STRADa LKB1-STRADα-MO25α: 0 0,1ug 32P-GST-STRADα Autoradiography 32P-LKB1 LKB1-STRADα: LKB1-HSP90-CDC37: 0 1 10ug 0 1 10ug 32P-LKB1 Autoradiography 32P-STRADα HSP90 LKB1 CDC37

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(Figure 4a). By comparison, LKB1 activation and consecutive phosphorylation of STRADa were detectable starting at 1 mg LKB1–STRADa complex. Besides, the intensity of the radioactive labeling drastically increased from 1 mg to 10 mg of this complex. In contrast, using the same amount of recombinant proteins, we did not detect any significant LKB1 autophosphorylation when associated with Hsp90 and Cdc37 (Figure 4a). Thus, these results indicate that LKB1 does not exhibit enzymatic activity when associated with Hsp90–Cdc37 chaperones. It has been shown that GA treatment induces PKR and Src activation on dissociation from Hsp90 (Donze´ et al., 2001; Koga et al., 2006). To study the effect of Hsp90 on LKB1 kinase activity, a time-course treatment with 2 mM GA was performed using Bosc cells expressing Flag-LKB1. As previously described (Nony et al., 2003; Boudeau et al., 2003a), the abundance of LKB1 was diminished 1 h after GA addition (Figure 4c), a phenomenon which persisted for at least 48 h (Figure 4c). In the same experiment, LKB1 was immunoprecipitated and its catalytic activity was assessed using the LKBtide peptide substrate (Lizcano et al., 2004). Following the immunokinase assay, quantification of the radioactive band normalized with the level of LKB1 protein revealed that GA rapidly increased LKB1 kinase activity (Figure 4b). The increase of catalytic activity reached a peak at 24 h and subsequently declined to the basal level 48 h after GA addition (Figure 4c). Thus, these data indicate that GA treatment activates LKB1, and suggest that Hsp90 not only has a role in the stabilization of LKB1 but also functions as a repressor of its activation. It further suggests that LKB1 activation relies on both dissociation from the Hsp90–Cdc37 complex and association with the STRAD–MO25 complex. Remodeling of the chaperone complex is associated with LKB1 degradation Xu et al. (2002) have postulated that Hsp/Hsc70 associated to the CHIP protein (carboxyl terminus of

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Figure 4 LKB1 kinase activity in purified complexes, and transient activation of LKB1 by GA. (a) LKB1–STRADa–MO25a complex was expressed by baculovirus and purified from insect cells (Upstate Biotechnology, Lake Placid, NY, USA). LKB1–Hsp90–Cdc37 complex and LKB1–STRADa complexes were purified from bacteria. Different quantities of each complex were incubated for 30 min at 30 1C with [g-32P] adenosine triphosphate. Proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and LKB1 autophosphorylation was visualized by autoradiography (upper panels). Recombinant proteins were visualized with Coomassie blue staining (bottom panels). (b, c) Bosc cells were transfected with a vector expressing Flag-LKB1 and treated with 2 mM GA for various times as indicated. Cell lysates were immunoblotted with anti-Flag and anti-actin antibodies (upper panel). LKB1 protein was immunoprecipitated using the anti-Flag antibody, incubated for 20 min with LKBtide peptide and [g-32P] adenosine triphosphate and then LKB1 kinase activity was measured (bottom panel). The scale of the chart is displayed in an arbitrary unit (U). The results represent the averages of four independent experiments. The LKB1 catalytic activity was normalized to the total LKB1 protein level. WB, western blotting.

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Hsc70-interacting protein) forms a prodegradation chaperone complex. To determine whether this complex is involved in the downregulation of LKB1 stability, HBL100 cells transfected with Flag-LKB1 were incubated with GA and LKB1 was immunoprecipitated. Time-course experiments showed that the fraction of LKB1 bound to Hsp90 was greatly reduced at 15 min after GA addition, and virtually undetectable after 90 min of treatment (Figure 5a). Conversely, although some Hsp/Hsc70 protein was found associated with LKB1 in non-treated cells, the amount of Hsp/Hsc70 associated to LKB1 sharply increased 5 min after the beginning of GA incubation (Figure 5a). To analyze whether Hsp90 and Hsp/Hsc70 bind to overlapping LKB1 regions, a series of LKB1 deletion mutants was expressed in HBL100 cells (Figure 5b and Nony et al., 2003). LKB1 immunoprecipitation followed by immunoblot analysis revealed that the carboxyterminal region of LKB1 is required for Hsp/Hsc70 fixation, whereas this domain is dispensable for Hsp90 binding (Figure 5b). Furthermore, deletion of either the N-terminal region of LKB1 (1–88) or a portion of the kinase domain (146–186) did not impair Hsp/Hsc70 interaction with LKB1. Immunoblotting with the antiFlag antibody confirmed that the mutant forms of FLAG-LKB1

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Figure 5 Differential recruitment of chaperones. (a) HBL100 cells were transfected with a vector expressing Flag-LKB1 and lysed after the indicated times of treatment with 2 mM GA. LKB1 protein was immunoprecipitated with the anti-Flag antibody. Cell lysates and the immunocomplexes were immunoblotted using the antiHsp90, anti-Hsp/Hsc70 and anti-Flag antibodies. (b) A series of five LKB1 deletion mutants was generated: 1–243, 1–317, 1–88, 146–186 and 88–317. The black rectangles represent the regions of the LKB1 protein retained in the deletion mutants. The kinase domain of LKB1 spans the region between amino acids 49 and 310. Wild-type or mutant Flag-LKB1 proteins were expressed in HBL100 cells and immunoprecipitated with the anti-Flag antibody. Immunoprecipitates were western blotted with anti-Hsp90 antibody and anti-Hsp/Hsc70 antibodies (upper panel). Cell lysates were western blotted with the anti-Flag antibody (bottom panel). IP, immunoprecipitation; WB, western blotting.

LKB1 were correctly expressed, even though the expression level of the 1–317 and the 88–317 mutants was weaker (Figure 5b). Taken together, these results show that LKB1 degradation is associated with preferential recruitment of the Hsp/Hsc70 chaperone and further indicate that Hsp90 and Hsp/Hsc70 recognize non-overlapping LKB1 motifs. CHIP interacts with LKB1 and mediates its degradation CHIP interacts with Hsp70/Hsp90 via its tetratricopeptide (TPR) motif (Figure 6a; Ballinger et al., 1999; Connell et al., 2001). CHIP also contains a U-box domain in its carboxy terminal tail that is endowed with an E3-ubiquitin ligase activity driving degradation of the chaperone substrates by the ubiquitin/proteasome system (Demand et al., 2001). To investigate whether CHIP binds to LKB1, HBL100 cells were co-transfected with vectors expressing Flag-LKB1 and Myc–CHIP. Immunoprecipitation of LKB1 followed by immunoblot analysis revealed that LKB1 interacts with CHIP (Figure 6a). The reverse experiment confirmed that CHIP and LKB1 form a complex (Figure 6a). We next investigated whether CHIP required association with chaperones to bind LKB1. It has been reported that a point mutation within the TPR domain (K30A) disrupts CHIP interaction with Hsp70/Hsp90 (Xu et al., 2002). As shown in Figure 6a, CHIP–K30A TPR mutant did not coimmunoprecipitate with LKB1, suggesting that CHIP is recruited on LKB1 as part of a chaperone complex. In the same experiment, we examined the functional consequences of CHIP expression on LKB1. Immunoblot analysis of cell lysate showed that expression of wild-type CHIP drastically decreased LKB1 protein levels. As a negative control, the level of the N-terminally flagged docking protein FRS2 remained unchanged on CHIP expression. Expression of CHIP– K30A or CHIP–H260Q (a mutant deficient in E3 ubiquitin ligase activity) did not impact on LKB1 protein level (Figure 6a and data not shown). To determine whether endogenous CHIP regulates LKB1 stability, HeLa cells were transfected with either control siRNA (green florescent protein) or an siRNA targeting CHIP (ch1), together with a plasmid encoding LKB1. As shown in Figure 6b, a significant reduction of CHIP protein level was achieved on RNAi transfection. Immunoblot analysis revealed that CHIP knockdown led to an increase of the level of exogenous LKB1 (Figure 6b). HBL100 cells were transfected either with control siRNA (green florescent protein) or with two distinct siRNAs targeting CHIP (Ch1 or Ch2). In the same way, downregulation of CHIP by RNAi led to the accumulation of endogenous LKB1 protein (Figure 6b), suggesting that endogenous CHIP is involved in LKB1 degradation. CHIP has been reported to be involved in GA-induced ubiquitination and degradation of Hsp70/Hsp90 client proteins, such as cystic fibrosis transmembrane conductance regulator (CFTR), ErbB2 and estrogen receptors (Meacham et al., 2001; Xu et al., 2002; Fan et al., 2005). Oncogene

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To determine whether this is also the case for LKB1, we first analyzed the capacity of GA to induce CHIP–LKB1 interaction. Addition of GA significantly enhanced the amount of wild-type CHIP immunoprecipitated with LKB1 but not that of the TPR mutant. We also observed that expression of ectopic CHIP enhanced GA-induced LKB1 degradation (Figure 6c). Additive effect of both GA treatment and CHIP expression suggests that LKB1 dissociation from Hsp90 promotes its interaction with CHIP and its subsequent degradation. To examine whether CHIP can functions as a bona fide E3 ubiquitin-ligase toward LKB1, we performed a ubiquitination assay using in vitro translated LKB1 and purified components including ubiquitin, the ubiquitinconjugating enzyme UbcH5b, and CHIP. In these experimental settings, the reticulocyte lysate used for LKB1 translation contained all the components of the TPR repeats

Discussion

U-box

CHIP 1

303 *K30A: TPR mutation FLAG-FRS2

FLAG-LKB1 -

-

WT TPR

WT TPR

IP: αMYC

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LKB1

IP: αFLAG

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CHIP

WB: αFLAG

LKB1

FRS2

WB: αMYC

CHIP

CHIP

WB: αActin

Actin

Actin

Lysate

ubiquitin-dependent proteolytic system. Ubiquitination of LKB1 was then detected by immunoblotting. As shown in Figure 6d, incubation of LKB1 with only CHIP and ubiquitin did not lead to detectable LKB1 ubiquitination (Figure 6d, lane 7). Interestingly, the overall intensity of the LKB1-ubiquitinated bands was significantly increased on both CHIP and UbcH5b addition (Figure 6d, lane 8). In presence of UbcH5b and ubiquitin, we also observed smear bands corresponding to the polyubiquitinated forms of LKB1 (lane 6), which probably resulted from combined activity of UbcH5b and an E3 ubiquitin-ligase present in the reticulocyte lysate. Thus, these data support the notion that CHIP is an E3 ligase that promotes LKB1 ubiquitination. Taken together, these results indicate that CHIP is a key effector in the mechanism leading to the degradation of LKB1.

Hsp90 controls the stability and activity of large array of protein kinases, and as such is a key factors in the kinome biogenesis (Whitesell and Lindquist, 2005). We and others have previously found that LKB1 stability depends on its interaction with Hsp90 (Nony et al., 2003; Boudeau et al., 2003a). Through the use of the chemical compound celastrol and of RNAi, we have now obtained evidence that LKB1 stabilization requires the binding of Cdc37. Interestingly, depletion of Cdc37 in the nematode Caenorhabditis elegans affects the level of a phosphorylated form of the LKB1 ortholog PAR-4, thus suggesting that the mechanisms controlling the

1

2 Ch

G

Ch

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Ch

RNAi:

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1

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+ LKB1

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LKB1

WB: αCHIP

CHIP

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-GA +GA FLAG-LKB1: - + + + + + + MYC-CHIP: WT WT - TPR WT WT - TPR IP: αFLAG

Lysate

WB: αMYC

* CHIP

WB: αFLAG

LKB1

WB: αFLAG

LKB1

WB: αMYC

CHIP

(kDa) 200 LKB1-ub

116 97 66

LKB1 1 LKB1 + ub./ub.-ald. UbcH5b CHIP Oncogene

2 + + -

3 + +

4 5 6 7 8 + + + + + - + + + + + - + - + + - - + +

9 + + +

Figure 6 Physical and functional interactions between LKB1 and CHIP. (a) CHIP protein structure and localization of the mutation (K30A) in a TPR repeat. Vector encoding either Flag-LKB1 or Flag-FRS2 was co-transfected in HBL100 cells with an empty vector, the wild-type (WT) CHIP or the TPR mutant CHIP expressing vectors. LKB1 protein was immunoprecipitated with the anti-Flag antibody and CHIP protein was immunoprecipitated with the anti-Myc antibody. Cell lysates and immunoprecipitates were immunoblotted using anti-Myc, anti-Flag and anti-actin antibodies. (b) HeLa cells were transiently co-transfected with a vector expressing LKB1 and with control siRNA (Ct) or CHIP siRNA (Chip1). Cells were treated with 2 mM of GA for 2 h and cell lysates were immunoblotted with antibodies recognizing LKB1 isoforms, CHIP and actin (left panels). HBL100 cells were transfected with control siRNA (Ct), green florescent protein (GFP)–siRNA or CHIP–siRNA (Ch1 and Ch2). Cell lysates were immunoblotted using anti-LKB1, anti-CHIP and anti-actin antibodies (right panels). (c) HBL100 cells were co-transfected with a vector expressing Flag-LKB1, and with vectors expressing either WT CHIP or TPR mutant CHIP. At 72 h post transfection, the cells were treated or not with 2 mM GA for 2 h. LKB1 protein was immunoprecipitated with the anti-Flag antibody. Cell lysates and the immunocomplexes were immunoblotted using anti-Flag and anti-Myc antibodies. Asterisk (*) represents immunoglobulin G. (d) An adenosine triphosphate regenerating system was added to LKB1 containing reticulocyte lysate. Next, in vitro translated LKB1 was incubated in a reaction buffer with purified ubiquity (ub.), ubiquitin aldehyde (ald.), UbcH5b and CHIP. Ubiquitinated LKB1 was detected using the anti-LKB1 antibody. IP, immunoprecipitation; WB, western blotting.

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maturation and folding of LKB1 have been conserved across evolution, even though PAR-4 stability appears less critically dependent on Cdc37 than its mammalian counterpart (Beers and Kemphues, 2006). The Hsp90–Cdc37 complex was found associated to the newly described LKB1-S isoform that differs in its C-terminal part from the LKB1-L full-length isoform, but not to the novel variant LKB1-DN. As previous reports indicated that the interaction between LKB1 and Hsp90 is mediated through the LKB1 catalytic domain (Nony et al., 2003; Boudeau et al., 2003a), these results indicate that the Hsp90–Cdc37 complex binds to the N-terminal part of the LKB1 catalytic domain that is missing in the LKB1-DN isoform. Consistently, the half-life of LKB1-DN is significantly shorter than that of LKB1-L and LKB1-S. However, as STRAD is also known to stabilize LKB1 and does not bind LKB1-DN (data not shown), it is possible that LKB1-DN instability is because of the additive effects resulting from the absence of interaction with Hsp90–Cdc37 and STRAD. Furthermore, structure–function studies have shown that surface electrostatics located within the aC-b4 loop of the catalytic domain define the capacity of the client kinases to be recognized by Hsp90 (Xu et al., 2005; Citri et al., 2006). Inspection of the LKB1 aC-b4 loop sequence confirmed that the neutral/positive surface charge fits with electrostatic features, which define the Hsp90 recognition site on kinases. In agreement with our data, this Hsp90/Cdc37 binding motif is missing in the LKB1-DN isoform. As we have previously shown that a naturally occurring mutant of LKB1 (G163D) does not bind to Hsp90, other regions within the LKB1 catalytic domain must be required for the binding of the Hsp90/Cdc37 complex (Nony et al., 2003). It is well documented that Hsp90 maintains client kinases, such as ErbB2, PKR and c-Src, in an inactive state. The dissociation of Hsp90 releases its repressor activity and leads to a transient activation of the client protein kinase (Donze´ et al., 2001; Koga et al, 2006; Xu et al., 2007; Yano et al., 2008). We have similarly established here that Hsp90 regulates both LKB1 stability and kinase activity. Using recombinant complexes produced in E. coli, we showed that LKB1 in complex with Hsp90–Cdc37 did not exhibit any detectable kinase activity in contrast to the LKB1– STRADa complex produced in the same conditions. In addition, we found that GA treatment resulted in transient LKB1 activation (and consecutive AMPK activation, data not shown). The recently solved structure of the LKB1–STRAD–MO25 heterotrimer indicates that the LKB1 aC-b4 loop is exposed on the protein surface and positioned at the interface between LKB1 and STRADa (Zeqiraj et al., 2009). Thus, Hsp90–Cdc37 and STRADa may bind overlapping sequences on LKB1, an idea consistent with the molecular model of the LKB1–Hsp90–Cdc37 complex (Figure 7). As the two endogenous complexes LKB1– Hsp90–Cdc37 and the LKB1 heterotrimeric holoenzyme were detected in cells, whereas STRADa alone was not found in complex with Hsp90 and Cdc37, we speculate

Figure 7 Molecular modeling of the Hsp90–Cdc37–LKB1 complex. The Hsp90–Cdc37 complex is shown as cyan, orange and green surface representing respectively each subunits of the Hsp90 dimer and Cdc37. LKB1 (gray shading) is represented bound to the Hsp90 cyan subunit and to STRAD (dark blue shading). The LKB1 N-lobe points downward with its ‘aC-b4’ strand-loop-helix structure colored in yellow. The N-terminal Cdc37 domain involved in kinase binding lies within the area delimited by a green line where it could bind to the LKB1 aC-b4 strand and competes with STRAD binding. In this model, the Hsp90–Cdc37 structure was adapted from the Hsp90–Cdc37–Cdk4 structure determined by electron microscopy (Vaughan et al., 2008). The LKB1–STRAD complex was extracted from the LKB1–STRAD–MO25 crystal structure (Zeqiraj et al., 2009, and PDB ID code 2WTK) and has been placed onto Hsp90–Cdc37 by manual superimposition of its LKB1 kinase domain onto Cdk4.

that the binding of chaperones assists the maturation and folding of LKB1 to adopt the appropriate conformation required for STRAD–MO25 interaction. A similar situation has been reported for the CDK4 kinase, which requires Hsp90–Cdc37 to be appropriately folded for cyclin D1 interaction (Stepanova et al., 1996). Moreover, in our hypothetical model, the repressor function of Hsp90 on LKB1 would ensure that the kinase activity is restricted to the holoenzyme complex. Our previous study and another study demonstrated that dissociation of Hsp90 leads to LKB1 degradation by the ubiquitin–proteasome pathway (Nony et al., 2003; Boudeau et al., 2003a). However, the molecular mechanism(s) regulating LKB1 degradation remained undefined. In the current study, time-course of GA treatment revealed that Hsp/Hsc70 replaces Hsp90 in the LKB1 complex as early as 5 min after drug addition. Mapping of the interaction regions with LKB1 deletion mutants showed that the C-terminus domain is required for Hsp/Hsc70 binding, whereas this domain is dispensable for the interaction with Hsp90. In agreement with these results, we found LKB1-DN in complex with Hsp/Hsc70 and not with Hsp90 (data not shown). These results indicate that Hsp/Hsc70 and Hsp90 recognize non-overlapping motifs on LKB1. A change in the composition of molecular chaperones during the course of GA treatment has been previously reported for ErbB2, and it was further shown that ectopic expression of CHIP induced a similar remodeling of the chaperone complex on ErbB2 (Xu et al., 2002). CHIP was originally identified as a co-chaperone, which binds Oncogene

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Hsp/Hsc70 and Hsp90 through its TPR domain and blocks their protein folding activity (Ballinger et al., 1999; Demand et al., 2001). A growing number of results support a role of CHIP as a protein quality control E3 ligase that ubiquinates misfolded substrates of Hsp chaperones (Cyr et al., 2002; Murata et al., 2003; McDonough and Patterson, 2003). We provide several lines of evidence indicating that CHIP is involved in LKB1 degradation. First, CHIP was co-immunoprecipitated with LKB1, whereas a TPR mutant fails to bind LKB1, thus indicating that CHIP recruitment is mediated either by Hsp/Hsc70 or Hsp90 proteins. Second, expression of CHIP promotes LKB1 downregulation, whereas the TPR mutant does not affect LKB1 stability. In addition, inhibition of CHIP expression by RNAi led to an increase of endogenous LKB1 steady-state levels. Third, in vitro reconstitution of ubiquitination reactions demonstrated that E1, the E2 ubiquitin-conjugating enzyme UbcH5b and CHIP act as a bona fide ubiquitin–ligase complex toward LKB1. Collectively, these results indicate that CHIP is an LKB1 E3-ligase inducing its degradation. However, we cannot exclude that additional E3 ligases might participate to LKB1 ubiquitination and contribute to its degradation, as documented for other cancer-associated proteins such as the ErbB2 oncogene and the p53 tumor suppressor (Ehrlich et al., 2009). Finally, we also demonstrated that depletion of CHIP by RNAi induces a significant augmentation of the polarization of LS174T cells (data not shown), which depends on the LKB1–AMPK pathway (Baas et al., 2004; Lee et al., 2007). These data suggest that CHIP-mediated LKB1 degradation has a crucial role in the control of its biological functions.

In conclusion, our study uncovers novel mechanisms that allow the chaperones Hsp90–Ccd37 and Hsp/ Hsc70–CHIP to finely control the balance between stabilization and degradation of LKB1. Moreover, therapeutic strategies that aim at targeting Hsp90– Cdc37 should take into account potential adverse oncogenic side effects due to the induced degradation of tumor suppressors such as LKB1, a point deserving careful attention with the recent demonstration that GA analogs promote bone metastasis in experimental models (Yano et al., 2008).

Materials and methods Materials and methods are listed in the Supplementary Information.

Conflict of interest The authors declare no conflict of interest.

Acknowledgements We thank Len Neckers and Dirk Bohmann for the kind gifts of the CHIP and HA-ubiquitin vectors, respectively. This work was supported by the Ligue Nationale Contre le Cancer, and Association de Recherche sur le Cancer. During this work HG, NA and CC have been the recipients of fellowship from the Ligue Nationale Contre le Cancer, Ligue Nationale Contre le Cancer, Comite´ de l’Ise`re, Association de Recherche sur le Cancer and Fondation pour la Recherche Me´dicale and CNRS, respectively.

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

Oncogene