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• W2046603670 abstract "We have shown previously that Akt exists in a signal complex with p38 MAPK, MAPK-activated protein kinase-2 (MK2), and heat shock protein 27 (Hsp27) and MK2 phosphorylates Akt on Ser-473. Additionally, dissociation of Hsp27 from Akt, prior to Akt activation, induced polymorphonuclear leukocyte (PMN) apoptosis. However, the role of Hsp27 in regulating Akt activation was not examined. This study tested the hypothesis that Hsp27 regulates Akt activation and promotes cell survival by scaffolding MK2 to the Akt signal complex. Here we show that loss of Akt/Hsp27 interaction by anti-Hsp27 antibody treatment resulted in loss of Akt/MK2 interaction, loss of Akt-Ser-473 phosphorylation, and induced PMN apoptosis. Transfection of myristoylated Akt (AktCA) in HK-11 cells induced Akt-Ser-473 phosphorylation, activation, and Hsp27-Ser-82 phosphorylation. Cotransfection of AktCA with Hsp27 short interfering RNA, but not scrambled short interfering RNA, silenced Hsp27 expression, without altering Akt expression in HK-11 cells. Silencing Hsp27 expression inhibited Akt/MK2 interaction, inhibited Akt phosphorylation and Akt activation, and induced HK-11 cell death. Deletion mutagenesis studies identified acidic linker region (amino acids 117–128) on Akt as an Hsp27 binding region. Deletion of amino acids 117–128 on Akt resulted in loss of its interaction with Hsp27 and MK2 but not with Hsp90 as demonstrated by immunoprecipitation and glutathione S-transferase pulldown studies. Co-transfection studies demonstrated that constitutively active MK2 (MK2EE) phosphorylated Aktwt (wild type) on Ser-473 but failed to phosphorylate AktΔ117–128 mutant in transfixed cells. These studies collectively define a novel role of Hsp27 in regulating Akt activation and cellular apoptosis by mediating interaction between Akt and its upstream activator MK2. We have shown previously that Akt exists in a signal complex with p38 MAPK, MAPK-activated protein kinase-2 (MK2), and heat shock protein 27 (Hsp27) and MK2 phosphorylates Akt on Ser-473. Additionally, dissociation of Hsp27 from Akt, prior to Akt activation, induced polymorphonuclear leukocyte (PMN) apoptosis. However, the role of Hsp27 in regulating Akt activation was not examined. This study tested the hypothesis that Hsp27 regulates Akt activation and promotes cell survival by scaffolding MK2 to the Akt signal complex. Here we show that loss of Akt/Hsp27 interaction by anti-Hsp27 antibody treatment resulted in loss of Akt/MK2 interaction, loss of Akt-Ser-473 phosphorylation, and induced PMN apoptosis. Transfection of myristoylated Akt (AktCA) in HK-11 cells induced Akt-Ser-473 phosphorylation, activation, and Hsp27-Ser-82 phosphorylation. Cotransfection of AktCA with Hsp27 short interfering RNA, but not scrambled short interfering RNA, silenced Hsp27 expression, without altering Akt expression in HK-11 cells. Silencing Hsp27 expression inhibited Akt/MK2 interaction, inhibited Akt phosphorylation and Akt activation, and induced HK-11 cell death. Deletion mutagenesis studies identified acidic linker region (amino acids 117–128) on Akt as an Hsp27 binding region. Deletion of amino acids 117–128 on Akt resulted in loss of its interaction with Hsp27 and MK2 but not with Hsp90 as demonstrated by immunoprecipitation and glutathione S-transferase pulldown studies. Co-transfection studies demonstrated that constitutively active MK2 (MK2EE) phosphorylated Aktwt (wild type) on Ser-473 but failed to phosphorylate AktΔ117–128 mutant in transfixed cells. These studies collectively define a novel role of Hsp27 in regulating Akt activation and cellular apoptosis by mediating interaction between Akt and its upstream activator MK2. Apoptosis or programmed cell death is a series of events in a cell that leads to its death. Human polymorphonuclear leukocytes (PMN) 3The abbreviations used are: PMN, polymorphonuclear leukocyte; Hsp27, heat shock protein 27; PDK1, phosphoinositide-dependent kinase-1; PDK2, phosphoinositide-dependent kinase-2; MAPK, mitogen-activated protein kinase; MK2, MAPK-activated protein kinase-2; HK-11 cells, human renal proximal tubular cells; HEK-293, human embryonic kidney cells; AktCA, c-Myc-tagged myristoylated constitutively active Akt; IEF, isoelectric focusing; MES, 4-morpholineethanesulfonic acid; PH, pleckstrin homology; PMSF, phenylmethylsulfonyl fluoride; fMLP, formylmethionylleucylphenylalanine; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide;FITC,fluoresceinisothiocyanate;siRNA,shortinterferingRNA;BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol. take part in host defense mechanisms against infection and inflammatory diseases. Inappropriate termination of PMN activation or failure to remove apoptotic PMNs results in inflammation. This apoptotic process has been suggested to represent an in vivo mechanism limiting oxidant-induced tissue injury caused by PMNs at the sites of inflammation. Although PMNs are constitutively committed to apoptosis from the time they enter circulation, the rate of apoptosis is not fixed. We reported that interleukin-8, granulocytemacrophage colony-stimulating factor, LTB4, and bacterial lipopolysaccharide (LPS) delay constitutive PMN apoptosis through the activation of the serine/threonine kinase Akt (1Klein J.B. Rane M.J. Scherzer J.A. Coxon P.Y. Kettritz R. Mathiesen J.M. Buridi A. McLeish K.R. J. Immunol. 2000; 164: 4286-4291Crossref PubMed Scopus (248) Google Scholar, 2Klein J.B. Buridi A. Coxon P.Y. Rane M.J. Manning T. Kettritz R. McLeish K.R. Cell. Signal. 2001; 13: 335-343Crossref PubMed Scopus (88) Google Scholar). We demonstrated that p38 mitogen-activated protein kinase (MAPK) activity is required for Akt phosphorylation and activation (3Rane M.J. Coxon P.Y. Powell D.W. Webster R. Klein J.B. Ping P. Pierce W. McLeish K.R. J. Biol. Chem. 2001; 276: 3517-3523Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar). Additionally, we showed that Akt exists in a signaling module with p38 MAPK, MAPK-activated protein kinase-2 (MK2), and heat shock protein 27 (Hsp27) (3Rane M.J. Coxon P.Y. Powell D.W. Webster R. Klein J.B. Ping P. Pierce W. McLeish K.R. J. Biol. Chem. 2001; 276: 3517-3523Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar). Heat shock proteins represent a group of chaperone proteins that protect the cells against a variety of stresses. Besides being involved in functioning as a chaperone, Hsp27 has also been shown to regulate stability of the cytoskeleton, cell motility (4Okamoto C.T. Am. J. 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Nelson C. Gleave M. Cancer Res. 2005; 65: 11083-11093Crossref PubMed Scopus (203) Google Scholar, 12Son G.H. Geum D. Chung S. Park E. Lee K.H. Choi S. Kim K. Biochem. Biophys. Res. Commun. 2005; 338: 1751-1758Crossref PubMed Scopus (30) Google Scholar, 13Sheth K. De A. Nolan B. Friel J. Duffy A. Ricciardi R. Miller-Graziano C. Bankey P. J. Surg. Res. 2001; 99: 129-133Abstract Full Text PDF PubMed Scopus (22) Google Scholar). When overexpressed in tumor cells, Hsp27 increases their tumorigenicity by overexpressing MMP-9 expression and down-regulating Src tyrosine kinase Yes expression (14Hansen R.K. Parra I. Hilsenbeck S.G. Himelstein B. Fuqua S.A. Biochem. Biophys. Res. Commun. 2001; 282: 186-193Crossref PubMed Scopus (29) Google Scholar, 15Garrido C. Fromentin A. Bonnotte B. Favre N. Moutet M. Arrigo A.P. Mehlen P. Solary E. Cancer Res. 1998; 58: 5495-5499PubMed Google Scholar, 16Bruey J.M. Paul C. Fromentin A. Hilpert S. Arrigo A.P. Solary E. Garrido C. Oncogene. 2000; 19: 4855-4863Crossref PubMed Scopus (128) Google Scholar) and protects against apoptotic cell death triggered by various stimuli, including cytotoxic drugs and ligation of the Fas/Apo-1/CD95 death receptor (17Garrido C. Ottavi P. Fromentin A. Hammann A. Arrigo A.P. Chauffert B. Mehlen P. Cancer Res. 1997; 57: 2661-2667PubMed Google Scholar, 18Garrido C. Mehlen P. Fromentin A. Hammann A. Assem M. Arrigo A.P. Chauffert B. Eur. J. Biochem. 1996; 237: 653-659Crossref PubMed Scopus (79) Google Scholar, 19Mehlen P. Schulze-Osthoff K. Arrigo A.P. J. Biol. Chem. 1996; 271: 16510-16514Abstract Full Text Full Text PDF PubMed Scopus (584) Google Scholar). Mice overexpressing Hsp27 were protected from lethal ischemia/reperfusion injury compared with their negative littermates (20Efthymiou C.A. Mocanu M.M. de Belleroche J. Wells D.J. Latchmann D.S. Yellon D.M. Basic Res. Cardiol. 2004; 99: 392-394Crossref PubMed Scopus (108) Google Scholar). Possible mechanisms of Hsp27 anti-apoptotic activity are proposed to result from its activity as a molecular chaperone. Hsp27 binds to and inactivates the pro-apoptotic molecules Smac, caspase 3, caspase 9, and cytochrome c (21Chauhan D. Li G. Hideshima T. Podar K. Mitsiades C. Mitsiades N. Catley L. Tai Y.T. Hayashi T. Shringarpure R. Burger R. Munshi N. Ohtake Y. Saxena S. Anderson K.C. Blood. 2003; 102: 3379-3386Crossref PubMed Scopus (135) Google Scholar, 22Pandey P. Farber R. Nakazawa A. Kumar S. Bharti A. Nalin C. Weichselbaum R. Kufe D. Kharbanda S. Oncogene. 2000; 19: 1975-1981Crossref PubMed Scopus (261) Google Scholar, 23Garrido C. Bruey J.M. Fromentin A. Hammann A. Arrigo A.P. Solary E. FASEB J. 1999; 13: 2061-2070Crossref PubMed Scopus (447) Google Scholar, 24Bruey J.M. Ducasse C. Bonniaud P. Ravagnan L. Susin S.A. DiazLatoud C. Gurbuxani S. Arrigo A.P. Kroemer G. Solary E. Garrido C. Nat. Cell Biol. 2000; 2: 645-652Crossref PubMed Scopus (840) Google Scholar, 25Paul C. Manero F. Gonin S. Kretz-Remy C. Virot S. Arrigo A.P. Mol. Cell. Biol. 2002; 22: 816-834Crossref PubMed Scopus (376) Google Scholar). Hsp27-mediated suppression of Bid translocation to the mitochondria correlates with an inhibition of cytochrome c release (25Paul C. Manero F. Gonin S. Kretz-Remy C. Virot S. Arrigo A.P. Mol. Cell. Biol. 2002; 22: 816-834Crossref PubMed Scopus (376) Google Scholar). Hsp27 has also been shown to promote survival pathways by modulating IKK complex stability and activity. Parcellier et al. (26Parcellier A. Schmitt E. Gurbuxani S. Seigneurin-Berny D. Pance A. Chantome A. Plenchette S. Khochbin S. Solary E. Garrido C. Mol. Cell. Biol. 2003; 23: 5790-5802Crossref PubMed Scopus (281) Google Scholar) demonstrated that Hsp27 mediates NF-KB activation and cell survival by promoting the proteasomal degradation of polyubiquitinated IKB. Phosphorylated Hsp27 has been shown to bind an adaptor protein Daxx and to inhibit Fas-mediated apoptosis (27Charette S.J. Lavoie J.N. Lambert H. Landry J. Mol. Cell. Biol. 2000; 20: 7602-7612Crossref PubMed Scopus (373) Google Scholar). Additionally, phosphorylation of Hsp27 has been shown to be required for proper maintenance of cell adhesion and inhibition of renal epithelial cell apoptosis (28de Graauw M. Tijdens I. Cramer R. Corless S. Timms J.F. van de Water B. J. Biol. Chem. 2005; 280: 29885-29898Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Furthermore, Sheth et al. (13Sheth K. De A. Nolan B. Friel J. Duffy A. Ricciardi R. Miller-Graziano C. Bankey P. J. Surg. Res. 2001; 99: 129-133Abstract Full Text PDF PubMed Scopus (22) Google Scholar) showed that introduction of recombinant Hsp27 caused delay of PMN apoptosis; however, mechanisms regulating this delay of PMN apoptosis were not determined. We recently demonstrated direct protein/protein interaction between Akt/Hsp27 (3Rane M.J. Coxon P.Y. Powell D.W. Webster R. Klein J.B. Ping P. Pierce W. McLeish K.R. J. Biol. Chem. 2001; 276: 3517-3523Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 8Rane M.J. Pan Y. Singh S. Powell D.W. Wu R. Cummins T. Chen Q. McLeish K.R. Klein J.B. J. Biol. Chem. 2003; 278: 27828-27835Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar). The physical association of Hsp27 with Akt is a critical determinant of PMN survival, as removal of Hsp27 from the Akt signal module prevented Akt phosphorylation and activation and resulted in accelerated PMN apoptosis suggesting an important role for Hsp27 in regulating Akt activity (8Rane M.J. Pan Y. Singh S. Powell D.W. Wu R. Cummins T. Chen Q. McLeish K.R. Klein J.B. J. Biol. Chem. 2003; 278: 27828-27835Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar). MK2 has been shown to bind and phosphorylate Hsp27 (29Stokoe D. Engel K. Campbell D.G. Cohen P. Gaestel M. FEBS Lett. 1992; 313: 307-313Crossref PubMed Scopus (472) Google Scholar), and MK2 is PDK2 for Akt in human PMNs (3Rane M.J. Coxon P.Y. Powell D.W. Webster R. Klein J.B. Ping P. Pierce W. McLeish K.R. J. Biol. Chem. 2001; 276: 3517-3523Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar). Recently, Zheng et al. (30Zheng C. Lin Z. Zhao Z.J. Yang Y. Niu H. Shen X. J. Biol. Chem. 2006; 281: 37215-37226Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar) demonstrated that MK2 is required for p38 MAPK and Hsp27 interaction; however, association of Hsp27 and Akt was not dependent on MK2. Hence we hypothesized that Hsp27 regulates Akt activation and apoptosis by scaffolding MK2 to the Akt signal complex. Akt contains an N-terminal pleckstrin homology (PH) domain and a catalytic kinase domain (residues 1–116 and 148–411 respectively) linked by a highly acidic linker region (residues 117–147). The C-terminal tail region lies between residues 412 and 480. Phosphoinositides are known to bind the PH domain of Akt and recruit it to the plasma membrane for full activation by PDK1 and PDK2 (28de Graauw M. Tijdens I. Cramer R. Corless S. Timms J.F. van de Water B. J. Biol. Chem. 2005; 280: 29885-29898Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 31Chan T.O. Tsichlis P.N. Sci. STKE 2001. 2001; : PE1Google Scholar, 32Franke T.F. Yang S.I. Chan T.O. Datta K. Kazlauskas A. Morrison D.K. Kaplan D.R. Tsichlis P.N. Cell. 1995; 81: 727-736Abstract Full Text PDF PubMed Scopus (1829) Google Scholar, 33Alessi D.R. James S.R. Downes C.P. Holmes A.B. Gaffney P.R. Reese C.B. Cohen P. Curr. Biol. 1997; 7: 261-269Abstract Full Text Full Text PDF PubMed Google Scholar). In the present study we show that amino acids 117–128 within the acidic linker region of Akt are required for interaction with Hsp27. An in-frame deletion mutant of Akt (AktΔ117–128), lacking the Hsp27 binding region, interacts with Hsp90 but not with Hsp27 and MK2. Disruption of Akt/Hsp27 interaction prevents MK2 association with Akt, resulting in loss of MK2-mediated Akt Ser-473 phosphorylation, activation, and induction of apoptosis. These studies demonstrate for the first time that Hsp27 regulates Akt activation and cellular apoptosis by scaffolding MK2 to the Akt signal complex. Anti-PH domain Akt, anti-Akt, and anti-p38 antisera were obtained from Cell Signaling Inc. (Beverly, MA). Anti-phospho-Ser-473-Akt was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-MK2 antibody was obtained from Sigma. Mouse isotype control antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Recombinant Hsp27 and anti-mouse Hsp27 were obtained from StressGen Biotechnologies Corp. (Victoria, British Columbia, Canada). Protein A-Sepharose and glutathione-Sepharose were obtained from Pharmingen. Histone H2B was obtained from Roche Applied Science. Recombinant GST-Akt-(1–149), recombinant active MK2, and recombinant active catalytic protein kinase A were obtained from Upstate Biotechnology Inc. (Lake Placid, NY). pUseAktwt (wild type) and pUseAktCA (myristoylated constitutively active Akt) constructs were obtained from Upstate Biotechnology, Inc. pUseAktwt was shuttled into GST vector pGEX-4T-2 as described previously (8Rane M.J. Pan Y. Singh S. Powell D.W. Wu R. Cummins T. Chen Q. McLeish K.R. Klein J.B. J. Biol. Chem. 2003; 278: 27828-27835Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar). pUseAktΔ117–128 was shuttled into GST vector pGEX-4T-2 as described under “Materials and Methods.” pcDNA3.1-Hsp27-wt was shuttled into pGEX-5X-2 (GE Healthcare) vector as described previously (8Rane M.J. Pan Y. Singh S. Powell D.W. Wu R. Cummins T. Chen Q. McLeish K.R. Klein J.B. J. Biol. Chem. 2003; 278: 27828-27835Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar). Constitutively active MK2EE cDNA construct was obtained from the Gaestel laboratory (Hannover, Germany). Isolation of PMNs and Culture Conditions—PMNs were isolated from venous blood obtained from healthy volunteers as described previously (3Rane M.J. Coxon P.Y. Powell D.W. Webster R. Klein J.B. Ping P. Pierce W. McLeish K.R. J. Biol. Chem. 2001; 276: 3517-3523Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 8Rane M.J. Pan Y. Singh S. Powell D.W. Wu R. Cummins T. Chen Q. McLeish K.R. Klein J.B. J. Biol. Chem. 2003; 278: 27828-27835Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar). PMN preparations routinely contained >95% PMNs, as determined by morphology, and were >99% viable by trypan blue dye exclusion. PMNs were suspended in RPMI 1640 medium supplemented with 10% fetal calf serum, l-glutamine, penicillin, and streptomycin and incubated for the indicated times at 37 °C in 5% CO2. Generation of pUseAktΔ117–128 Mutant by Site-directed Mutagenesis—In-frame deletion of amino acids 117–128 from pUseAktwt cDNA construct was carried out using the Transformer site-directed mutagenesis kit from BD Biosciences according to the manufacturer's instructions. The in-frame deletion primer was 5′-AGGCAGGAAGAAGAGTCAGGGGCTGAAGAG-3′, and the selection primer for pUseAktwt (mutating the KpnI site) was 5′-GTTAAGCTTGAATCCGAGCTCG-3′. Cloning and mutation were confirmed by DNA sequencing. Subcloning and Purification of GST Beads and Recombinant Proteins—AktΔ117–128 was excised from pUSEAktΔ117–128 with restriction enzymes BamHI/PmeI and ligated into BamHI/SmaI sites of pGEX-4T-2 (GE Healthcare) vector. Generation of GST-Hsp27pGEX-5X-2, GST-AktpGEX-4T-2, was described previously (8Rane M.J. Pan Y. Singh S. Powell D.W. Wu R. Cummins T. Chen Q. McLeish K.R. Klein J.B. J. Biol. Chem. 2003; 278: 27828-27835Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar). GST-pGEX-4T-2, GST-Hsp27pGEX-5X-2, GST-Akt-pGEX-4T-2, and GST-AktΔ117–128pGEX-4T-2 cDNAs were transformed into Escherichia coli BL21(DE3)PlysS, and the expression and purification of GST, GST-Hsp27, GST-Aktwt, and GST-AktΔ117–128 fusion proteins were performed as described previously (8Rane M.J. Pan Y. Singh S. Powell D.W. Wu R. Cummins T. Chen Q. McLeish K.R. Klein J.B. J. Biol. Chem. 2003; 278: 27828-27835Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar). pRSETA vector was digested with restriction enzyme EcoRI followed by generation of a blunt end by treatment with Klenow enzyme followed by digestion with BamHI restriction enzyme. This vector was then ligated to either Aktwt or AktΔ117–128, which were excised from pUSEAktwt or pUSEAktΔ117–128 with restriction enzymes BamHI/PmeI. All positive clones were confirmed by DNA sequencing. Expression of pRSET-Aktwt and pRSET-AktΔ117–128 plasmids was carried out in BL21(DE3)pLysS chemically competent E. coli cells, and protein was purified using the ProBond purification system (Invitrogen). Immunoblot Analysis—Immunoblotting procedures were performed as described previously (3Rane M.J. Coxon P.Y. Powell D.W. Webster R. Klein J.B. Ping P. Pierce W. McLeish K.R. J. Biol. Chem. 2001; 276: 3517-3523Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 8Rane M.J. Pan Y. Singh S. Powell D.W. Wu R. Cummins T. Chen Q. McLeish K.R. Klein J.B. J. Biol. Chem. 2003; 278: 27828-27835Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar). Fifty μg of protein was subjected to 10% SDS-PAGE and immunoblot analysis with anti-pAktSer-473 (1:1000, Santa Cruz Biotechnology), anti-Akt (1:1000, Santa Cruz Biotechnology), anti-Hsp27 (StressGen), anti-Ser(P)-82Hsp27 (Cell Signaling), anti-c-Myc (Cell Signaling), anti-MK2 (Sigma), and anti-Hsp90 (Santa Cruz Biotechnology) antisera (3Rane M.J. Coxon P.Y. Powell D.W. Webster R. Klein J.B. Ping P. Pierce W. McLeish K.R. J. Biol. Chem. 2001; 276: 3517-3523Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 8Rane M.J. Pan Y. Singh S. Powell D.W. Wu R. Cummins T. Chen Q. McLeish K.R. Klein J.B. J. Biol. Chem. 2003; 278: 27828-27835Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar). Gel Filtration Chromatography—The system used for the size exclusion chromatography experiments consisted of a HiPrep 26/60 Sephacryl S-300 HR prepacked chromatography column connected to an AKTA purifier 10 liquid chromatography system (Amersham Biosciences), equipped with a Frac-900 automated fraction collector, and controlled by the UNICORN version 4.00 software (Amersham Biosciences). Prior to chromatography, the column was equilibrated in 50 mm Tris-Cl, pH 7.4, 1 mm EDTA, 150 mm NaCl, 1.5 mm MgCl2, 5% glycerol, 0.5% Triton X-100. For the chromatographic separation of the samples, control and fMLP (0.3 μm)-stimulated PMNs were harvested and resuspended in Akt lysis buffer containing 20 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1% (v/v) Triton X-100, 0.5% (v/v) Nonidet P-40, 1 mm EDTA, 1 mm EGTA, 20 mm sodium orthovanadate, 10 μm p-nitrophenol phosphate, 20 mm NaF, 5 mm PMSF, 21 μg/ml aprotinin, and 5 μg/ml leupeptin. The cell lysate was cleared by centrifugation at 14,000 rpm for 15 min. Next, 3–5 ml of total cleared cell lysate was injected onto the column with a manual injection through a 50-ml capacity superloop system (Amersham Biosciences). Isocratic elution with 2.5 column volumes of the same buffer was performed. Both a constant flow rate of 1.0 ml/min and the absorbance profiles at A230nm and A280nm were monitored during the entire chromatographic procedure. Fractions of 1.0 ml were collected, and 100 μl of every fifth fraction was used for Western blot analysis. All procedures were performed at 4 °C. Isoelectric Focusing (IEF) Electrophoresis—PMN lysates (25 μg) were subjected to IEF electrophoresis. Proteins were separated based on their isoelectric point. Precast IEF (NOVEX) gels (Invitrogen) were run according to manufacturer's instructions. Gels were fixed in 100 ml of 20% methanol solution for 20 min. Gel was rinsed in 1× SDS-running buffer for 20 min and transferred onto nitrocellulose membrane using semidry transfer apparatus (Invitrogen) for 20 min at 20 V. Nitrocellulose membranes were washed with Krebs+ buffer and then immunoblotted with anti-Hsp27 and anti-Ser(P)-82Hsp27 antisera. Two-dimensional PAGE—Protein lysates (50 μg) were diluted into urea/thiourea rehydration buffer (Genomic Solutions), and proteins were separated by two-dimensional PAGE. IPG strips, pH 3–10 (Invitrogen), were rehydrated overnight with protein samples. Proteins were separated on the basis of their isoelectric point by IEF using the ZOOM IPG Runner (Invitrogen) with a maximal voltage of 2000 V and 50 μA per gel. Following IEF, IPG strips were incubated twice in equilibration buffer I (6 m urea, 130 mm dithiothreitol, 30% glycerol, 45 mm Tris base, 1.6% SDS, 0.002% bromphenol blue; Genomic Solutions) and once in equilibration buffer II (6 m urea, 135 mm iodoacetamide, 30% glycerol, 45 mm Tris base, 1.6% SDS, 0.002% bromphenol blue; Genomic Solutions) for 10 min. Equilibrated IPG strips were applied to 4–12% BisTris gradient gels (Invitrogen), and proteins were separated in the second dimension based on their molecular size using NuPAGE MES/SDS buffer (Invitrogen) at 200 V for 40 min. Following electrophoresis, gels were transferred onto nitrocellulose and immunoblotted with anti-Hsp27 antibody. GST Pulldown Assay—PMNs (2 × 107) were untreated or treated with isotype control antibody or Hsp27 antibody for 2 h at 37 °C. The cells were lysed with 200 μl of lysis buffer containing 1% (v/v) Nonidet P-40, 10% (v/v) glycerol, 137 mm NaCl, 20 mm Tris-HCl, pH 7.4, 1 μg/ml aprotinin, 1 μg/ml leupeptin, 5 mm phenylmethylsulfonyl fluoride, 20 mm NaF, 1 mm sodium pyrophosphate, 1 mm sodium orthovanadate, and 1% (v/v) Triton X-100. Appropriate GST beads (10 μl) were added to the lysates and incubated at 4 °C for 1 h with shaking. The beads were washed three times with Krebs buffer, and 15 μl of 2× Laemmli buffer was added to each tube. The samples were boiled for 3 min and then subjected to 10% SDS-PAGE. Proteins were transferred onto nitrocellulose and immunoblotted with appropriate antibodies. GST Pulldown Assay with Recombinant Proteins—Appropriate GST beads (10 μl) were added to 50 μl of kinase buffer (20 mm HEPES, 10 mm MgCl2, 10 mm MnCl2) containing 50 ng of appropriate recombinant protein. The samples were incubated at 4 °C for 1 h with shaking. The beads were washed three times with Krebs buffer, and 15 μl of 2× Laemmli buffer was added to each tube. The samples were boiled for 3 min and then subjected to 10% SDS-PAGE and immunoblotting. Recombinant GST-Akt-PH domain (1–149 amino acids) was first conjugated with glutathione-Sepharose beads by incubating the two at 4 °C for 1 h with shaking. These beads were then incubated with 50 ng of recombinant Hsp27 as described above. Cell Culture—HK-11 cells (human renal tubular epithelial cells) immortalized by transduction with adenovirus 12-SV40 were obtained from Dr. Racusen (34Racusen L.C. Monteil C. Sgrignoli A. Lucskay M. Marouillat S. Rhim J.G. Morin J.P. J. Lab. Clin. Med. 1997; 129: 318-329Abstract Full Text PDF PubMed Scopus (206) Google Scholar). Cells were cultured in Dulbecco's modified Eagle's medium/Ham's F-12 (Invitrogen) supplemented with 5% fetal calf serum (Sigma) and penicillin/streptomycin (100 units/ml) (Invitrogen). Fresh growth medium was added to cells every 3–4 days until confluent. HEK-293 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and penicillin/streptomycin (100 units/ml) (Invitrogen). cDNA Transfections—HK-11 or HEK-293 cells were plated on 6-well trays a day prior to performing transfections, to achieve 60% confluence. On the day of transfection appropriate cells were washed with serum-free RPMI 1640 medium. One μg of appropriate cDNA was transfected into these cells using GenePORTER reagent according to the manufacturer's protocol (Gene Therapy Systems). Twenty four hours after transfection cells were lysed in 100 μl of Akt lysis buffer containing 20 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1% (v/v) Triton X-100, 0.5% (v/v) Nonidet P-40, 1 mm EDTA, 1 mm EGTA, 20 mm sodium orthovanadate, 10 μm p-nitrophenol phosphate, 20 mm NaF, 5 mm PMSF, 21 μg/ml aprotinin, and 5 μg/ml leupeptin, and proteins concentrations were determined. Protein lysates (50 μg) were subjected to SDS-PAGE and immunoblot analysis or to immunoprecipitation studies. Construction of Hsp27 siRNA—Construction of siRNA was performed as described previously (35Khundmiri S.J. Weinman E.J. Steplock D. Cole J. Ahmad A. Baumann P.D. Barati M. Rane M.J. Lederer E. J. Am. Soc. Nephrol. 2005; 16: 2598-2607Crossref PubMed Scopus (30) Google Scholar). Hsp27 siRNA was generated using Silencer® siRNA construction kit (Ambion, Austin, TX). Hsp27 sequence 5′-AAGACCAAGGATGGCGTGGTG-3′ was targeted to generate Hsp27 siRNA. The sense and antisense siRNA oligonucleotides used to generate Hsp27 siRNA were 5′-AACACCACGCCATCCTTGGTCCCTGTCTC-3′ (sense) and 5′-AAGACCAAGGATGGCGTGGTGCCTGTCTC-3′ (antisense). The Hsp27 siRNA and a scrambled siRNA were transfected by using Gene-PORTER reagent as outlined above. Assessment of Cell Viability—Cell viability was measured by assessment of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) reduction (Sigma) as described previously (36Zhang S.X. Gozal D. Sachleben Jr., L.R. Rane M. Klein J.B. Gozal E. FASEB J. 2003; 17: 1709-1711Crossref PubMed Scopus (90) Google Scholar, 37Gozal E. Sachleben Jr., L.R. Rane M.J. Vega C. Gozal D. Am. J. Physiol. 2005; 288: C535-C542Crossref PubMed Scopus (60) Google Scholar). The soluble form of MTT was reduced by mitochondria of live cells, resulting in a water-insoluble salt. Product formation was monitored by reading absorbance at 540 nm using a microplate reader. Akt Immunoprecipitation Assays—Appropriate cells were lysed in Akt lysis buffer containing 20 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1% (v/v) Triton X-100, 0.5% (v/v) Nonidet P-40, 1 mm EDTA, 1 mm EGTA, 20 mm sodium orthovanadate, 10 μm p-ni-trophenol phosphate, 20 mm NaF, 5 mm PMSF, 21 μg/ml aprotinin, and 5 μg/ml leupeptin. Following centrifugation at 15,000 × g for 15 min at 4 °C, cleared lysates were incubated with 20 μl of anti-Akt PH domain agarose beads or with mouse isotype control antibody beads as described previously (8Rane M.J. Pan Y. Singh S. Powell D.W. Wu R. Cummins T. Chen Q. McLeish K.R. Klein J.B. J. Biol. Chem. 2003; 278: 27828-27835Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar). Immunoprecipitated proteins were eluted with 40 μl of 2× Laemmli dye. Samples were boiled for 2 min; beads were precipitated by a quick spin" @default.
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