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Stabilization of integrin-linked kinase by binding to Hsp90
BBRC Biochemical and Biophysical Research Communications 331 (2005) 1061–1068 www.elsevier.com/locate/ybbrc
Stabilization of integrin-linked kinase by binding to Hsp90 Yumiko Aoyagi a, Naoya Fujita a, Takashi Tsuruo a,b,* a b
Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, 3-10-6, Ariake Kohtoh-ku, Tokyo 135-8550, Japan Received 28 March 2005 Available online 11 April 2005
Abstract Integrin-linked kinase (ILK) is a serine/threonine kinase that interacts with the cytoplasmic domain of b-integrins and growth factor receptors in response to extracellular signals. It is a key molecule in cell adhesion, proliferation, and cell survival. We found that treating cells with specific inhibitors of the heat shock protein 90 (Hsp90) caused rapid cell detachment. Screening the responsible proteins revealed a decreased amount of ILK in Hsp90 inhibitor-treated cells. ILK was identified as a new Hsp90 client protein because it formed a complex with Hsp90 and Cdc37, and binding was suppressed by Hsp90 inhibitors. Experiments with a series of ILK-deletion mutants revealed that the amino acid residues 377–406 were required for Hsp90 binding. Dissociation of ILK from Hsp90 shortened its half-life by promoting proteasome-dependent degradation. These results indicate that Hsp90 plays an important role in the stability of ILK in cells. Ó 2005 Elsevier Inc. All rights reserved. Keywords: ILK; Hsp90; Cancer; 17-AAG; Geldanamycin
The 90-kDa heat-shock proteins (Hsp90a and Hsp90b) are highly conserved, ubiquitously expressed, and abundant, and are involved in a diverse array of cellular processes. Deletion studies in yeast have revealed that Hsp90 is essential for cell growth [1]. In contrast to other heat-shock proteins, Hsp90 is not required for maturation or maintenance of most proteins in vivo. Most of the cellular targets (known as clients) are signaling proteins. Hsp90 acts as a chaperone for unstable clients and keeps them poised until they are stabilized by conformational changes associated with signal transduction. The identified Hsp90 client proteins, such as Akt, Raf-1, and 3-phosphoinositide-dependent protein kinase-1 (PDK1), were important participants in signaling pathways that drive tumor cell proliferation and survival [2–4]. A recent report suggested that tumor-derived Hsp90 showed high ATPase activity when compared with normal cell-derived Hsp90 [5]. Thus, Hsp90-direct*
Corresponding author. Fax: +81 3 5841 8487. E-mail address: [email protected] (T. Tsuruo).
0006-291X/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.03.225
ed therapy has been viewed as a mechanism to target numerous oncogenic signaling pathways simultaneously and to sensitize cells to chemotherapeutic agents [6,7]. The interaction of Hsp90 with client proteins depends on its ability to bind and hydrolyze ATP. ATP-mimetic drugs, including radicicol and the ansamycin antibiotics geldanamycin [8], can effectively prevent completion of client protein refolding and lead to proteasome-dependent degradation of proteins that require Hsp90 for conformational maturation [9]. Therefore, Hsp90 inhibitors are expected to be potent anti-cancer drugs. A less-toxic derivative of geldanamycin, 17-(allylamino)-17-demethoxygeldanamycin (17-AAG), has now entered into phase I clinical trials for melanoma [10]. ILK is an ankyrin-repeat containing serine/threonine protein kinase, which has been determined to be a b1-integrin subunit interactor [11]. ILK forms a multiprotein complex in focal adhesions by interacting with a-parvin, b-parvin, paxillin, and PINCH, and is reported to play essential roles in cell spreading and actin organization [12–15]. ILK has been shown to induce the phosphory-
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lation of Akt and GSK-3b [16]. The phosphorylation leads to cell survival and proliferation [16,17], and to angiogenesis through HIF-1 [18]. ILK has been reported to phosphorylate transcriptional factor aNAC and to promote invasion by upregulating matrix metalloproteinase 9 expression through AP-1 activation [19]. These studies suggest that ILK plays a pivotal role in tumor development. In support of this notion, ILK overexpression and hyperactivity were reported to be correlated with tumor progression in prostate cancer, colon cancer, gastric cancer, ovarian cancer, malignant melanoma, breast cancer, and Ewing sarcoma [20]. Thus, ILK is believed to be a new cancer therapeutic target. Recently, small molecules that could inhibit ILK kinase activity were developed. These small molecules were reported to effectively suppress tumor growth in vitro and in vivo [18,21]. In this study, we revealed cell detachment and decrease in ILK expression when cells were treated with Hsp90 inhibitors. Thus, we investigated the possibility that ILK is one of the Hsp90 client proteins. We found the association of endogenous ILK with endogenous Hsp90 in cells and that kinase-specific co-chaperone Cdc37 was involved in ILK–Hsp90 complex. The amino acid residues 377–406 on ILK were identified as the Hsp90 binding domain. Hsp90 inhibitor treatment dissociated ILK from Hsp90 and promoted proteasome-dependent degradation. We, thus, concluded that ILK requires the association of Hsp90 for its intracellular stability. Materials and methods Cell lines and culture conditions. Human embryonic kidney 293T, human fibrosarcoma HT1080, and African green monkey kidney COS7 cells were cultured in DulbeccoÕs modified EagleÕs medium (DMEM; Nissui, Pharmaceutical, Tokyo, Japan) supplemented with 10% heatinactivated fetal bovine serum (FBS) (Gibco Laboratories, Grand Island, NY). Mouse colon adenocarcinoma NL-17 cells, human breast cancer MCF-7 cells, human lung cancer A549 cells, and mouse lymph node stroma CA-12 cells were cultured in RPMI1640 medium (Nissui) supplemented with 10% FBS. These cells were cultured at 37 °C in a humidified atmosphere of 95% air and 5% CO2. Reagents. Geldanamycin, chymotrypsin-like serine protease inhibitor N-a-tosyl-L-phenylalanyl chloromethyl ketone (TPCK), trypsinlike serine protease inhibitor N-a-tosyl-L-lysine chloromethyl ketone (TLCK) and translation inhibitor cycloheximide were purchased from Sigma (St. Louis, MO). Oxime derivative of radicicol, KF58333, was kindly provided by Kyowa Hakko Kogyo (Tokyo, Japan). 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) was obtained from Alomone Labs (Jerusalem, Israel). Caspase inhibitors benzyloxycarbonyl-Asp-CH2OCO-2,6,-dichlorobenzene (Z-Asp) and benzyloxycarbonyl-Val-Ala-Asp-CH2OCO-2,6,-dichlorobenzene (Z-VAD) were purchased from Funakoshi (Tokyo, Japan). Proteasome inhibitors benzyloxycarbonyl-Leu-Leu-Leu-aldehyde (MG132), benzyloxycarbonyl-Ile-Glu(OBut)-Ala-Leu-aldehyde (PSI), and serine and cysteine protease inhibitor acetyl-Leu-Leu-Arg-aldehyde (leupeptin) were obtained from the Peptide Institute (Osaka, Japan). Plasmids. Human wild-type ILK cDNA (WT-ILK) in a pFLAGCMV-2 vector and a pHM6 vector were established in our laboratory. Several ILK fragments that encompassed residues 1–406 (DC407-ILK)
and 1–375 (DC376-ILK) were generated by converting appropriate codons in WT-ILK cDNA to stop codons using a QuickChange sitedirected mutagenesis kit (Stratagene, La Jolla, CA). Another ILK fragment 170–451 (DN169-ILK) was produced by PCR with WT-ILK cDNA as the template and then subcloned into a pHM6 vector. The V5-tagged human WT-hsp90b cDNA in a pcDNA3.1/GS vector was purchased from Invitrogen (San Diego, CA). WT-cdc37 cDNA was amplified by PCR with a cdc37 IMAGE clone [22] (clone ID 3619769) as the template and then subcloned into a pHM6 vector. All the plasmids for transfection were purified using a Qiagen plasmid Maxi or Spin kit, according to the manufacturerÕs protocol (Qiagen, Chatsworth, CA). Transient transfection, immunoprecipitation, and Western blot analysis. Cells were transfected with appropriate plasmids using Superfect transfection reagent (Qiagen) according to the manufacturerÕs instructions. At the appropriate time points, cells were harvested and solubilized in lysis buffer (20 mM Tris-HCl, pH 7.5, 0.2% Nonidet P-40, 10% glycerol, 1 mM EDTA, 1.5 mM magnesium chloride, and 137 mM sodium chloride, 50 mM sodium fluoride, 1 mM sodium vanadate, 12 mM b-glycerophosphate, 1 mM PMSF, and 1 mM aprotinin). The cell lysates were centrifuged at 17,000g for 15 min. The supernatants were incubated with antibody-conjugated beads (anti-FLAG M2 agarose or anti-V5 agarose; Sigma) or Protein G–Sepharose that had been conjugated with control mouse IgG (BD PharMingen, San Diego, CA) or mouse anti-ILK antibody (BD Transduction Laboratories, Lexington, KY). Immunoprecipitated proteins were electrophoresed in a 4–20% gradient or 10% polyacrylamide gel. The electrophoresed proteins were transblotted onto a nitrocellulose membrane. After blocking, the membrane was subjected to Western blot analysis, as described below. Western blot analysis was performed as below. In brief, cells were solubilized with the lysis buffer as described above. The cell lysates were centrifuged at 17,000g for 15 min. The supernatants were electrophoresed and transblotted onto a nitrocellulose membrane. In some experiments, the supernatants were recovered as soluble fractions and the precipitates were solubilized by directly adding the diluted sodium dodecyl sulfate (SDS) sample buffer to obtain insoluble fractions. After blocking, the membranes were incubated with antibodies to Akt (Cell Signaling Technology, Beverly, MA), PDK1, c-Raf-1, FAK, Mena, ILK (BD Transduction Laboratories), ILK (Medical and Biological Laboratories, Nagoya, Japan), HA tag (Roche, Mannheim, Germany), Hsp90 (Stressgen, Victoria, Canada), V5 tag (Invitrogen), FLAG tag or b-actin (Sigma). Blots were scanned using an EPSON ES-2200 scanner (EPSON, Tokyo, Japan) supported by AdobePhotoshop 5.5 and were quantified using NIH Image 1.62 software. Cell adhesion assay. After treating cells with drugs for 6 h, floating cells were removed after rotating for 30 min at 150 min1 on a gel destain rotator (Marysol, Tokyo, Japan). Bound cells were counted by incubating with 3-(4,5-methylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) for 2.5 h. Formazon products were solubilized with dimethyl sulfoxide, and the optical density was measured at 525 nm with a reference at 650 nm, using a microplate spectrophotometer (Benchmark Plus, Bio-Rad, Hercules, CA). siRNA design and transfection. Two siRNAs targeting ILK mRNA were designed according to the previous report [23] and synthesized by Qiagen. The coding strand of siRNA named ILK-3 was 5 0 AATCTCAACCGTATTCCATAC-3 0 (directed to residues 472–492 of ILK mRNA) and the coding strand of siRNA named ILK-5 was 5 0 AATGTACTACATGAAGGCACC-3 0 (directed to residues 835–855). Non-silencing control siRNA was purchased from Qiagen. Transient transfections were carried out using LipofectAMINE 2000 reagent (Invitrogen), according to the manufacturerÕs guidelines. Immunostaining. Cells were plated onto glass-bottomed dishes coated with poly-D-lysine (MatTek, Ashland, MA). The cells were washed, fixed, and permeabilized in 4% paraformaldehyde with 0.2% Triton X-100 for 10 min. After blocking for 1 h in PBS supplemented with 10% bovine serum albumin (Sigma), the labeling was carried out
Y. Aoyagi et al. / Biochemical and Biophysical Research Communications 331 (2005) 1061–1068 by incubation for 1 h with a mouse monoclonal anti-ILK antibody (Medical and Biological Laboratories) followed by a 45-min incubation with Hoechst 33342, Alexa Flour 488-conjugated goat anti-mouse IgG, and Texas red-labeled phalloidin (Molecular Probes, Eugene, OR). After washing, cells were visualized using a fluorescence microscope (Olympus IX-70, Olympus, Tokyo, Japan) equipped with a CCD camera.
Results and discussion Treatment of the cells with Hsp90 inhibitors induces cell detachment and ILK downregulation Hsp90 plays an important role in cell signaling, cell proliferation, and apoptosis. We have previously found that Hsp90 interacts with Akt and PDK1 in cells [2,3]. When cells were treated with Hsp90 inhibitors, they underwent apoptosis, at least, in part by suppressing the Akt signaling pathway in 18–24 h. In experiments with the Hsp90-inhibitor treatment, we found that cells detached from the culture dishes before undergoing apoptosis. As shown in Fig. 1A (left panel), mouse lymph node stroma CA-12 cells tightly bound on the surface of cell culture dishes and formed starburst shapes. When they were treated with radicicol analogue
Fig. 1. Hsp90 inhibitor-mediated cell detachment. (A) Phase contrast microphotographs of CA-12 cells. Cells were plated for 12 h and then treated with (right) or without (left) 1 lM KF58333 for 6 h. (B) A549, MCF-7, HT1080, CA-12, and 293T cells were plated onto a 24-well plate and treated with vehicle, 1 lM KF58333 or 1 lM 17-AAG for 6 h. To remove detached cells, plates were rotated at 150 min1 for 30 min. MTT assay was performed to measure adherent cells. The value of control cells was taken as 100%.
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KF58333 (1 lM) [24], CA-12 cells round up in 6 h and finally lost their adhesiveness (Fig. 1A, right panel). We obtained similar results in human embryonic kidney 293T cells (data not shown). To confirm this observation, we checked the effect of Hsp90 inhibitors on cell detachment in several cell lines including cancer cell lines (Fig. 1B). Human lung cancer A549, human fibrosarcoma HT1080, human breast cancer MCF-7, CA-12, and 293T cells were plated on 24-well plate and treated with vehicle, 1 lM KF58333 or 1 lM 17-AAG. As shown in Fig. 1B, both Hsp90 inhibitors induced cell detachment in all the cell lines tested within 6 h, indicating that these effects on Hsp90 inhibitors are not restricted to particular cell lines. These results suggest that Hsp90 inhibitors have some effects on cell adhesion-associated molecules. Consistent with our previous findings [2,3], treatment of the cells with benzoquinone ansamycin geldanamycin, KF58333, and a geldanamycin analogue 17-AAG [25] resulted in Akt and PDK1 degradation in 293T cells (Fig. 2A). Under this condition, we checked the expression of adhesion-associated molecules in 293T cells that had been treated with Hsp90 inhibitors. We revealed that geldanamycin, KF58333, and 17-AAG drastically downregulated endogenous ILK expression in a dosedependent fashion in 293T cells (Fig. 2A). The ILK destabilization was also observed in HT1080, African green monkey kidney COS-7, mouse colon adenocarcinoma NL-17 (Fig. 2B), and CA-12 cells (data not shown), indicating that these effects of Hsp90 inhibitors are not restricted to one particular cell line. Moreover, destabilization of ILK was also observed in the transfected ILK protein (data not shown). Hsp90 inhibitors had no effect on the expression levels of other focal adhesion-associated proteins Mena, p130Cas, vasodilatorstimulated phosphoprotein (VASP), and PYK2/CAKb (Fig. 2A and data not shown). Consistent with the work of Ochel et al. [26], we observed a slight decrease in FAK expression in 17-AAG-treated cells. FAK downregulation, however, might not be the main cause of cell detachment because high doses of Hsp90 inhibitors were required for FAK degradation (Fig. 2A). One of the basic steps toward cell adhesion was the generation of actin stress fiber [27]. To visualize actin stress fiber, we stained HT1080 and CA-12 cells with Texas red-labeled phalloidin and checked the formation under the fluorescence microscope. As shown in Fig. 2C, KF58333 treatment induced the loss of actin stress fibers in HT1080 cells. We obtained similar results in CA-12 cells (data not shown). Since focal adhesions are essential structures for linking integrins to the actin cytoskeleton, KF58333 might promote cell detachment by inhibiting the formation of focal adhesion complexes. To confirm the ILK involvement in cell adhesion, we checked cell adhesion in 293T cells that had been transfected with ILK siRNAs (Fig. 3). We synthesized ILK-3
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Fig. 2. ILK was depleted by Hsp90 inhibitors. (A) 293T cells were treated with the indicated concentrations of geldanamycin (Geldana), KF58333 or 17-AAG for 12 h. Cells were harvested and lysed in lysis buffer for Western blot analysis. The cell lysates were electrophoresed and immunoblotted with an anti-ILK antibody, an anti-Akt antibody, an anti-PDK1 antibody, an anti-FAK antibody, an anti-Mena antibody, or an anti-b-actin antibody. (B) HT1080, COS-7, and NL-17 cells were treated with the indicated concentrations of KF58333 for 18 h. Cells were harvested and lysed in lysis buffer for Western blot analysis. The cell lysates were electrophoresed and immunoblotted with an anti-ILK antibody, an anti-Raf-1 antibody, an anti-Hsp90 antibody, or an anti-b-actin antibody. (C) HT1080 cells plated onto glass-bottomed dishes were treated with or without 1 lM KF58333. After incubation for 24 h, the cells were fixed and stained. The endogenous ILK proteins were observed in green. The nuclei were observed in blue. The actin stress fibers were observed in red.
Fig. 3. Treatment of the cells with ILK siRNA-induced cell detachment. (A) 293T and HT1080 cells were transfected with siRNAs using LipofectAMINE 2000 (Control siRNA, ILK-3 siRNA or ILK-5 siRNA). After transfection for 96 h, cells were harvested and lysed with lysis buffer. The cell lysates were electrophoresed and immunoblotted with an anti-ILK antibody or an anti-b-actin antibody. (B) 293T cells were transfected with siRNAs as in (A). After transfection for 24 h, cells were plated onto glass-bottomed dishes. The phase contrast microphotographs were taken 96 h after transfection. (C) HT1080 cells were transfected with siRNAs as in (A). After transfection for 24 h, the cells were plated onto glass-bottomed dishes. After incubation for additional 72 h, cells were fixed and stained. The endogenous ILK proteins were observed in green. The nuclei were observed in blue. The actin stress fibers were observed in red.
and ILK-5 siRNAs according to the previous report [23]. Both siRNAs to ILK downregulated endogenous ILK expression in 293T and HT1080 cells (Fig. 3A).
Under the conditions, ILK siRNAs promoted cell detachment (Fig. 3B), like Hsp90 inhibitor-treated cells (Fig. 1A). ILK downregulation also interrupted the
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formation of actin stress fibers (Fig. 3C). These results indicate that decrease in ILK expression plays an important role in Hsp90 inhibitor-induced cell detachment. Hsp90 binds to middle domain of ILK Treating cells with Hsp90 inhibitors induced ILK destabilization; it is possible that ILK is an Hsp90 client protein. To examine the possibility, we first studied the ILK–Hsp90 binding in 293T cells by immunoprecipitating endogenous ILK protein. Although the mouse anti-ILK antibody we used was not good enough to immunoprecipitate abundant amounts of endogenous ILK protein, endogenous ILK was specifically immuno-
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precipitated (Fig. 4A). Under the condition, endogenous Hsp90 protein was co-immunoprecipitated by an antiILK antibody but not by a control mouse IgG. This result indicates that ILK forms a complex with Hsp90 in cells. To confirm the result, we transfected FLAGtagged WT-ILK into 293T cells and immunoprecipitated it with an anti-FLAG antibody. As shown in Fig. 4B, endogenous Hsp90 was present in the FLAGILK immunoprecipitates. Endogenous Hsp90 was not co-immunoprecipitated when 293T cells had not been transfected with FLAG-ILK (Fig. 4B). Interestingly, treatment of the cells with 1 lM KF58333 suppressed the ILK–Hsp90 binding and decreased the amount of co-immunoprecipitated endogenous Hsp90 in a time-
Fig. 4. ILK forms a complex with Hsp90 and cdc37. (A) Subconfluent 293T cells were harvested and lysed with lysis buffer for immunoprecipitation. Then cell lysates were incubated in agarose beads conjugated with anti-mouse IgG antibody for 1 h to avoid non-specific association to immunoglobulin. The endogenous ILK proteins were immunoprecipitated, and the co-immunoprecipitated proteins were detected by Western blotting. (B) 293T cells were transfected with empty pFLAG-CMV-2 vector or pFLAG-CMV-2 vector containing WT-ILK cDNA. After transfection for 20, 22, or 23 h, cells were treated with 1 lM KF58333. After incubation for the indicated times, cells were harvested and lysed with lysis buffer. The FLAG-tagged ILK proteins were immunoprecipitated with anti-FLAG agarose beads. The immunoprecipitated proteins were electrophoresed and immunoblotted with an anti-FLAG antibody or an anti-Hsp90 antibody. Expression of FLAG-tagged ILK and endogenous Hsp90 proteins was confirmed by immunoblot analysis with an anti-FLAG antibody or an anti-Hsp90 antibody. (C) 293T cells were transfected with pcDNA3.1/GS vector encoding WT-hsp90b together with pFLAG-CMV-2 vector containing WT-ILK and empty pHM6 vector or pHM6 vector encoding WT-cdc37. After transfection for 46 h, cells were treated with or without 1 lM KF58333. After incubation for an additional 2 h, cells were harvested and lysed with lysis buffer. The FLAG-tagged ILK proteins were immunoprecipitated with an anti-FLAG agarose. The immunoprecipitated proteins were electrophoresed and immunoblotted with an anti-V5 antibody, an anti-FLAG antibody, or an anti-HA antibody. (D) 293T cells were transfected with empty pcDNA3.1/GS vector or pcDNA3.1/GS vector encoding WT-hsp90b together with pHM6 vector containing WTILK (WT), DC407-ILK (DC407), DC376-ILK (DC376), or DN169-ILK (DN169) cDNA. After transfection for 24 h, cells were harvested and lysed with lysis buffer for immunoprecipitation. The HA-tagged ILK mutant proteins were immunoprecipitated with an anti-V5 agarose. The immunoprecipitated proteins were electrophoresed and immunoblotted with an anti-V5 antibody or an anti-HA antibody.
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dependent manner, without affecting the Hsp90 expression levels (Fig. 4B). We confirmed the ILK–Hsp90b binding by transfecting both ILK and hsp90b cDNAs into 293T cells (Fig. 4C). Hsp90 has been reported to have two isoforms, Hsp90a and Hsp90b [28]. Thus, we also examined ILK–Hsp90a binding by transfecting both ILK and hsp90a cDNAs into 293T cells. We obtained similar results using Hsp90a (data not shown). The ILK binding to endogenous Hsp90 or exogenous Hsp90b was inhibited by adding KF58333 to the cell culture medium (Figs. 4B and C). These results clearly indicate that ILK is a novel member of Hsp90 client proteins. Hsp90 carries out its functions as an integral component of a multiprotein chaperone complex that contains additional chaperones and co-chaperones [6]. Cdc37 is thought to be a unique co-chaperone that is involved in the folding and regulation of protein kinases, such as cdk4 [29], Akt [30], IKK [31], and src [32]. To examine the role of cdc37 in Hsp90–ILK complex formation, we generated an expression plasmid containing HA-tagged human cdc37. We then transfected hsp90b and ILK cDNAs together with the cdc37 cDNA into 293T cells. As shown in Fig. 4C, cdc37 was co-immunoprecipitated with FLAG-ILK. Treatment of the cells with Hsp90 inhibitors decreased the amount of co-immunoprecipitated cdc37 and Hsp90. These results indicate that cdc37 is composed of ILK–Hsp90 complex and is associated with the complex formation, as reported in cdk4, Akt, IKK, and src [29–32]. To identify more precisely the binding site in ILK, we prepared several NH2-terminal and COOH-terminal deletion mutants. These mutants were co-transfected with V5-tagged hsp90b into 293T cells. Immunoprecipitation following Western blot analysis revealed that V5tagged Hsp90b could interact with WT-, DN169-, and DC407-ILK (Fig. 4D). As Hsp90 could hardly interact with DC376-ILK, amino acid residues around 377–406 of ILK (middle of kinase domain) were found to be involved in ILK binding to Hsp90. The identified ILKbinding domain was reported to be associated with the binding to other ILK-binding partners, such as CH-ILKBP [15] and paxillin [33]. It would be interesting and important to examine the effect of Hsp90 binding on other partnersÕ binding. That binding will be the subject of future studies. Proteasome is associated with Hsp90 inhibitor-mediated ILK destabilization To clarify the mechanisms of ILK downregulation by the Hsp90 inhibitor, we first tried to determine the halflife of ILK in the cells treated with KF58333 or not treated. The change of half-life of ILK was estimated in the presence of the translation inhibitor cycloheximide. To
dissociate Hsp90 from ILK, HT1080 cells were pre-treated with 1 lM KF58333 or vehicle control 2 h before adding 50 lg/ml cycloheximide. At the appropriate time points, cells were harvested. ILK expression level was estimated by immunoblot analysis with an anti-ILK antibody. As shown in Fig. 5A, KF58333 treatment promoted the destabilization of ILK. The decrease in ILK
Fig. 5. Hsp90 inhibitor-mediated ILK depletion was involved in ILK degradation. (A) HT1080 cells were treated with or without 1 lM KF58333 for 2 h and then treated with 25 lg/ml cycloheximide for the indicated time. After incubation, cells were harvested and lysed with lysis buffer for Western blot analysis. The cell lysates were electrophoresed and immunoblotted with an anti-ILK antibody, an antiHsp90 antibody or an anti-b-actin antibody. (B) The ILK amount was quantified using IMAGE 1.62 software. The ILK level at 0 h was taken as 1. (C) 293T cells were treated with or without 1 lM KF58333 in the presence or absence of Z-Asp (100 lg/ml), Z-VAD (100 lg/ml), PSI (10 lM), leupeptin (20 lM), TPCK (20 lM), or TLCK (20 lM) for 18 h. MG132 (10 lM) was added 4 h before cell harvest. Then cells were harvested and lysed with lysis buffer for Western blot analysis. The cell lysates were centrifuged at 17,000g for 15 min. The supernatants were recovered as soluble fractions (soluble) and the precipitates were solubilized by directly adding the diluted SDS-sample buffer to obtain insoluble fractions (insoluble). Each fraction was electrophoresed and immunoblotted with an anti-ILK antibody or an anti-b-actin antibody.
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was observed in 6 h. Quantification of the ILK amount using NIH Image 1.62 software revealed that its half-life was 8.34 h in non-treated cells but 5.58 h in KF58333treated cells (Fig. 5B). The half-life of ILK was shortened by about 33% by Hsp90 inhibitor treatment. This result indicates that ILK requires the association of Hsp90 for its intracellular stability. Treatment with Hsp90 inhibitors is known to stimulate the proteasome-dependent degradation of several Hsp90 client proteins such as Akt and MOK [34]. To estimate the roles of proteases in ILK degradation, in the absence of Hsp90 function, we examined the effects of a series of protease inhibitors on ILK downregulation. 293T cells were cultured in medium containing KF58333 with caspase inhibitors (Z-Asp or Z-VAD), a proteasome inhibitor (PSI), a serine and cysteine protease inhibitor (leupeptin), a chymotrypsin-like serine protease inhibitor (TPCK), or a trypsin-like serine protease inhibitor (TLCK) for 18 h. Because proteasome inhibitor MG132 exhibited toxicity when cells were incubated for 18 h, it was added to the cell culture 4 h before cell harvest. Cells were then harvested and lysed with lysis buffer containing 0.2% NP-40. The cell lysates were centrifuged at 17,000g for 15 min. The supernatants were recovered as soluble fractions, and the precipitates were solubilized by directly adding the diluted sodium dodecyl sulfate (SDS) sample buffer to obtain insoluble fractions. Western blot analysis revealed that KF58333mediated ILK degradation was partially blocked by PSI (Fig. 5C) and increased the ILK amount in the soluble fraction (1.5-fold average). In the insoluble fraction, a drastic increase in the ILK amount was observed as a result of a chymotrypsin-like protease inhibitor TPCK and a proteasome inhibitor PSI, even in the presence of KF58333. Moreover, MG132 increased the ILK amount (2- to 3-fold average) in the insoluble fraction. However, MG132 showed marginal effects on the rescue of ILK amount when compared to the effects of PSI. MG132 showed high toxicity when compared to PSI. So, we could not treat cells with MG132 for 18 h. MG132 was added 4 h before cell harvest. This might be the reason why MG132 could not fully inhibit ILK degradation. Because slight increase in the ILK amount in insoluble fractions was also observed in the TPCK, PSI, and MG132-treated cells that were not treated with KF58333 (data not shown), proteasomes were also involved in steady-state turn over of ILK. These results indicate that the proteasome system is associated with ILK degradation induced by Hsp90 inhibitors. In conclusion, we herein identified ILK as a new member of the Hsp90 client protein group. To support this notion we revealed that ILK binds to Hsp90 through its middle kinase domain (Fig. 4D). Hsp90 inhibitors inhibit their complex formation and lead to proteasome-dependent ILK degradation (Figs. 4 and
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5). The ILK depletion might be associated with Hsp90 inhibitor-mediated cell detachment. Ochel et al. [26] have previously reported that FAK is an Hsp90 client protein. We confirmed the decrease in FAK in Hsp90 inhibitor-treated cells (Fig. 2A). However, a higher dose of Hsp90 inhibitors was required for FAK degradation when compared with that needed for ILK degradation. These results suggest that ILK downregulation is the major cause of Hsp90 inhibitor-mediated cell detachment. ILK overexpression was observed in various kinds of cancer and was reported to be associated with cell proliferation, metastasis, and invasion [20]. Thus, ILK has emerged as an attractive target for cancer therapy. A small-molecule ILK inhibitor was reported to effectively suppress tumor growth in vitro and in vivo [18,21]. On the other hand, it has been suggested that ILK functions as not only a kinase but also an adaptor [20]. Besides inhibition of ILK activity, repression of ILK expression level would be effective in cancer therapy. Thus, our findings could be valuable for developing new treatments for cancer. The correlation between Hsp90 inhibitors and ILK decrease will be the subject of future research.
Acknowledgments This study was supported in part by a special grant for Advanced Research on Cancer from the Ministry of Education, Culture, Sports, Science and Technology, Japan (to T.T.), and by a grant from Uehara Memorial Foundation (to N.F.).
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Stabilization of integrin-linked kinase by binding to Hsp90 Yumiko Aoyagi a, Naoya Fujita a, Takashi Tsuruo a,b,* a b
Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, 3-10-6, Ariake Kohtoh-ku, Tokyo 135-8550, Japan Received 28 March 2005 Available online 11 April 2005
Abstract Integrin-linked kinase (ILK) is a serine/threonine kinase that interacts with the cytoplasmic domain of b-integrins and growth factor receptors in response to extracellular signals. It is a key molecule in cell adhesion, proliferation, and cell survival. We found that treating cells with specific inhibitors of the heat shock protein 90 (Hsp90) caused rapid cell detachment. Screening the responsible proteins revealed a decreased amount of ILK in Hsp90 inhibitor-treated cells. ILK was identified as a new Hsp90 client protein because it formed a complex with Hsp90 and Cdc37, and binding was suppressed by Hsp90 inhibitors. Experiments with a series of ILK-deletion mutants revealed that the amino acid residues 377–406 were required for Hsp90 binding. Dissociation of ILK from Hsp90 shortened its half-life by promoting proteasome-dependent degradation. These results indicate that Hsp90 plays an important role in the stability of ILK in cells. Ó 2005 Elsevier Inc. All rights reserved. Keywords: ILK; Hsp90; Cancer; 17-AAG; Geldanamycin
The 90-kDa heat-shock proteins (Hsp90a and Hsp90b) are highly conserved, ubiquitously expressed, and abundant, and are involved in a diverse array of cellular processes. Deletion studies in yeast have revealed that Hsp90 is essential for cell growth [1]. In contrast to other heat-shock proteins, Hsp90 is not required for maturation or maintenance of most proteins in vivo. Most of the cellular targets (known as clients) are signaling proteins. Hsp90 acts as a chaperone for unstable clients and keeps them poised until they are stabilized by conformational changes associated with signal transduction. The identified Hsp90 client proteins, such as Akt, Raf-1, and 3-phosphoinositide-dependent protein kinase-1 (PDK1), were important participants in signaling pathways that drive tumor cell proliferation and survival [2–4]. A recent report suggested that tumor-derived Hsp90 showed high ATPase activity when compared with normal cell-derived Hsp90 [5]. Thus, Hsp90-direct*
Corresponding author. Fax: +81 3 5841 8487. E-mail address: [email protected] (T. Tsuruo).
0006-291X/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.03.225
ed therapy has been viewed as a mechanism to target numerous oncogenic signaling pathways simultaneously and to sensitize cells to chemotherapeutic agents [6,7]. The interaction of Hsp90 with client proteins depends on its ability to bind and hydrolyze ATP. ATP-mimetic drugs, including radicicol and the ansamycin antibiotics geldanamycin [8], can effectively prevent completion of client protein refolding and lead to proteasome-dependent degradation of proteins that require Hsp90 for conformational maturation [9]. Therefore, Hsp90 inhibitors are expected to be potent anti-cancer drugs. A less-toxic derivative of geldanamycin, 17-(allylamino)-17-demethoxygeldanamycin (17-AAG), has now entered into phase I clinical trials for melanoma [10]. ILK is an ankyrin-repeat containing serine/threonine protein kinase, which has been determined to be a b1-integrin subunit interactor [11]. ILK forms a multiprotein complex in focal adhesions by interacting with a-parvin, b-parvin, paxillin, and PINCH, and is reported to play essential roles in cell spreading and actin organization [12–15]. ILK has been shown to induce the phosphory-
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lation of Akt and GSK-3b [16]. The phosphorylation leads to cell survival and proliferation [16,17], and to angiogenesis through HIF-1 [18]. ILK has been reported to phosphorylate transcriptional factor aNAC and to promote invasion by upregulating matrix metalloproteinase 9 expression through AP-1 activation [19]. These studies suggest that ILK plays a pivotal role in tumor development. In support of this notion, ILK overexpression and hyperactivity were reported to be correlated with tumor progression in prostate cancer, colon cancer, gastric cancer, ovarian cancer, malignant melanoma, breast cancer, and Ewing sarcoma [20]. Thus, ILK is believed to be a new cancer therapeutic target. Recently, small molecules that could inhibit ILK kinase activity were developed. These small molecules were reported to effectively suppress tumor growth in vitro and in vivo [18,21]. In this study, we revealed cell detachment and decrease in ILK expression when cells were treated with Hsp90 inhibitors. Thus, we investigated the possibility that ILK is one of the Hsp90 client proteins. We found the association of endogenous ILK with endogenous Hsp90 in cells and that kinase-specific co-chaperone Cdc37 was involved in ILK–Hsp90 complex. The amino acid residues 377–406 on ILK were identified as the Hsp90 binding domain. Hsp90 inhibitor treatment dissociated ILK from Hsp90 and promoted proteasome-dependent degradation. We, thus, concluded that ILK requires the association of Hsp90 for its intracellular stability. Materials and methods Cell lines and culture conditions. Human embryonic kidney 293T, human fibrosarcoma HT1080, and African green monkey kidney COS7 cells were cultured in DulbeccoÕs modified EagleÕs medium (DMEM; Nissui, Pharmaceutical, Tokyo, Japan) supplemented with 10% heatinactivated fetal bovine serum (FBS) (Gibco Laboratories, Grand Island, NY). Mouse colon adenocarcinoma NL-17 cells, human breast cancer MCF-7 cells, human lung cancer A549 cells, and mouse lymph node stroma CA-12 cells were cultured in RPMI1640 medium (Nissui) supplemented with 10% FBS. These cells were cultured at 37 °C in a humidified atmosphere of 95% air and 5% CO2. Reagents. Geldanamycin, chymotrypsin-like serine protease inhibitor N-a-tosyl-L-phenylalanyl chloromethyl ketone (TPCK), trypsinlike serine protease inhibitor N-a-tosyl-L-lysine chloromethyl ketone (TLCK) and translation inhibitor cycloheximide were purchased from Sigma (St. Louis, MO). Oxime derivative of radicicol, KF58333, was kindly provided by Kyowa Hakko Kogyo (Tokyo, Japan). 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) was obtained from Alomone Labs (Jerusalem, Israel). Caspase inhibitors benzyloxycarbonyl-Asp-CH2OCO-2,6,-dichlorobenzene (Z-Asp) and benzyloxycarbonyl-Val-Ala-Asp-CH2OCO-2,6,-dichlorobenzene (Z-VAD) were purchased from Funakoshi (Tokyo, Japan). Proteasome inhibitors benzyloxycarbonyl-Leu-Leu-Leu-aldehyde (MG132), benzyloxycarbonyl-Ile-Glu(OBut)-Ala-Leu-aldehyde (PSI), and serine and cysteine protease inhibitor acetyl-Leu-Leu-Arg-aldehyde (leupeptin) were obtained from the Peptide Institute (Osaka, Japan). Plasmids. Human wild-type ILK cDNA (WT-ILK) in a pFLAGCMV-2 vector and a pHM6 vector were established in our laboratory. Several ILK fragments that encompassed residues 1–406 (DC407-ILK)
and 1–375 (DC376-ILK) were generated by converting appropriate codons in WT-ILK cDNA to stop codons using a QuickChange sitedirected mutagenesis kit (Stratagene, La Jolla, CA). Another ILK fragment 170–451 (DN169-ILK) was produced by PCR with WT-ILK cDNA as the template and then subcloned into a pHM6 vector. The V5-tagged human WT-hsp90b cDNA in a pcDNA3.1/GS vector was purchased from Invitrogen (San Diego, CA). WT-cdc37 cDNA was amplified by PCR with a cdc37 IMAGE clone [22] (clone ID 3619769) as the template and then subcloned into a pHM6 vector. All the plasmids for transfection were purified using a Qiagen plasmid Maxi or Spin kit, according to the manufacturerÕs protocol (Qiagen, Chatsworth, CA). Transient transfection, immunoprecipitation, and Western blot analysis. Cells were transfected with appropriate plasmids using Superfect transfection reagent (Qiagen) according to the manufacturerÕs instructions. At the appropriate time points, cells were harvested and solubilized in lysis buffer (20 mM Tris-HCl, pH 7.5, 0.2% Nonidet P-40, 10% glycerol, 1 mM EDTA, 1.5 mM magnesium chloride, and 137 mM sodium chloride, 50 mM sodium fluoride, 1 mM sodium vanadate, 12 mM b-glycerophosphate, 1 mM PMSF, and 1 mM aprotinin). The cell lysates were centrifuged at 17,000g for 15 min. The supernatants were incubated with antibody-conjugated beads (anti-FLAG M2 agarose or anti-V5 agarose; Sigma) or Protein G–Sepharose that had been conjugated with control mouse IgG (BD PharMingen, San Diego, CA) or mouse anti-ILK antibody (BD Transduction Laboratories, Lexington, KY). Immunoprecipitated proteins were electrophoresed in a 4–20% gradient or 10% polyacrylamide gel. The electrophoresed proteins were transblotted onto a nitrocellulose membrane. After blocking, the membrane was subjected to Western blot analysis, as described below. Western blot analysis was performed as below. In brief, cells were solubilized with the lysis buffer as described above. The cell lysates were centrifuged at 17,000g for 15 min. The supernatants were electrophoresed and transblotted onto a nitrocellulose membrane. In some experiments, the supernatants were recovered as soluble fractions and the precipitates were solubilized by directly adding the diluted sodium dodecyl sulfate (SDS) sample buffer to obtain insoluble fractions. After blocking, the membranes were incubated with antibodies to Akt (Cell Signaling Technology, Beverly, MA), PDK1, c-Raf-1, FAK, Mena, ILK (BD Transduction Laboratories), ILK (Medical and Biological Laboratories, Nagoya, Japan), HA tag (Roche, Mannheim, Germany), Hsp90 (Stressgen, Victoria, Canada), V5 tag (Invitrogen), FLAG tag or b-actin (Sigma). Blots were scanned using an EPSON ES-2200 scanner (EPSON, Tokyo, Japan) supported by AdobePhotoshop 5.5 and were quantified using NIH Image 1.62 software. Cell adhesion assay. After treating cells with drugs for 6 h, floating cells were removed after rotating for 30 min at 150 min1 on a gel destain rotator (Marysol, Tokyo, Japan). Bound cells were counted by incubating with 3-(4,5-methylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) for 2.5 h. Formazon products were solubilized with dimethyl sulfoxide, and the optical density was measured at 525 nm with a reference at 650 nm, using a microplate spectrophotometer (Benchmark Plus, Bio-Rad, Hercules, CA). siRNA design and transfection. Two siRNAs targeting ILK mRNA were designed according to the previous report [23] and synthesized by Qiagen. The coding strand of siRNA named ILK-3 was 5 0 AATCTCAACCGTATTCCATAC-3 0 (directed to residues 472–492 of ILK mRNA) and the coding strand of siRNA named ILK-5 was 5 0 AATGTACTACATGAAGGCACC-3 0 (directed to residues 835–855). Non-silencing control siRNA was purchased from Qiagen. Transient transfections were carried out using LipofectAMINE 2000 reagent (Invitrogen), according to the manufacturerÕs guidelines. Immunostaining. Cells were plated onto glass-bottomed dishes coated with poly-D-lysine (MatTek, Ashland, MA). The cells were washed, fixed, and permeabilized in 4% paraformaldehyde with 0.2% Triton X-100 for 10 min. After blocking for 1 h in PBS supplemented with 10% bovine serum albumin (Sigma), the labeling was carried out
Y. Aoyagi et al. / Biochemical and Biophysical Research Communications 331 (2005) 1061–1068 by incubation for 1 h with a mouse monoclonal anti-ILK antibody (Medical and Biological Laboratories) followed by a 45-min incubation with Hoechst 33342, Alexa Flour 488-conjugated goat anti-mouse IgG, and Texas red-labeled phalloidin (Molecular Probes, Eugene, OR). After washing, cells were visualized using a fluorescence microscope (Olympus IX-70, Olympus, Tokyo, Japan) equipped with a CCD camera.
Results and discussion Treatment of the cells with Hsp90 inhibitors induces cell detachment and ILK downregulation Hsp90 plays an important role in cell signaling, cell proliferation, and apoptosis. We have previously found that Hsp90 interacts with Akt and PDK1 in cells [2,3]. When cells were treated with Hsp90 inhibitors, they underwent apoptosis, at least, in part by suppressing the Akt signaling pathway in 18–24 h. In experiments with the Hsp90-inhibitor treatment, we found that cells detached from the culture dishes before undergoing apoptosis. As shown in Fig. 1A (left panel), mouse lymph node stroma CA-12 cells tightly bound on the surface of cell culture dishes and formed starburst shapes. When they were treated with radicicol analogue
Fig. 1. Hsp90 inhibitor-mediated cell detachment. (A) Phase contrast microphotographs of CA-12 cells. Cells were plated for 12 h and then treated with (right) or without (left) 1 lM KF58333 for 6 h. (B) A549, MCF-7, HT1080, CA-12, and 293T cells were plated onto a 24-well plate and treated with vehicle, 1 lM KF58333 or 1 lM 17-AAG for 6 h. To remove detached cells, plates were rotated at 150 min1 for 30 min. MTT assay was performed to measure adherent cells. The value of control cells was taken as 100%.
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KF58333 (1 lM) [24], CA-12 cells round up in 6 h and finally lost their adhesiveness (Fig. 1A, right panel). We obtained similar results in human embryonic kidney 293T cells (data not shown). To confirm this observation, we checked the effect of Hsp90 inhibitors on cell detachment in several cell lines including cancer cell lines (Fig. 1B). Human lung cancer A549, human fibrosarcoma HT1080, human breast cancer MCF-7, CA-12, and 293T cells were plated on 24-well plate and treated with vehicle, 1 lM KF58333 or 1 lM 17-AAG. As shown in Fig. 1B, both Hsp90 inhibitors induced cell detachment in all the cell lines tested within 6 h, indicating that these effects on Hsp90 inhibitors are not restricted to particular cell lines. These results suggest that Hsp90 inhibitors have some effects on cell adhesion-associated molecules. Consistent with our previous findings [2,3], treatment of the cells with benzoquinone ansamycin geldanamycin, KF58333, and a geldanamycin analogue 17-AAG [25] resulted in Akt and PDK1 degradation in 293T cells (Fig. 2A). Under this condition, we checked the expression of adhesion-associated molecules in 293T cells that had been treated with Hsp90 inhibitors. We revealed that geldanamycin, KF58333, and 17-AAG drastically downregulated endogenous ILK expression in a dosedependent fashion in 293T cells (Fig. 2A). The ILK destabilization was also observed in HT1080, African green monkey kidney COS-7, mouse colon adenocarcinoma NL-17 (Fig. 2B), and CA-12 cells (data not shown), indicating that these effects of Hsp90 inhibitors are not restricted to one particular cell line. Moreover, destabilization of ILK was also observed in the transfected ILK protein (data not shown). Hsp90 inhibitors had no effect on the expression levels of other focal adhesion-associated proteins Mena, p130Cas, vasodilatorstimulated phosphoprotein (VASP), and PYK2/CAKb (Fig. 2A and data not shown). Consistent with the work of Ochel et al. [26], we observed a slight decrease in FAK expression in 17-AAG-treated cells. FAK downregulation, however, might not be the main cause of cell detachment because high doses of Hsp90 inhibitors were required for FAK degradation (Fig. 2A). One of the basic steps toward cell adhesion was the generation of actin stress fiber [27]. To visualize actin stress fiber, we stained HT1080 and CA-12 cells with Texas red-labeled phalloidin and checked the formation under the fluorescence microscope. As shown in Fig. 2C, KF58333 treatment induced the loss of actin stress fibers in HT1080 cells. We obtained similar results in CA-12 cells (data not shown). Since focal adhesions are essential structures for linking integrins to the actin cytoskeleton, KF58333 might promote cell detachment by inhibiting the formation of focal adhesion complexes. To confirm the ILK involvement in cell adhesion, we checked cell adhesion in 293T cells that had been transfected with ILK siRNAs (Fig. 3). We synthesized ILK-3
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Fig. 2. ILK was depleted by Hsp90 inhibitors. (A) 293T cells were treated with the indicated concentrations of geldanamycin (Geldana), KF58333 or 17-AAG for 12 h. Cells were harvested and lysed in lysis buffer for Western blot analysis. The cell lysates were electrophoresed and immunoblotted with an anti-ILK antibody, an anti-Akt antibody, an anti-PDK1 antibody, an anti-FAK antibody, an anti-Mena antibody, or an anti-b-actin antibody. (B) HT1080, COS-7, and NL-17 cells were treated with the indicated concentrations of KF58333 for 18 h. Cells were harvested and lysed in lysis buffer for Western blot analysis. The cell lysates were electrophoresed and immunoblotted with an anti-ILK antibody, an anti-Raf-1 antibody, an anti-Hsp90 antibody, or an anti-b-actin antibody. (C) HT1080 cells plated onto glass-bottomed dishes were treated with or without 1 lM KF58333. After incubation for 24 h, the cells were fixed and stained. The endogenous ILK proteins were observed in green. The nuclei were observed in blue. The actin stress fibers were observed in red.
Fig. 3. Treatment of the cells with ILK siRNA-induced cell detachment. (A) 293T and HT1080 cells were transfected with siRNAs using LipofectAMINE 2000 (Control siRNA, ILK-3 siRNA or ILK-5 siRNA). After transfection for 96 h, cells were harvested and lysed with lysis buffer. The cell lysates were electrophoresed and immunoblotted with an anti-ILK antibody or an anti-b-actin antibody. (B) 293T cells were transfected with siRNAs as in (A). After transfection for 24 h, cells were plated onto glass-bottomed dishes. The phase contrast microphotographs were taken 96 h after transfection. (C) HT1080 cells were transfected with siRNAs as in (A). After transfection for 24 h, the cells were plated onto glass-bottomed dishes. After incubation for additional 72 h, cells were fixed and stained. The endogenous ILK proteins were observed in green. The nuclei were observed in blue. The actin stress fibers were observed in red.
and ILK-5 siRNAs according to the previous report [23]. Both siRNAs to ILK downregulated endogenous ILK expression in 293T and HT1080 cells (Fig. 3A).
Under the conditions, ILK siRNAs promoted cell detachment (Fig. 3B), like Hsp90 inhibitor-treated cells (Fig. 1A). ILK downregulation also interrupted the
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formation of actin stress fibers (Fig. 3C). These results indicate that decrease in ILK expression plays an important role in Hsp90 inhibitor-induced cell detachment. Hsp90 binds to middle domain of ILK Treating cells with Hsp90 inhibitors induced ILK destabilization; it is possible that ILK is an Hsp90 client protein. To examine the possibility, we first studied the ILK–Hsp90 binding in 293T cells by immunoprecipitating endogenous ILK protein. Although the mouse anti-ILK antibody we used was not good enough to immunoprecipitate abundant amounts of endogenous ILK protein, endogenous ILK was specifically immuno-
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precipitated (Fig. 4A). Under the condition, endogenous Hsp90 protein was co-immunoprecipitated by an antiILK antibody but not by a control mouse IgG. This result indicates that ILK forms a complex with Hsp90 in cells. To confirm the result, we transfected FLAGtagged WT-ILK into 293T cells and immunoprecipitated it with an anti-FLAG antibody. As shown in Fig. 4B, endogenous Hsp90 was present in the FLAGILK immunoprecipitates. Endogenous Hsp90 was not co-immunoprecipitated when 293T cells had not been transfected with FLAG-ILK (Fig. 4B). Interestingly, treatment of the cells with 1 lM KF58333 suppressed the ILK–Hsp90 binding and decreased the amount of co-immunoprecipitated endogenous Hsp90 in a time-
Fig. 4. ILK forms a complex with Hsp90 and cdc37. (A) Subconfluent 293T cells were harvested and lysed with lysis buffer for immunoprecipitation. Then cell lysates were incubated in agarose beads conjugated with anti-mouse IgG antibody for 1 h to avoid non-specific association to immunoglobulin. The endogenous ILK proteins were immunoprecipitated, and the co-immunoprecipitated proteins were detected by Western blotting. (B) 293T cells were transfected with empty pFLAG-CMV-2 vector or pFLAG-CMV-2 vector containing WT-ILK cDNA. After transfection for 20, 22, or 23 h, cells were treated with 1 lM KF58333. After incubation for the indicated times, cells were harvested and lysed with lysis buffer. The FLAG-tagged ILK proteins were immunoprecipitated with anti-FLAG agarose beads. The immunoprecipitated proteins were electrophoresed and immunoblotted with an anti-FLAG antibody or an anti-Hsp90 antibody. Expression of FLAG-tagged ILK and endogenous Hsp90 proteins was confirmed by immunoblot analysis with an anti-FLAG antibody or an anti-Hsp90 antibody. (C) 293T cells were transfected with pcDNA3.1/GS vector encoding WT-hsp90b together with pFLAG-CMV-2 vector containing WT-ILK and empty pHM6 vector or pHM6 vector encoding WT-cdc37. After transfection for 46 h, cells were treated with or without 1 lM KF58333. After incubation for an additional 2 h, cells were harvested and lysed with lysis buffer. The FLAG-tagged ILK proteins were immunoprecipitated with an anti-FLAG agarose. The immunoprecipitated proteins were electrophoresed and immunoblotted with an anti-V5 antibody, an anti-FLAG antibody, or an anti-HA antibody. (D) 293T cells were transfected with empty pcDNA3.1/GS vector or pcDNA3.1/GS vector encoding WT-hsp90b together with pHM6 vector containing WTILK (WT), DC407-ILK (DC407), DC376-ILK (DC376), or DN169-ILK (DN169) cDNA. After transfection for 24 h, cells were harvested and lysed with lysis buffer for immunoprecipitation. The HA-tagged ILK mutant proteins were immunoprecipitated with an anti-V5 agarose. The immunoprecipitated proteins were electrophoresed and immunoblotted with an anti-V5 antibody or an anti-HA antibody.
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dependent manner, without affecting the Hsp90 expression levels (Fig. 4B). We confirmed the ILK–Hsp90b binding by transfecting both ILK and hsp90b cDNAs into 293T cells (Fig. 4C). Hsp90 has been reported to have two isoforms, Hsp90a and Hsp90b [28]. Thus, we also examined ILK–Hsp90a binding by transfecting both ILK and hsp90a cDNAs into 293T cells. We obtained similar results using Hsp90a (data not shown). The ILK binding to endogenous Hsp90 or exogenous Hsp90b was inhibited by adding KF58333 to the cell culture medium (Figs. 4B and C). These results clearly indicate that ILK is a novel member of Hsp90 client proteins. Hsp90 carries out its functions as an integral component of a multiprotein chaperone complex that contains additional chaperones and co-chaperones [6]. Cdc37 is thought to be a unique co-chaperone that is involved in the folding and regulation of protein kinases, such as cdk4 [29], Akt [30], IKK [31], and src [32]. To examine the role of cdc37 in Hsp90–ILK complex formation, we generated an expression plasmid containing HA-tagged human cdc37. We then transfected hsp90b and ILK cDNAs together with the cdc37 cDNA into 293T cells. As shown in Fig. 4C, cdc37 was co-immunoprecipitated with FLAG-ILK. Treatment of the cells with Hsp90 inhibitors decreased the amount of co-immunoprecipitated cdc37 and Hsp90. These results indicate that cdc37 is composed of ILK–Hsp90 complex and is associated with the complex formation, as reported in cdk4, Akt, IKK, and src [29–32]. To identify more precisely the binding site in ILK, we prepared several NH2-terminal and COOH-terminal deletion mutants. These mutants were co-transfected with V5-tagged hsp90b into 293T cells. Immunoprecipitation following Western blot analysis revealed that V5tagged Hsp90b could interact with WT-, DN169-, and DC407-ILK (Fig. 4D). As Hsp90 could hardly interact with DC376-ILK, amino acid residues around 377–406 of ILK (middle of kinase domain) were found to be involved in ILK binding to Hsp90. The identified ILKbinding domain was reported to be associated with the binding to other ILK-binding partners, such as CH-ILKBP [15] and paxillin [33]. It would be interesting and important to examine the effect of Hsp90 binding on other partnersÕ binding. That binding will be the subject of future studies. Proteasome is associated with Hsp90 inhibitor-mediated ILK destabilization To clarify the mechanisms of ILK downregulation by the Hsp90 inhibitor, we first tried to determine the halflife of ILK in the cells treated with KF58333 or not treated. The change of half-life of ILK was estimated in the presence of the translation inhibitor cycloheximide. To
dissociate Hsp90 from ILK, HT1080 cells were pre-treated with 1 lM KF58333 or vehicle control 2 h before adding 50 lg/ml cycloheximide. At the appropriate time points, cells were harvested. ILK expression level was estimated by immunoblot analysis with an anti-ILK antibody. As shown in Fig. 5A, KF58333 treatment promoted the destabilization of ILK. The decrease in ILK
Fig. 5. Hsp90 inhibitor-mediated ILK depletion was involved in ILK degradation. (A) HT1080 cells were treated with or without 1 lM KF58333 for 2 h and then treated with 25 lg/ml cycloheximide for the indicated time. After incubation, cells were harvested and lysed with lysis buffer for Western blot analysis. The cell lysates were electrophoresed and immunoblotted with an anti-ILK antibody, an antiHsp90 antibody or an anti-b-actin antibody. (B) The ILK amount was quantified using IMAGE 1.62 software. The ILK level at 0 h was taken as 1. (C) 293T cells were treated with or without 1 lM KF58333 in the presence or absence of Z-Asp (100 lg/ml), Z-VAD (100 lg/ml), PSI (10 lM), leupeptin (20 lM), TPCK (20 lM), or TLCK (20 lM) for 18 h. MG132 (10 lM) was added 4 h before cell harvest. Then cells were harvested and lysed with lysis buffer for Western blot analysis. The cell lysates were centrifuged at 17,000g for 15 min. The supernatants were recovered as soluble fractions (soluble) and the precipitates were solubilized by directly adding the diluted SDS-sample buffer to obtain insoluble fractions (insoluble). Each fraction was electrophoresed and immunoblotted with an anti-ILK antibody or an anti-b-actin antibody.
Y. Aoyagi et al. / Biochemical and Biophysical Research Communications 331 (2005) 1061–1068
was observed in 6 h. Quantification of the ILK amount using NIH Image 1.62 software revealed that its half-life was 8.34 h in non-treated cells but 5.58 h in KF58333treated cells (Fig. 5B). The half-life of ILK was shortened by about 33% by Hsp90 inhibitor treatment. This result indicates that ILK requires the association of Hsp90 for its intracellular stability. Treatment with Hsp90 inhibitors is known to stimulate the proteasome-dependent degradation of several Hsp90 client proteins such as Akt and MOK [34]. To estimate the roles of proteases in ILK degradation, in the absence of Hsp90 function, we examined the effects of a series of protease inhibitors on ILK downregulation. 293T cells were cultured in medium containing KF58333 with caspase inhibitors (Z-Asp or Z-VAD), a proteasome inhibitor (PSI), a serine and cysteine protease inhibitor (leupeptin), a chymotrypsin-like serine protease inhibitor (TPCK), or a trypsin-like serine protease inhibitor (TLCK) for 18 h. Because proteasome inhibitor MG132 exhibited toxicity when cells were incubated for 18 h, it was added to the cell culture 4 h before cell harvest. Cells were then harvested and lysed with lysis buffer containing 0.2% NP-40. The cell lysates were centrifuged at 17,000g for 15 min. The supernatants were recovered as soluble fractions, and the precipitates were solubilized by directly adding the diluted sodium dodecyl sulfate (SDS) sample buffer to obtain insoluble fractions. Western blot analysis revealed that KF58333mediated ILK degradation was partially blocked by PSI (Fig. 5C) and increased the ILK amount in the soluble fraction (1.5-fold average). In the insoluble fraction, a drastic increase in the ILK amount was observed as a result of a chymotrypsin-like protease inhibitor TPCK and a proteasome inhibitor PSI, even in the presence of KF58333. Moreover, MG132 increased the ILK amount (2- to 3-fold average) in the insoluble fraction. However, MG132 showed marginal effects on the rescue of ILK amount when compared to the effects of PSI. MG132 showed high toxicity when compared to PSI. So, we could not treat cells with MG132 for 18 h. MG132 was added 4 h before cell harvest. This might be the reason why MG132 could not fully inhibit ILK degradation. Because slight increase in the ILK amount in insoluble fractions was also observed in the TPCK, PSI, and MG132-treated cells that were not treated with KF58333 (data not shown), proteasomes were also involved in steady-state turn over of ILK. These results indicate that the proteasome system is associated with ILK degradation induced by Hsp90 inhibitors. In conclusion, we herein identified ILK as a new member of the Hsp90 client protein group. To support this notion we revealed that ILK binds to Hsp90 through its middle kinase domain (Fig. 4D). Hsp90 inhibitors inhibit their complex formation and lead to proteasome-dependent ILK degradation (Figs. 4 and
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5). The ILK depletion might be associated with Hsp90 inhibitor-mediated cell detachment. Ochel et al. [26] have previously reported that FAK is an Hsp90 client protein. We confirmed the decrease in FAK in Hsp90 inhibitor-treated cells (Fig. 2A). However, a higher dose of Hsp90 inhibitors was required for FAK degradation when compared with that needed for ILK degradation. These results suggest that ILK downregulation is the major cause of Hsp90 inhibitor-mediated cell detachment. ILK overexpression was observed in various kinds of cancer and was reported to be associated with cell proliferation, metastasis, and invasion [20]. Thus, ILK has emerged as an attractive target for cancer therapy. A small-molecule ILK inhibitor was reported to effectively suppress tumor growth in vitro and in vivo [18,21]. On the other hand, it has been suggested that ILK functions as not only a kinase but also an adaptor [20]. Besides inhibition of ILK activity, repression of ILK expression level would be effective in cancer therapy. Thus, our findings could be valuable for developing new treatments for cancer. The correlation between Hsp90 inhibitors and ILK decrease will be the subject of future research.
Acknowledgments This study was supported in part by a special grant for Advanced Research on Cancer from the Ministry of Education, Culture, Sports, Science and Technology, Japan (to T.T.), and by a grant from Uehara Memorial Foundation (to N.F.).
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