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• W2040372727 abstract "G protein-coupled receptor kinase 2 (GRK2) is a serine/threonine-specific protein kinase that mediates agonist-dependent phosphorylation of numerous G protein-coupled receptors. In an effort to identify proteins that regulate GRK2 function, we searched for interacting proteins by immunoprecipitation of endogenous GRK2 from HL60 cells. Subsequent analysis by gel electrophoresis and mass spectrometry revealed that GRK2 associates with heat shock protein 90 (Hsp90). GRK2 interaction with Hsp90 was confirmed by co-immunoprecipitation and was effectively disrupted by geldanamycin, an Hsp90-specific inhibitor. Interestingly, geldanamycin treatment of HL60 cells decreased the expression of endogenous GRK2 in a dose- and time-dependent manner, and metabolic labeling demonstrated that geldanamycin rapidly accelerated the degradation of newly synthesized GRK2. The use of various protease inhibitors suggested that GRK2 degradation induced by geldanamycin was predominantly through the proteasome pathway. To test whether Hsp90 plays a general role in regulating GRK maturation, additional GRKs were studied by transient expression in COS-1 cells and subsequent treatment with geldanamycin. These studies demonstrate that GRK3, GRK5, and GRK6 are also stabilized by interaction with Hsp90. Taken together, our work revealed that GRK interaction with heat shock proteins plays an important role in regulating GRK maturation. G protein-coupled receptor kinase 2 (GRK2) is a serine/threonine-specific protein kinase that mediates agonist-dependent phosphorylation of numerous G protein-coupled receptors. In an effort to identify proteins that regulate GRK2 function, we searched for interacting proteins by immunoprecipitation of endogenous GRK2 from HL60 cells. Subsequent analysis by gel electrophoresis and mass spectrometry revealed that GRK2 associates with heat shock protein 90 (Hsp90). GRK2 interaction with Hsp90 was confirmed by co-immunoprecipitation and was effectively disrupted by geldanamycin, an Hsp90-specific inhibitor. Interestingly, geldanamycin treatment of HL60 cells decreased the expression of endogenous GRK2 in a dose- and time-dependent manner, and metabolic labeling demonstrated that geldanamycin rapidly accelerated the degradation of newly synthesized GRK2. The use of various protease inhibitors suggested that GRK2 degradation induced by geldanamycin was predominantly through the proteasome pathway. To test whether Hsp90 plays a general role in regulating GRK maturation, additional GRKs were studied by transient expression in COS-1 cells and subsequent treatment with geldanamycin. These studies demonstrate that GRK3, GRK5, and GRK6 are also stabilized by interaction with Hsp90. Taken together, our work revealed that GRK interaction with heat shock proteins plays an important role in regulating GRK maturation. G protein-coupled receptor kinases (GRKs) 1The abbreviations used are: GRKG protein-coupled receptor kinaseGAgeldanamycinHsp90heat shock protein 90GPCRG protein-coupled receptormAbmonoclonal antibody.1The abbreviations used are: GRKG protein-coupled receptor kinaseGAgeldanamycinHsp90heat shock protein 90GPCRG protein-coupled receptormAbmonoclonal antibody. are a family of serine/threonine protein kinases that specifically phosphorylate the agonist-activated form of G protein-coupled receptors (GPCRs). Receptor phosphorylation functions to promote arrestin binding resulting in receptor desensitization and trafficking (1Pitcher J.A. Freedman N.J. Lefkowitz R.J. Annu. Rev. Biochem. 1998; 67: 653-692Crossref PubMed Scopus (1059) Google Scholar, 2Krupnick J.G. Benovic J.L. Annu. Rev. Pharmacol. Toxicol. 1998; 38: 289-319Crossref PubMed Scopus (855) Google Scholar, 3Perry S.J. Lefkowitz R.J. Trends Cell Biol. 2002; 12: 130-138Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). Recent studies have revealed that GRKs themselves are also subject to various regulatory processes including phosphorylation and ubiquitination (4Penn R.B. Pronin A.N. Benovic J.L. Trends Cardiovasc. Med. 2000; 10: 81-89Crossref PubMed Scopus (185) Google Scholar, 5Kohout T.A. Lefkowitz R.J. Mol. Pharmacol. 2003; 63: 9-18Crossref PubMed Scopus (351) Google Scholar). For example, GRK2 phosphorylation by cAMP-dependent protein kinase (6Cong M. Perry S.J. Lin F.T. Fraser I.D. Hu L.A. Chen W. Pitcher J.A. Scott J.D. Lefkowitz R.J. J. Biol. Chem. 2001; 276: 15192-15199Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar), protein kinase C (7Winstel R. Freund S. Krasel C. Hoppe E. Lohse M.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2105-2109Crossref PubMed Scopus (141) Google Scholar), and c-Src (8Sarnago S. Elorza A. Mayor Jr., F. J. Biol. Chem. 1999; 274: 34411-34416Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar) results in activation, whereas phosphorylation by ERK1/2 effectively inhibits GRK2 activity (9Pitcher J.A. Tesmer J.J. Freeman J.L. Capel W.D. Stone W.C. Lefkowitz R.J. J. Biol. Chem. 1999; 274: 34531-34534Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 10Elorza A. Sarnago S. Mayor Jr., F. Mol. Pharmacol. 2000; 57: 778-783Crossref PubMed Scopus (77) Google Scholar). Interestingly, c-Src phosphorylation also promotes the degradation of GRK2 via a process that is regulated by receptor stimulation and may involve GRK2 ubiquitination and targeting to the proteasome pathway (11Penela P. Elorza A. Sarnago S. Mayor Jr., F. EMBO J. 2001; 20: 5129-5138Crossref PubMed Scopus (107) Google Scholar). GRKs also interact with numerous additional proteins including G protein α (12Carman C.V. Parent J.L. Day P.W. Pronin A.N. Sternweis P.M. Wedegaertner P.B. Gilman A.G. Benovic J.L. Kozasa T. J. Biol. Chem. 1999; 274: 34483-34492Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar, 13Usui H. Nishiyama M. Moroi K. Shibasaki T. Zhou J. Ishida J. Fukamizu A. Haga T. Sekiya S. Kimura S. Int. J. Mol. Med. 2000; 5: 335-340PubMed Google Scholar, 14Sallese M. Mariggio S. D'Urbano E. Iacovelli L. De Blasi A. Mol. Pharmacol. 2000; 57: 826-831Crossref PubMed Scopus (121) Google Scholar) and βγ (15Pitcher J.A. Inglese J. Higgins J.B. Arriza J.L. Casey P.J. Kim C. Benovic J.L. Kwatra M.M. Caron M.G. Lefkowitz R.J. Science. 1992; 257: 1264-1267Crossref PubMed Scopus (563) Google Scholar, 16Carman C.V. Barak L.S. Chen C. Liu-Chen L.Y. Onorato J.J. Kennedy S.P. Caron M.G. Benovic J.L. J. Biol. Chem. 2000; 275: 10443-10452Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar) subunits, clathrin (17Shiina T. Arai K. Tanabe S. Yoshida N. Haga T. Nagao T. Kurose H. J. Biol. Chem. 2001; 276: 33019-33026Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar), the GRK-interacting protein GIT1 (18Premont R.T. Claing A. Vitale N. Freeman J.L. Pitcher J.A. Patton W.A. Moss J. Vaughan M. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14082-14087Crossref PubMed Scopus (250) Google Scholar), caveolin-1 (19Carman C.V. Lisanti M.P. Benovic J.L. J. Biol. Chem. 1999; 274: 8858-8864Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar), phosphoinositide 3-kinase-α and -γ (20Naga Prasad S.V. Barak L.S. Rapacciuolo A. Caron M.G. Rockman H.A. J. Biol. Chem. 2001; 276: 18953-18959Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar), cytoskeletal proteins such as tubulin and actin (21Pitcher J.A. Hall R.A. Daaka Y. Zhang J. Ferguson S.S. Hester S. Miller S. Caron M.G. Lefkowitz R.J. Barak L.S. J. Biol. Chem. 1998; 273: 12316-12324Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 22Carman C.V. Som T. Kim C.M. Benovic J.L. J. Biol. Chem. 1998; 273: 20308-20316Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 23Freeman J.L. De La Cruz E.M. Pollard T.D. Lefkowitz R.J. Pitcher J.A. J. Biol. Chem. 1998; 273: 20653-20657Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar), and various calcium-binding proteins (reviewed in Ref. 24Iacovelli L. Sallese M. Mariggio S. de Blasi A. FASEB J. 1999; 13: 1-8Crossref PubMed Scopus (68) Google Scholar). Many of these interactions are thought to be important for regulating the localization and enzymatic activity of GRKs. In an effort to identify proteins that interact with endogenous GRKs, here we used immunoprecipitation and mass spectrometry analysis and demonstrated that GRK2 interacts with heat shock protein 90 (Hsp90).Hsp90 is a highly conserved protein chaperone that interacts with a diverse group of regulatory and signaling proteins including various protein kinases (25Whitesell L. Mimnaugh E.G. De Costa B. Myers C.E. Neckers L.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8324-8328Crossref PubMed Scopus (1317) Google Scholar, 26Stancato L.F. Silverstein A.M. Owens-Grillo J.K. Chow Y.H. Jove R. Pratt W.B. J. Biol. Chem. 1997; 272: 4013-4020Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 27Sato S. Fujita N. Tsuruo T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10832-10837Crossref PubMed Scopus (826) Google Scholar), heterotrimeric G proteins (28Busconi L. Guan J. Denker B.M. J. Biol. Chem. 2000; 275: 1565-1569Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 29Waheed A.A. Jones T.L. J. Biol. Chem. 2002; 277: 32409-32412Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar) and G protein-coupled receptors (30Pai K.S. Mahajan V.B. Lau A. Cunningham D.D. J. Biol. Chem. 2001; 276: 32642-32647Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). The functional role of Hsp90 interaction is protein-dependent. For example, Hsp90 can maintain the active state of the serine/threonine kinase Akt by preventing its dephosphorylation (27Sato S. Fujita N. Tsuruo T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10832-10837Crossref PubMed Scopus (826) Google Scholar), whereas Hsp90 interaction with the tyrosine kinases c-Src (31Xu Y. Singer M.A. Lindquist S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 109-114Crossref PubMed Scopus (164) Google Scholar) and Lck (32Hartson S.D. Barrett D.J. Burn P. Matts R.L. Biochemistry. 1996; 35: 13451-13459Crossref PubMed Scopus (65) Google Scholar) functions in protein maturation. Hsp90 contains three functional domains: an N-terminal region that binds ATP, a middle segment that has been implicated in binding client proteins, and a conserved C-terminal region (33Buchner J. Trends Biochem. Sci. 1999; 24: 136-141Abstract Full Text Full Text PDF PubMed Scopus (579) Google Scholar, 34Meyer P. Prodromou C. Hu B. Vaughan C. Roe S.M. Panaretou B. Piper P.W. Pearl L.H. Mol. Cell. 2003; 11: 647-658Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar). The interaction of Hsp90 with client proteins is dependent on its ability to bind and hydrolyze ATP and can be effectively disrupted by ATP-mimetic drugs including the antibiotics geldanamycin (GA) and herbimycin-A (25Whitesell L. Mimnaugh E.G. De Costa B. Myers C.E. Neckers L.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8324-8328Crossref PubMed Scopus (1317) Google Scholar, 33Buchner J. Trends Biochem. Sci. 1999; 24: 136-141Abstract Full Text Full Text PDF PubMed Scopus (579) Google Scholar). These antibiotics bind tightly to the Hsp90 ATP/ADP binding pocket often resulting in proteasome-dependent degradation of proteins that require Hsp90 for conformational maturation (35An W.G. Schulte T.W. Neckers L.M. Cell Growth & Differ. 2000; 11: 355-360PubMed Google Scholar, 36Schulte T.W. An W.G. Neckers L.M. Biochem. Biophys. Res. Commun. 1997; 239: 655-659Crossref PubMed Scopus (171) Google Scholar).In our search for proteins that regulate the function of endogenous GRK2, we found that GRK2 interacts with Hsp90 in HL60 cells. Disruption of GRK2/Hsp90 interaction with geldanamycin resulted in increased degradation of GRK2 mainly through the proteasome pathway. Moreover, additional GRK family members are also regulated by interaction with Hsp90. These studies suggest that heat shock proteins likely play a general role in regulating GRK maturation.EXPERIMENTAL PROCEDURESMaterials—HL60 and COS-1 cells were from the American Type Culture Collection (Manassas, VA). Anti-GRK2/3 and anti-GRK4-6 mouse monoclonal antibodies were from Upstate Biotechnology (Lake Placid, NY) and an anti-GRK2 mouse monoclonal antibody (3A10) was purified from a hybridoma cell culture supernatant. Anti-Hsp90 monoclonal antibodies were from Stressgen Biotechnologies (Victoria, Canada) and Sigma, whereas purified Hsp90 was from Stressgen Biotechnologies. Geldanamycin was from Sigma, and [35S]methionine/cysteine labeling mixture was from Amersham Biosciences. SYPRO ruby protein gel stain was from Molecular Probes (Eugene, OR), lactacystin and calpeptin were from Calbiochem (La Jolla, CA), and FuGENE 6™ was from Roche Applied Science.Cell Culture and Transfection—HL60 cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin G, and 100 μg/ml streptomycin sulfate at 37 °C in a humidified atmosphere containing 5% CO2. COS-1 cells were maintained in Dulbecco's modified Eagle's medium and were transfected at a confluence of ∼70% with 1 μg of total DNA/6-cm plate using FuGENE 6™ following the manufacturer's instructions.Immunoprecipitation of GRK2—HL60 cells (∼108) grown in 15-cm plates to a density of ∼2 × 106 cells/ml were harvested by centrifugation, washed two times with cold phosphate-buffered saline, and homogenized with 2 ml of buffer A (20 mm HEPES, pH 7.5, 10 mm EDTA, 150 mm NaCl, 10 μg/ml leupeptin, 2 μg/ml pepstatin, 10 μg/ml aprotinin, 10 μg/ml benzamidine, and 0.1% Triton) using a Brinkman Polytron (14,000 rpm, 2 × 30 s). The homogenate was centrifuged at 100,000 × g for 30 min, and the supernatant was pre-cleared by incubation with protein G-agarose for 1 h at 4 °C. The supernatant was then incubated on a rocker with 20 μl of anti-GRK2/3 or anti-GRK4-6 mAb for 1 h at 4 °C followed by the addition of 100 μl of 50% protein G-agarose pre-equilibrated in lysis buffer and a 1-h incubation at 4 °C. Samples were then centrifuged, and the pellets were washed three times with 1 ml of buffer A and one time with 20 mm Tris-HCl, pH 7.5, 2 mm EDTA on a rocker for 15 min at 4 °C. Bound proteins were eluted by addition of 50 μl of SDS sample buffer followed by boiling for 10 min. Samples were electrophoresed on a 7.5% polyacrylamide gel and stained with Coomassie Blue or SYPRO ruby stain following the manufacturer's instructions. Stained protein bands were excised from the polyacrylamide gel and stored at -20 °C until further analysis.Mass Spectrometry Analysis—Proteins in polyacrylamide gel slices were digested with 20 ng/μl trypsin (Promega, Madison, WI) in 25 mm NH4HCO3 buffer for 16 h at 37 °C. Mass spectra of tryptic peptides were acquired using surface-enhanced laser desorption/ionization on a hydrophobic H4 chip using a Ciphergen PBS II Instrument (Fremont, CA). Proteins were identified by comparing observed peptide mass fingerprints with those theoretically derived from the NCBInr data base using the Profound data base searching algorithm (Rockefeller University).Immunoblotting—To analyze co-immunoprecipitation of GRK2 and Hsp90, lysates from HL60 or transfected COS-1 cells were immunoprecipitated with anti-GRK2/3 mAb as described above, electrophoresed on a 7.5% SDS-polyacrylamide gel, transferred to nitrocellulose, and immunoblotted using anti-Hsp90 mAb, horseradish peroxidase-labeled goat anti-mouse secondary antibodies, and chemiluminescence (Pierce). To analyze GRK levels in transfected COS-1 cells, cells were homogenized in buffer A and centrifuged, and the supernatants were electrophoresed on a 7.5% SDS-polyacrylamide gel, transferred to nitrocellulose, and immunoblotted using anti-GRK2/3 or anti-GRK4-6 mAbs, horseradish peroxidase-labeled goat anti-mouse secondary antibodies, and chemiluminescence. Some blots were stripped and reprobed using an α-tubulin mAb (Sigma).Metabolic Labeling—HL60 cells (4 × 107) were harvested and washed two times with 10 ml of Dulbecco's modified Eagle's medium without methionine and cysteine and then incubated in 10 ml of the same medium with or without 1 μm geldanamycin for 1 h. The cells were then incubated in 4 ml of medium containing 200 μCi/ml [35S]methionine/cysteine labeling mixture with or without 1 μm geldanamycin for 30 min (pulse), washed two times with RPMI 1640 containing 10% fetal bovine serum, and chased with the same medium in the presence of geldanamycin or vehicle for 2-18 h. In an additional series of experiments, geldanamycin was not added until the beginning of the chase period. The cells were then lysed, and GRK2 was immunoprecipitated, electrophoresed on a 10% polyacrylamide gel, and detected by fluorography. GRK2 levels were quantified using liquid scintillation counting (PerkinElmer Life Sciences).Substrate Phosphorylation—Wild-type GRK2 was overexpressed and purified from Sf9 cells as described in Ref. 37Kim C.M. Dion S.B. Onorato J.J. Benovic J.L. Receptor. 1993; 3: 39-55PubMed Google Scholar. GRK2 activity was assayed by incubating 22 nm kinase and purified Hsp90 (0, 15, 150, or 1500 nm) with either rod outer segment membranes (4 μm rhodopsin) or tubulin (1 μm) in 20 mm Tris-HCl, pH 8.0, 4 mm MgCl2, and 0.1 mm [γ-32P]ATP (∼1,000 cpm/pmol). Samples were incubated for 5 min (rhodopsin) or 10 min (tubulin) at 30 °C in room light, quenched with SDS buffer, and electrophoresed on a 10% polyacrylamide gel. Gels were dried and autoradiographed, and and 32P-labeled proteins were excised and counted to determine pmol of phosphate transferred.RESULTS AND DISCUSSIONIdentification of Proteins Associated with Endogenous GRK2 in HL60 Cells—GRKs were initially identified via their ability to phosphorylate and regulate the activity of agonist-occupied GPCRs (1Pitcher J.A. Freedman N.J. Lefkowitz R.J. Annu. Rev. Biochem. 1998; 67: 653-692Crossref PubMed Scopus (1059) Google Scholar, 2Krupnick J.G. Benovic J.L. Annu. Rev. Pharmacol. Toxicol. 1998; 38: 289-319Crossref PubMed Scopus (855) Google Scholar). Although GRKs themselves are also subject to numerous regulatory interactions and post-translational modifications (4Penn R.B. Pronin A.N. Benovic J.L. Trends Cardiovasc. Med. 2000; 10: 81-89Crossref PubMed Scopus (185) Google Scholar, 5Kohout T.A. Lefkowitz R.J. Mol. Pharmacol. 2003; 63: 9-18Crossref PubMed Scopus (351) Google Scholar), the majority of studies have utilized over-expression or in vitro analysis to analyze these processes. Here we attempted to identify proteins that interact with and potentially regulate the function of endogenous GRKs. The strategy used involved immunoprecipitation of endogenous GRKs from cell lysates followed by SDS-PAGE and mass spectrometry. In the present study, we used HL60 cells because they have high endogenous levels of GRK2 (∼170,000 molecules/cell) and GRK6 (∼27,000 molecules/cell) (38Loudon R.P. Perussia B. Benovic J.L. Blood. 1996; 88: 4547-4557Crossref PubMed Google Scholar). An HL60 cell lysate (from ∼108 cells) was incubated with either anti-GRK2/3 monoclonal antibodies that selectively immunoprecipitate GRK2 and -3, or anti-GRK4-6 monoclonal antibodies that immunoprecipitate GRK4, -5, and -6 (39Oppermann M. Diverse-Pierluissi M. Drazner M.H. Dyer S.L. Freedman N.J. Peppel K.C. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7649-7654Crossref PubMed Scopus (81) Google Scholar). The resulting protein complexes were electrophoresed on a 7.5% polyacrylamide gel and then stained with Coomassie Blue or SYPRO ruby stain. Protein bands of ∼90, ∼80, and ∼52 kDa were clearly observed in the GRK2/3 immunoprecipitation that were not evident in the GRK4-6 lane (Fig. 1). In contrast, there were no protein bands selectively immunoprecipitated in the GRK4-6 lane. This may reflect the 7-fold lower level of GRK6 compared with GRK2 in HL60 cells (38Loudon R.P. Perussia B. Benovic J.L. Blood. 1996; 88: 4547-4557Crossref PubMed Google Scholar) and the lower efficiency of the GRK4-6 antibodies in immunoprecipitation. 2A. Pronin and J. L. Benovic, unpublished observation. The 80-kDa band in the GRK2/3 lane was identified as GRK2 by immunoblotting using an anti-GRK2-specific monoclonal antibody. The 90- and 52-kDa bands were excised from the gel, digested with trypsin, and analyzed by mass spectrometry. The 90-kDa protein was identified as Hsp90-β (19 matching peptides and 31% overall sequence coverage), whereas the 52-kDa protein was a proteolytic fragment of GRK2.To confirm the interaction of Hsp90 and GRK2, we used co-immunoprecipitation and immunoblotting. GRK2 from HL60 cells was immunoprecipitated using the anti-GRK2/3 mAb, electrophoresed on a 10% polyacrylamide gel, and analyzed for Hsp90 using an anti-Hsp90 monoclonal antibody (Fig. 2A). Interestingly, a 90-kDa band that was slightly larger than the Hsp90 band in the HL60 lysate was specifically detected in the anti-GRK2/3 mAb immunoprecipitation. This band was not detected using Hsp90 antibodies specific for either the α or β isoforms (not shown), suggesting that the ∼90-kDa Hsp90 that co-immunoprecipitates with GRK2 may be a different variant. Indeed, a number of Hsp90 variants have been identified including various polymorphisms of Hsp90-α and -β (40Passarino G. Cavalleri G.L. Stecconi R. Franceschi C. Altomare K. Dato S. Greco V. Luca Cavalli Sforza L. Underhill P.A. de Benedictis G. Hum. Mutat. 2003; 21: 554-555Crossref PubMed Scopus (28) Google Scholar) and Hsp90N, a 75-kDa Hsp90 that lacks the ansamycin-binding domain (41Grammatikakis N. Vultur A. Ramana C.V. Siganou A. Schweinfest C.W. Watson D.K. Raptis L. J. Biol. Chem. 2002; 277: 8312-8320Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). In addition, analysis of the NCBI protein data base reveals a 737-amino acid Hsp90 variant (NCBI accession number T46243) that is 99.6% identical to Hsp90-β over the first 709 residues but contains 13 additional amino acids within a divergent C-terminal tail. It is also possible that the Hsp90 that co-immunoprecipitates with GRK2 is post-translationally modified. However, treatment of the GRK2 immunoprecipitate with alkaline phosphatase did not change the mobility of the Hsp90 band, suggesting that it is not phosphorylated (not shown).Fig. 2Characterization of GRK2/Hsp90 interaction by immunoprecipitation.A, the interaction of GRK2 with Hsp90 was also demonstrated by immunoprecipitation of GRK2 from HL60 cell lysates using anti-GRK2/3 mAb and detection of Hsp90 using an anti-Hsp90 antibody as described under “Experimental Procedures.” No Hsp90 co-immunoprecipitated with the anti-GRK4-6 mAb (right lane). B, to further verify that Hsp90 co-immunoprecipitated with GRK2, HL60 cells were treated with the Hsp90-specific inhibitor geldanamycin for 2 h at 37 °C. The cells were then lysed, immunoprecipitated with anti-GRK2/3 mAb, and analyzed by SDS-PAGE and Western blotting (WB) using an anti-Hsp90 mAb. The blot was also stripped and probed for GRK2 using an anti-GRK2 mAb, whereas the lysate was blotted for total Hsp90 and GRK2. IP, immunoprecipitate.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Geldanamycin specifically binds to the ATP/ADP pocket of Hsp90 and effectively disrupts Hsp90 interaction with a number of proteins (42Young J.C. Moarefi I. Hartl F.U. J. Cell Biol. 2001; 154: 267-273Crossref PubMed Scopus (705) Google Scholar). As further evidence that Hsp90 interacts with GRK2, we incubated HL60 cells with 1 μm geldanamycin for 2 h at 37 °C. Geldanamycin treatment was found to effectively block Hsp90 association with GRK2 as assessed by GRK2 immunoprecipitation (Fig. 2B). Thus, endogenous GRK2 appears to specifically associate with Hsp90 in HL60 cells.Effect of Geldanamycin on the Expression, Maturation, and Degradation of Endogenous GRK2—An in vivo requirement for Hsp90 has been established for steroid hormone receptors (43Pratt W.B. Toft D.O. Endocr. Rev. 1997; 18: 306-360Crossref PubMed Scopus (1522) Google Scholar), various protein kinases such as v-Src, Wee-1, Cdk4, Raf, and Akt (27Sato S. Fujita N. Tsuruo T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10832-10837Crossref PubMed Scopus (826) Google Scholar, 44Stepanova L. Leng X. Parker S.B. Harper J.W. Genes Dev. 1996; 10: 1491-1502Crossref PubMed Scopus (443) Google Scholar, 45Csermely P. Schnaider T. Soti C. Prohaszka Z. Nardai G. Pharmacol. Ther. 1998; 79: 129-168Crossref PubMed Scopus (889) Google Scholar, 46Xu Y. Lindquist S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7074-7078Crossref PubMed Scopus (377) Google Scholar), and additional proteins such as nitric-oxide synthase (47Garcia-Cardena G. Fan R. Shah V. Sorrentino R. Cirino G. Papapetropoulos A. Sessa W.C. Nature. 1998; 392: 821-824Crossref PubMed Scopus (854) Google Scholar) and calcineurin (48Imai J. Yahara I. Mol. Cell. Biol. 2000; 20: 9262-9270Crossref PubMed Scopus (106) Google Scholar). Geldanamycin destabilizes the interaction between Hsp90 and its associated client proteins leading to accelerated degradation and loss of function. To assess the effect of Hsp90 interaction on GRK2 expression, HL60 cells were treated with various concentrations of geldanamycin for 24 h, and GRK2 levels were analyzed by immunoblotting. Geldanamycin induced down-regulation of GRK2 expression in a dose-dependent manner with 50% inhibition observed at ∼0.3 μm and ∼65% lower GRK2 levels at 10 μm geldanamycin (Fig. 3, A and B). This effect was time-dependent with 50% of the maximal reduction in GRK2 levels observed after an ∼8-h treatment with 5 μm geldanamycin (Fig. 3C). The reduction in GRK2 levels suggests that association of GRK2 with Hsp90 is required for GRK2 stability.Fig. 3Inhibition of GRK2 expression in HL60 cells by geldanamycin. HL60 cells were treated with 0, 0.08, 0.4, 2, or 10 μm geldanamycin for 24 h at 37 °C, and total GRK2 levels were then analyzed by Western blotting (WB) using an anti-GRK2 mAb. A, shows a representative immunoblot of GRK2 and α-tubulin expression after geldanamycin treatment. B, shows the relative levels of GRK2 expression from three independent experiments quantified by densitometry. C, shows the time course of geldanamycin treatment. HL60 cells were incubated with 5 μm geldanamycin for 0, 1, 3, 9, or 27 h, harvested, and immunoblotted for total GRK2 and reprobed for α-tubulin as described under “Experimental Procedures.”View Large Image Figure ViewerDownload Hi-res image Download (PPT)One important function for Hsp90 is to catalyze the proper folding of newly synthesized client proteins. In the absence of Hsp90 action, the protein may be subject to rapid degradation. Thus, a decline in the steady state level of GRK2 following geldanamycin treatment could result from changes in the rate of GRK2 synthesis and/or degradation. To characterize these processes, HL60 cells were metabolically labeled with [35S]methionine/cysteine for 30 min to label newly synthesized proteins, chased for 0, 2, 6, or 18 h with complete medium, and then analyzed for GRK2 levels by immunoprecipitation, SDS-PAGE, and autoradiography.To assess whether disrupting Hsp90/GRK2 interaction affects de novo GRK2 synthesis, geldanamycin was present during the entire pulse and chase periods (GA-1), whereas to assess whether Hsp90 affects GRK2 degradation, geldanamycin was present only during the chase period (GA-2). The half-life of GRK2 in HL60 cells in the absence of geldanamycin treatment was ∼20 h (Fig. 4), similar to our previous studies that reported a half-life of 24 h in HL60 cells (38Loudon R.P. Perussia B. Benovic J.L. Blood. 1996; 88: 4547-4557Crossref PubMed Google Scholar). When geldanamycin was present only during the chase period, the half-life of GRK2 was decreased to ∼12 h, suggesting that Hsp90 interaction with GRK2 has a relatively modest stabilizing effect on the rate of GRK2 degradation (Fig. 4). In contrast, when geldanamycin was present during the pulse and chase periods, there was a dramatic increase in the rate of GRK2 degradation, with the half-life of GRK2 decreasing to <2 h (Fig. 4). In addition, although a single protein band of ∼80 kDa was observed in the untreated and GA-2 samples, multiple protein bands were observed in the GA-1 samples. Our data also showed that degradation of GRK2 was mainly accelerated at the early chase times (Fig. 4B), suggesting that disruption of Hsp90 interaction during the synthesis of GRK2 results in proteolytic degradation of GRK2. Thus, the interaction of GRK2 and Hsp90 appears to inhibit proteolytic degradation of newly synthesized GRK2 and likely aids in GRK2 folding and maturation. It is interesting that the half-life for newly synthesized GRK2 in geldanamycin-treated HL60 cells (<2 h) is similar to the ∼1-h half-life reported by Penela et al. (11Penela P. Elorza A. Sarnago S. Mayor Jr., F. EMBO J. 2001; 20: 5129-5138Crossref PubMed Scopus (107) Google Scholar, 49Penela P. Ruiz-Gomez A. Castano J.G. Mayor Jr., F. J. Biol. Chem. 1998; 273: 35238-35244Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar) in untreated C6 glioma, Jurkat, and transfected HEK-293 cells. This may reflect differences in co- and/or post-translational processes between these cell types, or it might suggest that Hsp90 interaction with GRK2 is lacking in some cells.Fig. 4Pulse-chase analysis of GRK2 expression in HL60 cells.A, HL60 cells were incubated for 30 min at 37 °C with medium containing [35S]methionine and [35S]cysteine and then chased with nonradioactive medium for the indicated time. Geldanamycin (1 μm) was either not present (Untreated) or was present during the en" @default.
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• W2040372727 title "G Protein-coupled Receptor Kinase Interaction with Hsp90 Mediates Kinase Maturation" @default.
• W2040372727 cites W1514666013 @default.
• W2040372727 cites W153442697 @default.
• W2040372727 cites W1553826095 @default.
• W2040372727 cites W1886070575 @default.
• W2040372727 cites W1966434897 @default.
• W2040372727 cites W1966619635 @default.
• W2040372727 cites W1971113488 @default.
• W2040372727 cites W1976980728 @default.
• W2040372727 cites W1979197099 @default.
• W2040372727 cites W1983401747 @default.
• W2040372727 cites W1987674945 @default.
• W2040372727 cites W1999530386 @default.
• W2040372727 cites W2000097800 @default.
• W2040372727 cites W2004134377 @default.
• W2040372727 cites W2010849424 @default.
• W2040372727 cites W2015893386 @default.
• W2040372727 cites W2030205108 @default.
• W2040372727 cites W2032157282 @default.
• W2040372727 cites W2032597695 @default.
• W2040372727 cites W2032848541 @default.
• W2040372727 cites W2033999405 @default.
• W2040372727 cites W2049546697 @default.
• W2040372727 cites W2054712075 @default.
• W2040372727 cites W2057648252 @default.
• W2040372727 cites W2059308387 @default.
• W2040372727 cites W2059761731 @default.
• W2040372727 cites W2061248581 @default.
• W2040372727 cites W2064727002 @default.
• W2040372727 cites W2071440144 @default.
• W2040372727 cites W2074579328 @default.
• W2040372727 cites W2077322645 @default.
• W2040372727 cites W2077785228 @default.
• W2040372727 cites W2082390299 @default.
• W2040372727 cites W2085643470 @default.
• W2040372727 cites W2086617442 @default.
• W2040372727 cites W2087958744 @default.
• W2040372727 cites W2088938008 @default.
• W2040372727 cites W2091014584 @default.
• W2040372727 cites W2094065595 @default.
• W2040372727 cites W2097840941 @default.
• W2040372727 cites W2102417288 @default.
• W2040372727 cites W2108255072 @default.
• W2040372727 cites W2125468282 @default.
• W2040372727 cites W2133508703 @default.
• W2040372727 cites W2138281171 @default.
• W2040372727 cites W2153195868 @default.
• W2040372727 cites W2155331383 @default.
• W2040372727 cites W2160042367 @default.
• W2040372727 cites W2160364430 @default.
• W2040372727 cites W2175029576 @default.
• W2040372727 cites W2341277428 @default.
• W2040372727 cites W2398478012 @default.
• W2040372727 cites W2419438832 @default.
• W2040372727 cites W2614496215 @default.
• W2040372727 cites W4297631428 @default.
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