Tumor suppressor Tsc1 is a new Hsp90 co‐chaperone that facilitates folding of kinase and non‐kinase clients
2017; Springer Nature; Volume: 36; Issue: 24 Linguagem: Inglês
10.15252/embj.201796700
ISSN1460-2075
AutoresMark R. Woodford, Rebecca Sager, Elijah Marris, Diana M. Dunn, Adam R. Blanden, Ryan L. Murphy, Nicholas Rensing, Oleg Shapiro, Barry Panaretou, Chrisostomos Prodromou, Stewart N. Loh, David H. Gutmann, Dimitra Bourboulia, Gennady Bratslavsky, Michael Wong, Mehdi Mollapour,
Tópico(s)DNA Repair Mechanisms
ResumoArticle10 November 2017Open Access Source DataTransparent process Tumor suppressor Tsc1 is a new Hsp90 co-chaperone that facilitates folding of kinase and non-kinase clients Mark R Woodford Mark R Woodford Department of Urology, SUNY Upstate Medical University, Syracuse, NY, USA Upstate Cancer Center, SUNY Upstate Medical University, Syracuse, NY, USA Search for more papers by this author Rebecca A Sager Rebecca A Sager Department of Urology, SUNY Upstate Medical University, Syracuse, NY, USA Upstate Cancer Center, SUNY Upstate Medical University, Syracuse, NY, USA Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, NY, USA Search for more papers by this author Elijah Marris Elijah Marris Department of Urology, SUNY Upstate Medical University, Syracuse, NY, USA Upstate Cancer Center, SUNY Upstate Medical University, Syracuse, NY, USA Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, NY, USA Search for more papers by this author Diana M Dunn Diana M Dunn Department of Urology, SUNY Upstate Medical University, Syracuse, NY, USA Upstate Cancer Center, SUNY Upstate Medical University, Syracuse, NY, USA Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, NY, USA Search for more papers by this author Adam R Blanden Adam R Blanden Upstate Cancer Center, SUNY Upstate Medical University, Syracuse, NY, USA Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, NY, USA Search for more papers by this author Ryan L Murphy Ryan L Murphy Department of Urology, SUNY Upstate Medical University, Syracuse, NY, USA Upstate Cancer Center, SUNY Upstate Medical University, Syracuse, NY, USA Search for more papers by this author Nicholas Rensing Nicholas Rensing Department of Neurology, Washington University School of Medicine, St. Louis, MO, USA Hope Center for Neurological Disorders, Washington University School of Medicine, St. Louis, MO, USA Search for more papers by this author Oleg Shapiro Oleg Shapiro Department of Urology, SUNY Upstate Medical University, Syracuse, NY, USA Upstate Cancer Center, SUNY Upstate Medical University, Syracuse, NY, USA Search for more papers by this author Barry Panaretou Barry Panaretou Institute of Pharmaceutical Science, King's College London, London, UK Search for more papers by this author Chrisostomos Prodromou Chrisostomos Prodromou Genome Damage and Stability Centre, University of Sussex, Brighton, UK Search for more papers by this author Stewart N Loh Stewart N Loh Upstate Cancer Center, SUNY Upstate Medical University, Syracuse, NY, USA Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, NY, USA Search for more papers by this author David H Gutmann David H Gutmann Department of Neurology, Washington University School of Medicine, St. Louis, MO, USA Search for more papers by this author Dimitra Bourboulia Dimitra Bourboulia Department of Urology, SUNY Upstate Medical University, Syracuse, NY, USA Upstate Cancer Center, SUNY Upstate Medical University, Syracuse, NY, USA Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, NY, USA Search for more papers by this author Gennady Bratslavsky Gennady Bratslavsky Department of Urology, SUNY Upstate Medical University, Syracuse, NY, USA Upstate Cancer Center, SUNY Upstate Medical University, Syracuse, NY, USA Search for more papers by this author Michael Wong Michael Wong Department of Neurology, Washington University School of Medicine, St. Louis, MO, USA Hope Center for Neurological Disorders, Washington University School of Medicine, St. Louis, MO, USA Search for more papers by this author Mehdi Mollapour Corresponding Author Mehdi Mollapour [email protected] orcid.org/0000-0002-2144-1361 Department of Urology, SUNY Upstate Medical University, Syracuse, NY, USA Upstate Cancer Center, SUNY Upstate Medical University, Syracuse, NY, USA Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, NY, USA Search for more papers by this author Mark R Woodford Mark R Woodford Department of Urology, SUNY Upstate Medical University, Syracuse, NY, USA Upstate Cancer Center, SUNY Upstate Medical University, Syracuse, NY, USA Search for more papers by this author Rebecca A Sager Rebecca A Sager Department of Urology, SUNY Upstate Medical University, Syracuse, NY, USA Upstate Cancer Center, SUNY Upstate Medical University, Syracuse, NY, USA Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, NY, USA Search for more papers by this author Elijah Marris Elijah Marris Department of Urology, SUNY Upstate Medical University, Syracuse, NY, USA Upstate Cancer Center, SUNY Upstate Medical University, Syracuse, NY, USA Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, NY, USA Search for more papers by this author Diana M Dunn Diana M Dunn Department of Urology, SUNY Upstate Medical University, Syracuse, NY, USA Upstate Cancer Center, SUNY Upstate Medical University, Syracuse, NY, USA Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, NY, USA Search for more papers by this author Adam R Blanden Adam R Blanden Upstate Cancer Center, SUNY Upstate Medical University, Syracuse, NY, USA Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, NY, USA Search for more papers by this author Ryan L Murphy Ryan L Murphy Department of Urology, SUNY Upstate Medical University, Syracuse, NY, USA Upstate Cancer Center, SUNY Upstate Medical University, Syracuse, NY, USA Search for more papers by this author Nicholas Rensing Nicholas Rensing Department of Neurology, Washington University School of Medicine, St. Louis, MO, USA Hope Center for Neurological Disorders, Washington University School of Medicine, St. Louis, MO, USA Search for more papers by this author Oleg Shapiro Oleg Shapiro Department of Urology, SUNY Upstate Medical University, Syracuse, NY, USA Upstate Cancer Center, SUNY Upstate Medical University, Syracuse, NY, USA Search for more papers by this author Barry Panaretou Barry Panaretou Institute of Pharmaceutical Science, King's College London, London, UK Search for more papers by this author Chrisostomos Prodromou Chrisostomos Prodromou Genome Damage and Stability Centre, University of Sussex, Brighton, UK Search for more papers by this author Stewart N Loh Stewart N Loh Upstate Cancer Center, SUNY Upstate Medical University, Syracuse, NY, USA Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, NY, USA Search for more papers by this author David H Gutmann David H Gutmann Department of Neurology, Washington University School of Medicine, St. Louis, MO, USA Search for more papers by this author Dimitra Bourboulia Dimitra Bourboulia Department of Urology, SUNY Upstate Medical University, Syracuse, NY, USA Upstate Cancer Center, SUNY Upstate Medical University, Syracuse, NY, USA Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, NY, USA Search for more papers by this author Gennady Bratslavsky Gennady Bratslavsky Department of Urology, SUNY Upstate Medical University, Syracuse, NY, USA Upstate Cancer Center, SUNY Upstate Medical University, Syracuse, NY, USA Search for more papers by this author Michael Wong Michael Wong Department of Neurology, Washington University School of Medicine, St. Louis, MO, USA Hope Center for Neurological Disorders, Washington University School of Medicine, St. Louis, MO, USA Search for more papers by this author Mehdi Mollapour Corresponding Author Mehdi Mollapour [email protected] orcid.org/0000-0002-2144-1361 Department of Urology, SUNY Upstate Medical University, Syracuse, NY, USA Upstate Cancer Center, SUNY Upstate Medical University, Syracuse, NY, USA Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, NY, USA Search for more papers by this author Author Information Mark R Woodford1,2,‡, Rebecca A Sager1,2,3,‡, Elijah Marris1,2,3, Diana M Dunn1,2,3, Adam R Blanden2,3, Ryan L Murphy1,2, Nicholas Rensing4,5, Oleg Shapiro1,2, Barry Panaretou6, Chrisostomos Prodromou7, Stewart N Loh2,3, David H Gutmann4, Dimitra Bourboulia1,2,3, Gennady Bratslavsky1,2, Michael Wong4,5 and Mehdi Mollapour *,1,2,3 1Department of Urology, SUNY Upstate Medical University, Syracuse, NY, USA 2Upstate Cancer Center, SUNY Upstate Medical University, Syracuse, NY, USA 3Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, NY, USA 4Department of Neurology, Washington University School of Medicine, St. Louis, MO, USA 5Hope Center for Neurological Disorders, Washington University School of Medicine, St. Louis, MO, USA 6Institute of Pharmaceutical Science, King's College London, London, UK 7Genome Damage and Stability Centre, University of Sussex, Brighton, UK ‡These authors contributed equally to this work *Corresponding author. Tel: +1 315 464 8749; E-mail: [email protected] The EMBO Journal (2017)36:3650-3665https://doi.org/10.15252/embj.201796700 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The tumor suppressors Tsc1 and Tsc2 form the tuberous sclerosis complex (TSC), a regulator of mTOR activity. Tsc1 stabilizes Tsc2; however, the precise mechanism involved remains elusive. The molecular chaperone heat-shock protein 90 (Hsp90) is an essential component of the cellular homeostatic machinery in eukaryotes. Here, we show that Tsc1 is a new co-chaperone for Hsp90 that inhibits its ATPase activity. The C-terminal domain of Tsc1 (998–1,164 aa) forms a homodimer and binds to both protomers of the Hsp90 middle domain. This ensures inhibition of both subunits of the Hsp90 dimer and prevents the activating co-chaperone Aha1 from binding the middle domain of Hsp90. Conversely, phosphorylation of Aha1-Y223 increases its affinity for Hsp90 and displaces Tsc1, thereby providing a mechanism for equilibrium between binding of these two co-chaperones to Hsp90. Our findings establish an active role for Tsc1 as a facilitator of Hsp90-mediated folding of kinase and non-kinase clients—including Tsc2—thereby preventing their ubiquitination and proteasomal degradation. Synopsis Besides stabilizing Tsc2 and forming the mTOR-regulating TSC complex, Tsc1 plays a more general role as a co-chaperone for Hsp90 on a range of client proteins. Tsc1 is a new co-chaperone for the molecular chaperone heat shock protein-90 (Hsp90). The interaction between Tsc1 and Hsp90 stabilises mTOR-regulator Tsc2. The co-chaperone Tsc1 binds to both subunits of the Hsp90 dimer and inhibits its ATPase activity. Tsc1 blocks the interaction between Hsp90 and the activating co-chaperone Aha1. Tsc1 is a facilitator of Hsp90-mediated folding of kinase and non-kinase clients. Introduction Molecular chaperones, as indicated by their names, are involved in folding, stability, and activity of many proteins also known as “client proteins” (Schopf et al, 2017). The molecular chaperone heat-shock protein 90 (Hsp90) looks after many clients (> 200 known) that are involved in tumor initiation, progression, and metastasis. Therefore, Hsp90 is recognized as a suitable target for cancer therapy (Neckers & Workman, 2012). Hsp90 chaperone function is linked to its ability to bind and hydrolyze ATP and depends on a cycle of Hsp90 conformational changes (Prodromou, 2012). Hsp90 drugs bind to the ATP-binding pocket of Hsp90 and inhibit its chaperone activity. In turn, this prevents Hsp90 association with client proteins, causing their proteasomal degradation. Unlike other anti-cancer drugs, Hsp90 inhibitors target multiple drivers of oncogenesis simultaneously. Co-chaperones regulate Hsp90 chaperone cycle by directly interacting with Hsp90 and providing directionality to the cycle (Cox & Johnson, 2011). Additionally, certain co-chaperones, for example, HOP and Cdc37p50, decelerate or inhibit the Hsp90 chaperone cycle, loading distinct sets of clients such as steroid hormone receptors and kinases, respectively, to the Hsp90 (Verba et al, 2016). Conversely, the Aha1 co-chaperone aids in high-energy conformational modulations necessary for Hsp90 ATPase competence. This markedly increases the weak endogenous enzymatic activity of Hsp90 and establishes Aha1 as a crucial component of active Hsp90 chaperone complexes (Panaretou et al, 2002; Li et al, 2013a). Tuberous sclerosis complex (TSC) is a rare autosomal dominant syndrome that is characterized by the development of benign tumors in various organs, including skin, brain, heart, kidneys, and bladder (Henske et al, 2016). TSC is also frequently associated with epilepsy, intellectual disability, and autism (Banerjee et al, 2011; Zeng et al, 2011). Mutations in one of the tumor suppressor genes, TSC1 or TSC2, cause TSC (Huang & Manning, 2008). Tsc2 protein (tuberin) of approximately 200 kDa in size behaves as a GTPase-activating protein (GAP), and Tsc1 (hamartin) with a molecular weight of 130 kDa is required to stabilize Tsc2 and prevent its ubiquitin-mediated degradation (Benvenuto et al, 2000). More specifically, Tsc1 prevents the interaction between Tsc2 and the E3 ubiquitin ligase HERC1 (Chong-Kopera et al, 2006). However, the precise protective role of Tsc1 toward Tsc2 remains elusive. Here, we show Tsc1 is a new co-chaperone of Hsp90. Tsc1 carboxy-domain forms a homodimer and it binds to the Hsp90 middle domain where it inhibits Hsp90 ATPase activity and also blocks interaction of the activating co-chaperone Aha1 with the Hsp90 middle domain. Tsc1 also increases the binding of Hsp90 to ATP and inhibitors. Tsc1 has a higher affinity for binding to Hsp90 than Aha1. However, this affinity is reversed when c-Abl-mediated phosphorylation of Aha1-Y223 increases its binding to Hsp90 and displaces Tsc1. This provides a mechanism of equilibrium between these two co-chaperones binding to Hsp90. Tsc1 functions as a facilitator of Hsp90 in chaperoning the kinase and non-kinase clients including Tsc2, therefore preventing their ubiquitination and degradation in the proteasome. Results Tsc2 is a client of Hsp70 and Hsp90 Molecular chaperones are generally involved in the folding and stability of proteins. We therefore tested whether the integrity of Tsc2 depended on the Hsp70 and Hsp90 chaperones. Endogenous Tsc2 was immunoprecipitated from HEK293 cells and it was shown to interact with both Hsp70 and Hsp90 and their co-chaperones including HOP, Cdc37p50, PP5, and p23, but not Aha1 (Figs 1A and EV1). We next showed that treating HEK293 cells with the Hsp70 inhibitor JG-98 (Fig 1B) (Li et al, 2013b), or Hsp90 inhibitors ganetespib GB (Fig 1C) (Ying et al, 2012), or SNX2112 (Fig 1D) (Barrott et al, 2013) cause the degradation of Tsc2. Surprisingly, inhibition of Hsp70 or Hsp90 did not affect the stability of Tsc1 (Fig 1B–D). It is noteworthy that we used the degradation of Akt and phospho-S473-Akt as a positive control for Hsp90 inhibition in cells, since Akt is a bona fide Hsp90 client protein. Figure 1. Tsc2 is a new client of Hsp90 A. Tsc2 was immunoprecipitated from HEK293 cell lysates using anti-Tsc2 antibody or IgG (control) and immunoblotted with indicated antibodies to confirm chaperone and co-chaperone interaction. B. HEK293 cells were treated with 10 μM JG-98 (Hsp70 inhibitor) for the indicated times. Tsc1 protein stability and Tsc2 protein stability were assessed by Western blotting. C, D. HEK293 cells were treated with either (C) 1 μM GB or (D) 2 μM SNX-2112 (Hsp90 inhibitors) for the indicated times. Tsc1 protein stability and Tsc2 protein stability were assessed by immunoblotting. Akt and phospho-S473-Akt were used as positive controls. E. HEK293 cells were treated with 50 nM bortezomib (BZ) for the indicated times, and Tsc1 and Tsc2 proteins were evaluated by immunoblotting. F. 50 nM BZ was added to HEK293 cells for 1 h followed by addition of 1 μM GB for 8 h. HEK293 cells were also treated individually with BZ or GB. UN represents untreated cells. Stability of Tsc2 was examined by immunoblotting. G. Empty vector (EV) or Tsc2-FLAG was used to transiently transfect HEK293 cells for 24 h followed by no treatment (UN), or treatment with either 50 nm BZ, 1 μM GB, or 10 μM JG-98 for 4 h. Tsc2-FLAG was immunoprecipitated, and ubiquitination was examined by immunoblotting with a anti-pan-ubiquitin antibody. Source data are available online for this figure. Source Data for Figure 1 [embj201796700-sup-0003-SDataFig1.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Tsc2 interaction with the chaperone and co-chaperone machinery (related to Fig 1)Tsc2 was immunoprecipitated from HEK293 cell lysates using anti-Tsc2 antibody and immunoblotted with indicated antibodies. IgG was used as control. These samples are the same as those presented in Fig 1A. Source data are available online for this figure. Download figure Download PowerPoint Inhibition of Hsp70 or Hsp90 generally leads to ubiquitination and degradation of its client proteins in the proteasome (Xu et al, 1999). Treating HEK293 cells with 50 nM proteasome inhibitor bortezomib (BZ) for 2 h stabilizes Tsc2 (Fig 1E). These data suggest that Tsc2 experiences protein turnover at steady state. We next showed that treating HEK293 cells with BZ for 1 h prior to addition of GB (Hsp90 inhibitor) blocked the degradation of Tsc2 (Fig 1F). Finally, to determine whether Hsp70 or Hsp90 inhibition leads to ubiquitination of Tsc2 prior to its degradation in the proteasome, Tsc2-FLAG was transiently expressed, immunoprecipitated, and salt-stripped (with 0.5 M NaCl) from HEK293 cells treated with either 50 nM BZ, 1 μM GB, or 10 μM JG-98 for 4 h. Tsc2 is ubiquitinated upon inhibition of Hsp70, Hsp90, or the proteasome (Fig 1G). Taken together, Tsc2 appears to be a new client of Hsp70 and Hsp90, and pharmacologic inhibition of Hsp90 leads to ubiquitination and degradation of Tsc2 in the proteasome. The new co-chaperone Tsc1 inhibits Hsp90 ATPase activity Tsc1 stability does not depend on Hsp70 and Hsp90; however, because of its relationship with Tsc2, we asked whether Tsc1 interacts with these two molecular chaperone machineries. We first immunoprecipitated endogenous Tsc1 from HEK293 cells and detected both Hsp70 and Hsp90 (Fig 2A). We also observed Tsc1 interaction with Hsp90 co-chaperones PP5 and Cdc37p50 (Fig 2A) but not Aha1, HOP, or p23 (Fig EV2A). We also co-immunoprecipitated Hsp90 client proteins Raf-1, Akt, Cdk4, glucocorticoid receptor (GR), and Tsc2 (Fig 2A). Hsp90 consists of N (amino)-, M (middle)-, and C (carboxy)-domains. To determine Tsc1 and Tsc2 interaction with these Hsp90 domains, we transiently transfected and expressed each domain with FLAG-tag in HEK293 cells. Using anti-FLAG M2 affinity gel, each Hsp90 domain was immunoprecipitated and co-immunoprecipitation of Tsc1 and Tsc2 was observed by immunoblotting. Tsc1 interacts with the Hsp90α M- and C-domains (Fig 2B), whereas Tsc2 bound only to the Hsp90α C-domain (Fig 2B). The MEEVD motif at the extreme C-terminus of Hsp90 is a highly conserved tetratricopeptide repeat (TPR) domain-binding site, which mediates interaction with many co-chaperones (Chadli et al, 2008). Deletion of MEEVD from Hsp90α (α∆TPR) or Hsp90β (β∆TPR) completely abrogated the interaction of Hsp90 with Tsc1 (Fig 2C). Figure 2. Tsc1 co-chaperone inhibits Hsp90 chaperone function Tsc1 was immunoprecipitated from HEK293 cell lysates using anti-Tsc1 antibody and immunoblotted with indicated antibodies to confirm chaperone, co-chaperone, and client protein interaction. IgG was used as control. FLAG-tagged Hsp90α N-, M-, and C-domains were transiently expressed and immunoprecipitated from HEK293 cells. Co-immunoprecipitation (Co-IP) of endogenous Tsc1 and Tsc2 was examined by immunoblotting. Empty vector (EV) was used as a control. Hsp90α-FLAG, Hsp90β-FLAG, and their deleted TPR domain-binding site constructs were transiently expressed and immunoprecipitated from HEK293 cells. Interaction of Tsc1 was assessed by immunoblotting. HOP was used as a positive control. Empty vector (EV) was used as a control. FLAG-tagged Tsc1 domains were transiently expressed and isolated from HEK293 cells. Co-IP of endogenous Hsp70 and Hsp90 was assessed by immunoblotting. Empty vector (EV) was used as a control. Tsc1-D-HA was co-expressed with FLAG-tagged Hsp90α N-, M-, or C- domains in HEK293 cells. Different domains of Hsp90α-FLAG were immunoprecipitated, and Tsc1-D-HA interaction was assessed by immunoblotting. Empty vector (EV) was used as a control. Bacterially expressed and purified Tsc1-D-His6 and Hsp90α binding affinity in the presence of AMPPNP, ADP, and GB were measured by fluorescence anisotropy. The error bars represent mean ± SD of three independent experiments. ATPase activity of Hsp90α in vitro. Inhibitory effects of purified Tsc1-D-His6 on the ATPase activity of Hsp90α are shown. All the data represent mean ± SD of three biological replicates. A Student's t-test was performed to assess statistical significance (**P < 0.005, ****P < 0.0001). Hsp90 was isolated and ATPase activity was measured from five Tsc1GFAPCKO mice with conditional inactivation of the TSC1 gene predominantly in glia and five Tsc1flox/+-GFAP-Cre and Tsc1flox/flox littermate controls (Tsc1-WT). A Student's t-test was performed to assess statistical significance (**P < 0.005). Tsc1, Hsp90, and client proteins from samples in (H) were examined by immunoblotting. SE (short exposure) and LE (long exposure) of the radiographic film. Source data are available online for this figure. Source Data for Figure 2 [embj201796700-sup-0004-SDataFig2.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Inhibition of the Hsp90 function by the co-chaperone Tsc1 (related to Fig 2) Tsc1 was immunoprecipitated from HEK293 cell lysates using anti-Tsc1 antibody and immunoblotted with indicated antibodies. IgG was used as control. These samples are the same as those presented in Fig 2A. BSA binding affinity to bacterially expressed and purified Hsp90α was determined by fluorescence anisotropy. The error bars represent mean ± SD of three independent experiments. Inorganic phosphate (Pi) standard curve. The x-axis shows μM of Pi per assay and the y-axis shows absorbance at 565 nm. Mean ± SD from values obtained in three independent experiments. ATPase activity of Hsp90α in vitro. Inhibitory effects of purified Tsc1-D-His6 on the ATPase activity of Hsp90α are shown. All the data represent mean ± SD of three biological replicates. A Student's t-test was performed to assess statistical significance (**P < 0.005, ****P < 0.0001). Different amounts of BSA have no effect on Hsp90 ATPase activity in vitro. All the data represent mean ± SD of three biological replicates. ATPase activity of Hsp90 from five Tsc1GFAPCKO (K1–K5) mice with conditional inactivation of the TSC1 gene in glia and five Tsc1flox/+-GFAP-Cre and Tsc1flox/flox littermate controls (C1–C5). All the data represent mean ± SD of three biological replicates. A Student's t-test was performed to assess statistical significance (**P < 0.005); 50 ng of the isolated Hsp90 was resolved on the SDS–PAGE gel and stained with Coomassie stain. Source data are available online for this figure. Download figure Download PowerPoint We next determined the region in Tsc1 that interacts with Hsp90. Based on previous work, we decided to express FLAG-tag Tsc1-fragment-A (N-domain; amino acid 1–301), Tsc1-fragment-B (Tsc2-binding domain; amino acid 302–430), and Tsc1-fragment-D (C-domain; amino acid 998–1,164) in HEK293 cells. Our data indicate that the carboxy-domain of Tsc1 (amino acid 998–1,164 or fragment D) interacts with Hsp90α (Fig 2D). In fact, Tsc1-D-HA interacts with the middle domain of Hsp90α-FLAG (Fig 2E). It is noteworthy that we were unable to express Tsc1-fragment-C (amino acid 431–718) in HEK293 cells. We next bacterially expressed and purified fragment D (Tsc1-D-His6) and examined its affinity for binding to Hsp90α in the presence of ADP, AMPPNP, and GB in vitro by fluorescently labeling Tsc1-D-His6 with Texas Red maleimide, and measuring the Kd by fluorescence anisotropy (Fig 2F). Our bacterially expressed and purified Hsp90α was functional based on its ATPase activity. The titration fit to a single-site binding equation with a Kd of 0.48 ± 0.19 μM (Fig 2F). This Kd was not significantly affected by the presence of ADP or non-hydrolyzing AMPPNP (Fig 2F). However, the presence of GB produced a Kd of > 5 μM, therefore suggesting Tsc1-D-His6 binding to Hsp90 is significantly reduced in the presence of GB. Binding of bovine serum albumin (BSA) was used as a negative control (Fig EV2B). The chaperone function of Hsp90 is linked to its ATPase activity (Panaretou et al, 1998). We therefore tested the impact of Tsc1 on Hsp90 ATPase activity. Recombinant Hsp90α and Tsc1-D-His6 were used in the molar ratio indicated (Figs 2G and EV2C and D) in the PiPer Phosphate Assay Kit (Thermo Fisher Scientific), in the presence of ATP as substrate. Hsp90α ATPase activity was measured in vitro as previously described (see Materials and Methods; Dunn et al, 2015; Figs 2G and EV2C and D). 10 μM GB inhibited Hsp90α ATPase activity (Fig 2G). Addition of Tsc1-D also significantly inhibited the ATPase activity of Hsp90α (Figs 2G and EV2D). Percentage ATPase activity was based on mmol Pi per mol Hsp90α per minute for Hsp90α alone and Tsc1-D was titrated until inhibition was achieved (Hsp90α = 300 nM, Tsc1-D = 1000 nM). We repeated the experiments in Fig 2G using BSA as a negative control (Fig EV2E). To obtain further evidence that Tsc1 is a regulator of Hsp90 ATPase activity, we used previously created Cre knockout (Tsc1GFAPCKO) mice with conditional inactivation of the TSC1 gene predominantly in glia (Uhlmann et al, 2002; Zeng et al, 2008) Brain tissue was harvested from Tsc1GFAPCKO (Tsc1flox/flox-GFAP-Cre) mice, as well as Tsc1flox/+-GFAP-Cre and Tsc1flox/flox littermate controls (Uhlmann et al, 2002; Zeng et al, 2008). Hsp90 was isolated from Tsc1GFAPCKO and control brains (Fig EV2F). PiPer Phosphate Assay was used to measure the isolated Hsp90 ATPase activity. Our data show that the conditional knockout of TSC1 in mouse brain caused a significant increase in ATPase activity compared to the control samples (Fig 2H). Compromised Hsp90 chaperone function impacts the stability and/or the activity of its client proteins. We therefore examined the stability of a selection of client proteins in lysates prepared from Tsc1GFAPCKO and control mouse brains by immunoblotting. Conditional inactivation of Tsc1 in mouse brains caused a down-regulation of the kinase clients such ErbB2 and Ulk1 and non-kinase client proteins such as estrogen receptor (ER) and GR (Fig 2I). Our data suggest that Tsc1 is a co-chaperone of Hsp90 and a potent inhibitor of the chaperone cycle. Furthermore, Tsc1 co-chaperone can influence and impact the stability and activity of a large number of Hsp90 client proteins. Tsc1 co-chaperone enables Tsc2 binding to Hsp90 To gain further insight into Tsc1 function as a new co-chaperone of Hsp90, we examined a selection of its bona fide clients in wild-type and TSC1-deficient (Tsc1−/−) murine embryo fibroblast (MEF) cell lines. Absence of Tsc1 significantly reduced both the activity and the stability of kinase clients phospho-Y416-c-Src, total c-Src, ErbB2, Ulk1, Raf-1, and Cdk4, and non-kinase clients phospho-S211-GR, total GR, FLCN, and Tsc2 (Fig 3A). To determine the effects of Tsc1 over-expression on the Hsp90 clients, HEK293 cells were transiently transfected with either 2 or 4 μg of Tsc1-HA-tagged plasmid. Over-expression of Tsc1 also caused a reduction in the stability of the Hsp90 kinase clients such as ErbB2, Ulk1, and Raf-1 (Fig 3B). Conversely, high levels of Tsc1 increased the total amount of GR (Fig 3B), but did not impact the stability of Cdk4 kinase. It also had a slight positive impact on the stability of both FLCN and Tsc2 (Fig 3B). We next examined the effects of Tsc1 on Hsp90 chaperone function by assessing the steady-state expression of cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is a client protein of Hsp90 (Youker et al, 2004; Wang et al, 2006) and relies on the Hsp90 chaperone cycle for correct folding (Youker et al, 2004; Wang et al, 2006). HEK293 cells were transiently co-transfected with CFTR and either pcDNA3 (control) or Tsc1-FLAG plasmids. Immunoblot analysis of these samples using anti-CFTR antibody showed a doublet (Fig 3C). The upper band is the mature Golgi-processed glycoform of CFTR located at the cell surface and the lower band an immature core-glycosylated protein (Fig 3C). Over-expression of Tsc1 significantly increased CFTR protein, suggesting a deceleration in Hsp90 chaperone activity and, consequently, an increase in CFTR expression. Our data provide further evidence that Tsc1 functions as a new co-chaperone of Hsp90, and its levels compromise Hsp90 chaperoning of client proteins. Figure 3. Tsc1 facilitates the chaperoning of Hsp90 clients Endogenous protein levels of Hsp90 kinase and non-kinase clients from wild-type and Tsc1−/− MEF cells were assessed by immunoblotting. Transi
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