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• W2007284051 abstract "Protein phosphatase 5 (PP5) is auto-inhibited by intramolecular interactions with its tetratricopeptide repeat (TPR) domain. Hsp90 has been shown to bind PP5 to activate its phosphatase activity. However, the functional implications of binding Hsp70 to PP5 are not yet clear. In this study, we find that both Hsp90 and Hsp70 bind to PP5 using a luciferase fragment complementation assay. A fluorescence polarization assay shows that Hsp90 (MEEVD motif) binds to the TPR domain of PP5 almost 3-fold higher affinity than Hsp70 (IEEVD motif). However, Hsp70 binding to PP5 stimulates higher phosphatase activity of PP5 than the binding of Hsp90. We find that PP5 forms a stable 1:1 complex with Hsp70, but the interaction appears asymmetric with Hsp90, with one PP5 binding the dimer. Solution NMR studies reveal that Hsc70 and PP5 proteins are dynamically independent in complex, tethered by a disordered region that connects the Hsc70 core and the IEEVD-TPR contact area. This tethered binding is expected to allow PP5 to carry out multi-site dephosphorylation of Hsp70-bound clients with a range of sizes and shapes. Together, these results demonstrate that Hsp70 recruits PP5 and activates its phosphatase activity which suggests dual roles for PP5 that might link chaperone systems with signaling pathways in cancer and development. Protein phosphatase 5 (PP5) is auto-inhibited by intramolecular interactions with its tetratricopeptide repeat (TPR) domain. Hsp90 has been shown to bind PP5 to activate its phosphatase activity. However, the functional implications of binding Hsp70 to PP5 are not yet clear. In this study, we find that both Hsp90 and Hsp70 bind to PP5 using a luciferase fragment complementation assay. A fluorescence polarization assay shows that Hsp90 (MEEVD motif) binds to the TPR domain of PP5 almost 3-fold higher affinity than Hsp70 (IEEVD motif). However, Hsp70 binding to PP5 stimulates higher phosphatase activity of PP5 than the binding of Hsp90. We find that PP5 forms a stable 1:1 complex with Hsp70, but the interaction appears asymmetric with Hsp90, with one PP5 binding the dimer. Solution NMR studies reveal that Hsc70 and PP5 proteins are dynamically independent in complex, tethered by a disordered region that connects the Hsc70 core and the IEEVD-TPR contact area. This tethered binding is expected to allow PP5 to carry out multi-site dephosphorylation of Hsp70-bound clients with a range of sizes and shapes. Together, these results demonstrate that Hsp70 recruits PP5 and activates its phosphatase activity which suggests dual roles for PP5 that might link chaperone systems with signaling pathways in cancer and development. Protein phosphatase 5 (PP5) 2The abbreviations used are: PP5protein phosphatase 5TPRtetratricopeptide repeatCRLC-terminal Renilla luciferaseNRLN-terminal Renilla luciferaseSEC-MALSsize exclusion chromatography and multi-angle light scatteringSNRsignal-to-noise ratioNBDnucleotide-binding domainSBDsubstrate-binding domainSRL-PFACsplit Renilla luciferase protein fragment-assisted complementation. is a member of the PPP family of serine/threonine-specific phosphatases and has been linked to signaling pathways that control growth arrest, apoptosis, and DNA damage repair (1Zuo Z. Urban G. Scammell J.G. Dean N.M. McLean T.K. Aragon I. Honkanen R.E. Ser/Thr protein phosphatase type 5 (PP5) is a negative regulator of glucocorticoid receptor-mediated growth arrest.Biochemistry. 1999; 38: 8849-8857Crossref PubMed Scopus (110) Google Scholar, 2Morita K. Saitoh M. Tobiume K. Matsuura H. Enomoto S. Nishitoh H. Ichijo H. Negative feedback regulation of ASK1 by protein phosphatase 5 (PP5) in response to oxidative stress.EMBO J. 2001; 20: 6028-6036Crossref PubMed Scopus (249) Google Scholar, 3Ali A. Zhang J. Bao S. Liu I. Otterness D. Dean N.M. Abraham R.T. Wang X.F. Requirement of protein phosphatase 5 in DNA-damage-induced ATM activation.Genes Dev. 2004; 18: 249-254Crossref PubMed Scopus (134) Google Scholar). Specifically, PP5 plays important roles in regulating the dynamic phosphorylation of p53, ASK-1, MAPK, and many other signaling components (2Morita K. Saitoh M. Tobiume K. Matsuura H. Enomoto S. Nishitoh H. Ichijo H. Negative feedback regulation of ASK1 by protein phosphatase 5 (PP5) in response to oxidative stress.EMBO J. 2001; 20: 6028-6036Crossref PubMed Scopus (249) Google Scholar, 5Zhou G. Golden T. Aragon I.V. Honkanen R.E. Ser/Thr protein phosphatase 5 inactivates hypoxia-induced activation of an apoptosis signal-regulating kinase 1/MKK-4/JNK signaling cascade.J. Biol. Chem. 2004; 279: 46595-46605Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 6Zuo Z. Dean N.M. Honkanen R.E. Serine/threonine protein phosphatase type 5 acts upstream of p53 to regulate the induction of p21(WAF1/Cip1) and mediate growth arrest.J. Biol. Chem. 1998; 273: 12250-12258Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). PP5 also has been implicated in the regulation of glucocorticoid receptor, although the mechanism is controversial (1Zuo Z. Urban G. Scammell J.G. Dean N.M. McLean T.K. Aragon I. Honkanen R.E. Ser/Thr protein phosphatase type 5 (PP5) is a negative regulator of glucocorticoid receptor-mediated growth arrest.Biochemistry. 1999; 38: 8849-8857Crossref PubMed Scopus (110) Google Scholar, 7Chen M.S. Silverstein A.M. Pratt W.B. Chinkers M. The tetratricopeptide repeat domain of protein phosphatase 5 mediates binding to glucocorticoid receptor heterocomplexes and acts as a dominant negative mutant.J. Biol. Chem. 1996; 271: 32315-32320Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar). Additionally, PP5 levels are elevated in human breast cancer (8Golden T. Aragon I.V. Rutland B. Tucker J.A. Shevde L.A. Samant R.S. Zhou G. Amable L. Skarra D. Honkanen R.E. Elevated levels of Ser/Thr protein phosphatase 5 (PP5) in human breast cancer.Biochim. Biophys. Acta. 2008; 1782: 259-270Crossref PubMed Scopus (56) Google Scholar). Together, these studies have suggested that PP5 may be a novel target for anti-cancer therapies (9Golden T. Swingle M. Honkanen R.E. The role of serine/threonine protein phosphatase type 5 (PP5) in the regulation of stress-induced signaling networks and cancer.Cancer Metastasis Rev. 2008; 27: 169-178Crossref PubMed Scopus (71) Google Scholar). However, the catalytic subunit of PPP phosphatases is highly conserved, and it has been difficult to develop selective, competitive inhibitors of these enzymes (10McConnell J.L. Wadzinski B.E. Targeting protein serine/threonine phosphatases for drug development.Mol. Pharmacol. 2009; 75: 1249-1261Crossref PubMed Scopus (148) Google Scholar). In addition to its catalytic domain, PP5 is the only member of the PPP family that contains an N-terminal tetratricopeptide repeat (TPR) domain (11Bollen M. Stalmans W. The structure, role, and regulation of type-1 protein phosphatases.Crit. Rev. Biochem. Mol. Biol. 1992; 27: 227-281Crossref PubMed Scopus (260) Google Scholar, 12Cohen P.T. Novel protein serine/threonine phosphatases: Variety is the spice of life.Trends Biochem. Sci. 1997; 22: 245-251Abstract Full Text PDF PubMed Scopus (460) Google Scholar). TPR domains are assembled from repeats of an amphipathic antiparallel helix that assemble into superhelical structures bearing a concave central groove (13Zhang M.H. Windheim M. Roe S.M. Peggie M. Cohen P. Prodromou C. Pearl L.H. Chaperoned ubiquitylation - crystal structures of the CHIPU box E3 ubiquitin ligase and a CHIP-Ubc13-Uev1a complex.Mol. Cell. 2005; 20: 525-538Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar, 14Yang J. Roe S.M. Cliff M.J. Williams M.A. Ladbury J.E. Cohen P.T. Barford D. Molecular basis for TPR domain-mediated regulation of protein phosphatase.EMBO J. 2005; 24: 1-10Crossref PubMed Scopus (161) Google Scholar, 15Wang L. Liu Y.T. Hao R. Chen L. Chang Z. Wang Z.X. Wang H.R. Wu J.W. Molecular mechanism of the negative regulation of Smad1/5 protein by carboxyl terminus of Hsc70-interacting protein (CHIP).J. Biol. Chem. 2011; 286: 15883-15894Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 16Scheufler C. Brinker A. Bourenkov G. Pegoraro S. Moroder L. Bartunik H. Hartl F.U. Moarefi I. Structure of TPR domain-peptide complexes: Critical elements in the assembly of the Hsp70-Hsp90 multichaperone machine.Cell. 2000; 101: 199-210Abstract Full Text Full Text PDF PubMed Scopus (1010) Google Scholar). PP5 has been shown to interact with the molecular chaperones heat shock protein 70 (Hsp70) and heat shock protein 90 (Hsp90) (17Chen L. Qi H. Korenberg J. Garrow T.A. Choi Y.J. Shane B. Purification and properties of human cytosolic folylpoly-γ-glutamate synthetase and organization, localization, and differential splicing of its gene.J. Biol. Chem. 1996; 271: 13077-13087Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 18Silverstein A.M. Galigniana M.D. Chen M.S. Owens-Grillo J.K. Chinkers M. Pratt W.B. Protein phosphatase 5 is a major component of glucocorticoid receptor.hsp90 complexes with properties of an FK506-binding immunophilin.J. Biol. Chem. 1997; 272: 16224-16230Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 19Cliff M.J. Harris R. Barford D. Ladbury J.E. Williams M.A. Conformational diversity in the TPR domain-mediated interaction of protein phosphatase 5 with Hsp90.Structure. 2006; 14: 415-426Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 20Cliff M.J. Williams M.A. Brooke-Smith J. Barford D. Ladbury J.E. Molecular recognition via coupled folding and binding in a TPR domain.J. Mol. Biol. 2005; 346: 717-732Crossref PubMed Scopus (75) Google Scholar, 21Zeke T. Morrice N. Vázquez-Martin C. Cohen P.T. Human protein phosphatase 5 dissociates from heat-shock proteins and is proteolytically activated in response to arachidonic acid and the microtubule-depolymerizing drug nocodazole.Biochem. J. 2005; 385: 45-56Crossref PubMed Scopus (31) Google Scholar). Specifically, the TPR domain of PP5 binds to cytoplasmic Hsp90 homologs, Hsp90α (stress-inducible) and Hsp90β (constitutively active), through a conserved MEEVD motif that is located at the end of the C termini of these chaperones (17Chen L. Qi H. Korenberg J. Garrow T.A. Choi Y.J. Shane B. Purification and properties of human cytosolic folylpoly-γ-glutamate synthetase and organization, localization, and differential splicing of its gene.J. Biol. Chem. 1996; 271: 13077-13087Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 18Silverstein A.M. Galigniana M.D. Chen M.S. Owens-Grillo J.K. Chinkers M. Pratt W.B. Protein phosphatase 5 is a major component of glucocorticoid receptor.hsp90 complexes with properties of an FK506-binding immunophilin.J. Biol. Chem. 1997; 272: 16224-16230Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 19Cliff M.J. Harris R. Barford D. Ladbury J.E. Williams M.A. Conformational diversity in the TPR domain-mediated interaction of protein phosphatase 5 with Hsp90.Structure. 2006; 14: 415-426Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 20Cliff M.J. Williams M.A. Brooke-Smith J. Barford D. Ladbury J.E. Molecular recognition via coupled folding and binding in a TPR domain.J. Mol. Biol. 2005; 346: 717-732Crossref PubMed Scopus (75) Google Scholar). Although biochemical data illustrates that an MEEVD peptide has high affinity for the TPR domain of PP5 (∼50 nm), solution phase NMR studies revealed that this interaction is highly dynamic with only few enduring contacts (19Cliff M.J. Harris R. Barford D. Ladbury J.E. Williams M.A. Conformational diversity in the TPR domain-mediated interaction of protein phosphatase 5 with Hsp90.Structure. 2006; 14: 415-426Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 20Cliff M.J. Williams M.A. Brooke-Smith J. Barford D. Ladbury J.E. Molecular recognition via coupled folding and binding in a TPR domain.J. Mol. Biol. 2005; 346: 717-732Crossref PubMed Scopus (75) Google Scholar). Comparatively less is known about how PP5 interacts with Hsp70. Co-immunoprecipitation studies suggest that PP5 binds Hsp70 (21Zeke T. Morrice N. Vázquez-Martin C. Cohen P.T. Human protein phosphatase 5 dissociates from heat-shock proteins and is proteolytically activated in response to arachidonic acid and the microtubule-depolymerizing drug nocodazole.Biochem. J. 2005; 385: 45-56Crossref PubMed Scopus (31) Google Scholar), but it is not yet clear how PP5 interacts with this chaperone or whether the TPR domain is involved. Based on the Hsp90-PP5 complex, it is likely that this interaction occurs through the IEEVD motif at the C termini of the cytoplasmic Hsp70 family members, including heat shock cognate 70 (Hsc70; HSPA8) and heat shock protein 72 (Hsp72; HSPA1A). protein phosphatase 5 tetratricopeptide repeat C-terminal Renilla luciferase N-terminal Renilla luciferase size exclusion chromatography and multi-angle light scattering signal-to-noise ratio nucleotide-binding domain substrate-binding domain split Renilla luciferase protein fragment-assisted complementation. PP5 belongs to a family of TPR domain-containing co-chaperones that includes Hop (Hsp70/90 organizing protein), CHIP (carboxyl terminus of Hsp70 interacting protein) and a number of immunophilins such as FKBP52 (FK506 binding protein 52 kDa). Members of this co-chaperone family bind to Hsp70 and/or Hsp90 at these chaperones' C-terminal EEVD motifs. In turn, the TPR co-chaperones are important regulators of chaperone function (16Scheufler C. Brinker A. Bourenkov G. Pegoraro S. Moroder L. Bartunik H. Hartl F.U. Moarefi I. Structure of TPR domain-peptide complexes: Critical elements in the assembly of the Hsp70-Hsp90 multichaperone machine.Cell. 2000; 101: 199-210Abstract Full Text Full Text PDF PubMed Scopus (1010) Google Scholar) (22Höhfeld J. Cyr D.M. Patterson C. From the cradle to the grave: molecular chaperones that may choose between folding and degradation.EMBO Rep. 2001; 2: 885-890Crossref PubMed Scopus (288) Google Scholar, 23Mayer M.P. Bukau B. Hsp70 chaperones: Cellular functions and molecular mechanism.Cell Mol. Life Sci. 2005; 62: 670-684Crossref PubMed Scopus (2065) Google Scholar). For example, complexes between CHIP and either Hsp70 or Hsp90 are linked to the ubiquitination and therefore the proteasomal degradation of chaperone-bound clients. Likewise, a complex between these chaperones and HOP is critical to the folding of some clients such as nuclear hormone receptors (24Connell P. Ballinger C.A. Jiang J. Wu Y. Thompson L.J. Höhfeld J. Patterson C. The co-chaperone CHIP regulates protein triage decisions mediated by heat-shock proteins.Nat. Cell Biol. 2001; 3: 93-96Crossref PubMed Scopus (0) Google Scholar, 25Qian S.B. McDonough H. Boellmann F. Cyr D.M. Patterson C. CHIP-mediated stress recovery by sequential ubiquitination of substrates and Hsp70.Nature. 2006; 440: 551-555Crossref PubMed Scopus (287) Google Scholar, 26Chen S. Smith D.F. Hop as an adaptor in the heat shock protein 70 (Hsp70) and Hsp90 chaperone machinery.J. Biol. Chem. 1998; 273: 35194-35200Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar). Additionally, FKBP52 couples clients of Hsp70 and Hsp90 to the cytoskeleton (27Heinlein C.A. Chang C. Role of chaperones in nuclear translocation and transactivation of steroid receptors.Endocrine. 2001; 14: 143-149Crossref PubMed Scopus (34) Google Scholar). However, less is known about the Hsp70-PP5 and Hsp90-PP5 complexes and their potential roles in the protein homeostasis network. One important clue comes from observations that the TPR domain and the C-terminal catalytic subunit of PP5 have an auto-inhibitory function, suppressing phosphatase activity. Indeed, binding of Hsp90 to the TPR domain has been reported to weakly activate PP5 (14Yang J. Roe S.M. Cliff M.J. Williams M.A. Ladbury J.E. Cohen P.T. Barford D. Molecular basis for TPR domain-mediated regulation of protein phosphatase.EMBO J. 2005; 24: 1-10Crossref PubMed Scopus (161) Google Scholar). However, it is not yet clear whether Hsp70 also binds the TPR domain or whether this interaction activates PP5. Toward these questions, we characterized the interaction of Hsp70 and Hsp90 with PP5, using a panel of cell-based assays and biophysical methods. These studies confirmed that PP5 binds Hsp70 and Hsp90 through the canonical EEVD motifs. However, we found that C-terminal peptides derived from Hsp90α/β bind to PP5 10-fold tighter than C-terminal peptides derived from Hsc70/Hsp72. Despite the weaker affinity of Hsp70 for PP5, this chaperone was far more effective at stimulating the phosphatase activity of PP5. Additionally, solution phase NMR studies showed that Hsp70 and PP5 move independently of each other in the bound complex, suggesting that the disordered C terminus of Hsp70 allows the activated PP5 to “sample” a relatively large area around the chaperone. This ultra-structure might be important in allowing PP5 to act on a wide range of chaperone clients. Together, these results suggest that the Hsp70-PP5 complex is a potent phosphatase that might link chaperone systems with signaling pathways in cancer and development. Reagents were obtained from the following sources: pLentilox vectors (University of Michigan Vector Core); pMSCG9 vector (Clay Brown, Center for Structural Biology, University of Michigan); restriction endonucleases (New England Biolabs); HEK293 (American Type Culture Collection); Dulbecco's modified Eagle's medium (Invitrogen, 11965-092); fetal bovine serum (10082-147); antibiotic-antimycotic (Invitrogen, 15240–062); six-well tissue culture plate (BD Falcon, 3046); polybrene linker (Santa Cruz Biotechnology, sc-134220) Renilla GLO luciferase kit (Promega, Madison, WI); and p-nitrophenyl phosphate phosphatase substrate kit (Thermo Scientific, 37620). C-terminal Renilla luciferase (CRL, residues 1–229) and full-length Hsp70 or Hsp90 (upstream of CRL) were PCR-amplified and subcloned into a pLentilox RSV-2 dsRed vector using the following restriction site design: BamHI-Hsp72-Xba1-CRL-Not1 and Xma1-HSP90α-BamHI-CRL-Xba1. In these fusion constructs, the stop codon of Hsp70/90 was deleted. Similarly, N-terminal Renilla luciferase (NRL, resides 230–311) and full-length PP5 (downstream of NRL) were amplified and subcloned into the pLentilox RSV vector using the following restriction site design: BamHI-NRL-Xba1-PP5-Not1. In this construct, the NRL stop codon was deleted. All fusion constructs (Hsp70/90-CRL and NRL-PP5) contained a GGGGSGGGGS (G4S)2 peptide linker between the protein of interest and the Renilla luciferase reporter (28Paulmurugan R. Massoud T.F. Huang J. Gambhir S.S. Molecular imaging of drug-modulated protein-protein interactions in living subjects.Cancer Res. 2004; 64: 2113-2119Crossref PubMed Scopus (112) Google Scholar). After all sequences were confirmed at the University of Michigan DNA sequencing core, lentiviral particles containing these constructs were purchased (University of Michigan Vector Core) to create a stable cell lines for additional studies. HEK293 cells were plated using DMEM (Invitrogen) with 10% FBS and no antibiotics into a six-well tissue culture plate. Cells were allowed to adhere and grow for 1 day (∼70% confluent) before transduction. The next day, fresh media without antibiotics (1.35 ml), 0.15 ml of 10× lentiviral particles (either Hsp70-CRL, Hsp90-CRL, or NRL-PP5), and polybrene linker to a final concentration of 8 μg/ml was added to each well. After an incubation period of 8 h at 37 °C and 5% CO2, the medium was replaced with DMEM with 10% FBS and 1% antibiotic-antimycotic. For cells that contained both HSP70/90-CRL and NRL-PP5 viral particles, the above procedure first performed with Hsp90/Hsp70-CRL viral particles. Using these cells, the same procedure was repeated a second time; however, NRL-PP5 lentiviral particle was added. The HSP70/90-CRL and PP5-NRL viral particles contained dsRED and GFP, respectively. Cells containing Hsp90-CRL, Hsp70-CRL, NRL-PP5, Hsp90-CRL + NRL-PP5, and Hsp70-CRL + NRL-PP5 constructs were then seeded into a 24-well plate at a density of 5,000 cell/wells and allowed to grow overnight at 37 °C and 5% CO2. The following day, the medium was removed, and the cells were washed with phosphate-buffered saline. Luciferase activity determined using the Renilla-GLO luciferase assay system kit. Briefly, following washing, 100 μl of 1× passive lysis buffer was added to each well and plates were allowed to shake for 15 min at room temperature. Afterward, 1× luciferase substrate was added, and the luminescence was measured using Biotek Synergy 2 plate reader. Full-length PP5 and Hsp90 were expressed in Escherichia coli BL21(DE3) cells from pMCSG9 plasmids. A fresh colony was grown in terrific broth medium supplemented with 50 mg/liter ampicillin at 37 °C with shaking at 250 rpm until A600 reached ∼0.8 and protein expression was induced by the addition of isopropyl 1-thio-β-d-galactopyranoside (final concentration of 1 mm). The temperature was reduced to 18 °C, and the culture was allowed to shake overnight. Cells were harvested by centrifugation (4000 × g, 10 min, 4 °C). Cell pellets was suspended in lysis buffer (50 mm NaH2PO4, 300 mm NaCl, 10 mm imidazole (pH 8.0)) and sonicated on ice, and clarified by centrifugation at 15,000 × g for 30 min. The His-tagged proteins were purified using a nickel-nitrilotriacetic acid (Qiagen) column. The eluted protein was subjected to dialysis (PP5 buffer, 40 mm Tris-HCl, pH 7.4, 10% glycerol, 1 mm DTT, and HSP90 buffer, 20 mm Tris-HCl, pH 7.4, 20 mm NaCl, 10% glycerol, 1 mm DTT). Proteins were treated with His-tagged tobacco etch virus protease TEV protease (1 μm) overnight at 4 °C to remove tags. This process was repeated a second time prior to extensive dialysis and removal of any residual His-tagged protein by nickel-nitrilotriacetic acid column. Human Hsp70s (pMCSG7 vector) were expressed in E. coli BL21(DE3) cells using terrific broth medium supplemented with 50 mg/liter ampicillin at 37 °C with shaking at 250 rpm until A600 of ∼0.8 was reached. Additionally, Hsc70 protein was isotopically labeled for all NMR experiments. For labeled Hsc70, the BL21 cells were grown M9 media with15NH4Cl (Sigma Aldrich). The temperature was reduced to 18 °C, and the culture was allowed to shake overnight. Cells were harvested by centrifugation (4000 × g, 10 min, 4 °C). All Hsp70s were purified as described (29Chang L. Thompson A.D. Ung P. Carlson H.A. Gestwicki J.E. Mutagenesis reveals the complex relationships between ATPase rate and the chaperone activities of Escherichia coli heat shock protein 70 (Hsp70/DnaK).J. Biol. Chem. 2010; 285: 21282-21291Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar), and His tags were removed via TEV protease. Final purification was performed on an ATP agarose column. All protein concentrations were measure using the Pierce BCA protein assay kit according to the manufacturer's protocol. To verify Hsc70 was not aggregated, the raw fluorescence values of the parallel and perpendicular intensity using fluorescence polarization shows no indication of aggregation. In addition, non-binding and binding tracer in fluorescence polarization was also used to confirm Hsc70 was not aggregated. NMR structure showed no sign of Hsc70 aggregation. All peptides were synthesized manually or with an ABI 433 peptide synthesizer using Fmoc chemistry with 2-chlorotrityl resin as the solid support. Either DIC/HOAt or HOBt/HBTU was used as the coupling reagent. Following completion of the peptide, a cleavage mixture composed of TFA/triisopropylsilane/H2O (19 ml:0.5 ml:0.5 ml) removed the peptide from the resin as well as any side chain protecting groups. The resulting solution was evaporated, and the crude peptide was precipitated with diethyl ether. Peptides were purified via RP-HPLC (Waters, Sunfire Prep C18, 19 mm × 150 mm, 5 μm) and confirmed by electrospray ionization mass spectroscopy (30Amblard M. Fehrentz J.A. Martinez J. Subra G. Methods and protocols of modern solid phase peptide synthesis.Mol. Biotechnol. 2006; 33: 239-254Crossref PubMed Scopus (331) Google Scholar). All fluorescence polarization experiments were conducted in 384-well, black, low volume, round-bottomed plates (Corning) using a BioTeck Synergy 2 plate reader (Winooski, VT). For binding experiments, to each well was added increasing amounts of protein and the 5-carboxyfluorescein-labeled Hsp70/90 C-terminal probe/tracer (20 nm). For competition studies, each well had PP5 protein at a concentration equivalent to the Kd, 5-carboxyfluorescein-labeled peptide was held constant at 20 nm, and varying concentrations of unlabeled peptide was added to compete off labeled peptide. All wells had a final volume of 20 μl in the assay buffer (40 mm Tris-HCl, pH 7.4, 10% glycerol, 1 mm DTT). The plate was allowed to incubate at room temperature for 5 min to reach equilibrium. The polarization values in millipolarization units were measured at an excitation wavelength at 485 nm and an emission wavelength at 528 nm. An equilibrium binding isotherm was constructed by plotting the fluorescence polarization reading as a function of the protein concentration at a fixed concentration of tracer (20 nm). All experimental data were analyzed using Prism software (version 5.0, Graphpad Software, San Diego, CA) and WinNonlin (version 5.3). NMR data were collected using an Agilent/Varian NMR System with a room temperature triple resonance probe, interfaced to an Oxford instruments 18.7 tesla magnet (1H 800 MHz). Backbone assignments for the C terminus of Hsc70 were reported previously (38Smith M.C. Scaglione K.M. Assimon V.A. Patury S. Thompson A.D. Dickey C.A. Southworth D.R. Paulson H.L. Gestwicki J.E. Zuiderweg E.R. The E3 ubiquitin ligase CHIP and the molecular chaperone Hsc70 form a dynamic, tethered complex.Biochemistry. 2013; 52: 5354-5364Crossref PubMed Scopus (44) Google Scholar). Experiments for studying the interaction of PP5 with Hsc70 were carried out using full-length PP5 and full-length 15N-labeled Hsc70 in the following buffer: 50 mm HEPES, 75 mm NaCl, 1 mm ADP, 5 mm MgCl2, 0.02% NaN3, 0.01% Triton, pH 7.4, 30 °C. The TROSY spectrum with 1:0 Hsc70/PP5 was recorded in 10 h with a sample of 254 μm Hsc70. The spectrum with 1:1 Hsc70/PP5 was recorded in 22 h with a sample of 169 μm Hsc70 and 149 μm PP5. The two spectra have the same intrinsic signal to noise ratio ((254/169)2 = 2.25). PP5-Hsp70 and PP5-Hsp90 complexes were formed by incubating proteins at equal molar concentrations (10 μm) in binding buffer (100 mm KCl, 20 mm HEPES (pH 7.5), 7 mm β-mercaptoethanol) at room temperature for 30 min. Identification and molecular weight determination of complexes was achieved through SEC (Wyatt WTC-050S5 and WTC-030S5 columns) with an Akta micro FPLC (GE Healthcare) and in-line DAWN HELEOS MALS and Optilab rEX differential refractive index detectors (Wyatt Technology Corp.). SEC was performed in 100 mm KCl, 20 mm HEPES (pH 7.5). Data were analyzed by the ASTRA software package (version 6, Wyatt Technology Corp.). The two spectra have the same intrinsic signal to noise ratio ((254/169)2 = 2.25). Purified PP5, Hsp70, and Hsp90 were immobilized in 4HBX 96-well plates (Thermo Scientific) and diluted with ELISA buffer (BioLegend). Equal molar concentration of protein was added to each well, and the plate was incubated overnight at 4 °C. Protein concentrations ranged from 50 to 0.5 μm. The following day, p-nitrophenyl phosphate (Fisher Scientific) was used according to the manufacturer's instructions. Once p-nitrophenyl phosphate substrates were added to the plates, they were incubated at 37 °C for 1 h. After color change, the OD405 was measured on the Biomatrix Plate Reader Synergy 2. The enzymatic activity was calculated using the following equation, Enzyme activityμmolminμg=volume×OD405path lengthεϵenzyme×time of incubation(Eq. 1) where ϵ is the molar extinction coefficient, which equals 1.78 × 104 m−1 × cm−1. Previous co-immunoprecipitation studies have suggested that PP5 interacts with both Hsp70 and Hsp90 in cells (21Zeke T. Morrice N. Vázquez-Martin C. Cohen P.T. Human protein phosphatase 5 dissociates from heat-shock proteins and is proteolytically activated in response to arachidonic acid and the microtubule-depolymerizing drug nocodazole.Biochem. J. 2005; 385: 45-56Crossref PubMed Scopus (31) Google Scholar). To confirm this result, we utilized the SRL-PFAC system, which has proven to be a powerful method for studying protein-protein interactions in cells (31Jiang Y. Bernard D. Yu Y. Xie Y. Zhang T. Li Y. Burnett J.P. Fu X. Wang S. Sun D. Split Renilla luciferase protein fragment-assisted complementation (SRL-PFAC) to characterize Hsp90-Cdc37 complex and identify critical residues in protein/protein interactions.J. Biol. Chem. 2010; 285: 21023-21036Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). In this assay, the full-length Renilla luciferase gene is divided into inactive halves, the NRL (residues 1–229) and the CRL (residues 230–311). The NRL and CRL will reconstitute functional luciferase if they are brought into close proximity. To explore whether Hsp90 and Hsp70 bind PP5, we created constructs that would express NRL-PP5, Hsp70-CRL, or Hsp90-CRL fusion proteins (Fig. 1A). We anticipated that a luminescence signal would be detected only if Hsp90 or Hsp70 interacts with PP5 (Fig. 1B). When HEK293 cells were transduced with viral vectors expressing either NRL-PP5, Hsp70-CRL, or Hsp90-CRL alone, low luciferase activity was measured (Fig. 1C). However, co-transduction with either the NRL-PP5+Hsp70-CRL pair or the NRL-PP5+Hsp90-CRL pair led to enhanced luciferase activity (Fig. 1C), consistent with the interaction of PP5 with both chaperones in cells. We also examined at a control pair (NRL-PP5+HOP-CRL) if nonspecific complementation would occur." @default.
• W2007284051 created "2016-06-24" @default.
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• W2007284051 date "2014-01-01" @default.
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• W2007284051 title "The Molecular Chaperone Hsp70 Activates Protein Phosphatase 5 (PP5) by Binding the Tetratricopeptide Repeat (TPR) Domain" @default.
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