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• W2086248882 abstract "Hypoxia-inducible factor 1 (HIF-1) regulates transcription in response to changes in O2 concentration. O2-dependent degradation of the HIF-1α subunit is mediated by prolyl hydroxylase (PHD), the von Hippel-Lindau (VHL)/Elongin-C/Elongin-B E3 ubiquitin ligase complex, and the proteasome. Inhibition of heat-shock protein 90 (HSP90) leads to O2/PHD/VHL-independent degradation of HIF-1α. We have identified the receptor of activated protein kinase C (RACK1) as a HIF-1α-interacting protein that promotes PHD/VHL-independent proteasomal degradation of HIF-1α. RACK1 competes with HSP90 for binding to the PAS-A domain of HIF-1α in vitro and in human cells. HIF-1α degradation induced by the HSP90 inhibitor 17-allylaminogeldanamycin is abolished by RACK1 loss of function. RACK1 binds to Elongin-C and promotes ubiquitination of HIF-1α. Elongin-C-binding sites in RACK1 and VHL show significant sequence similarity. Thus, RACK1 is an essential component of an O2/PHD/VHL-independent mechanism for regulating HIF-1α stability through competition with HSP90 and recruitment of the Elongin-C/B ubiquitin ligase complex. Hypoxia-inducible factor 1 (HIF-1) regulates transcription in response to changes in O2 concentration. O2-dependent degradation of the HIF-1α subunit is mediated by prolyl hydroxylase (PHD), the von Hippel-Lindau (VHL)/Elongin-C/Elongin-B E3 ubiquitin ligase complex, and the proteasome. Inhibition of heat-shock protein 90 (HSP90) leads to O2/PHD/VHL-independent degradation of HIF-1α. We have identified the receptor of activated protein kinase C (RACK1) as a HIF-1α-interacting protein that promotes PHD/VHL-independent proteasomal degradation of HIF-1α. RACK1 competes with HSP90 for binding to the PAS-A domain of HIF-1α in vitro and in human cells. HIF-1α degradation induced by the HSP90 inhibitor 17-allylaminogeldanamycin is abolished by RACK1 loss of function. RACK1 binds to Elongin-C and promotes ubiquitination of HIF-1α. Elongin-C-binding sites in RACK1 and VHL show significant sequence similarity. Thus, RACK1 is an essential component of an O2/PHD/VHL-independent mechanism for regulating HIF-1α stability through competition with HSP90 and recruitment of the Elongin-C/B ubiquitin ligase complex. Oxygen homeostasis represents an essential organizing principle of metazoan evolution and biology. Hypoxia-inducible factor 1 (HIF-1) has been identified as a critical mediator of adaptive responses to reduced O2 availability in many developmental, physiological, and pathological contexts through its transcriptional regulation of genes that encode proteins required for tissue O2 delivery and energy metabolism (Hu et al., 2003Hu C.J. Wang L.Y. Chodosh L.A. Keith B. Simon M.C. Differential roles of hypoxia-inducible factor 1α (HIF-1α) and HIF-2α in hypoxic gene regulation.Mol. Cell. Biol. 2003; 23: 9361-9374Crossref PubMed Scopus (956) Google Scholar, Manalo et al., 2005Manalo D.J. Rowan A. Lavoie T. Natarajan L. Kelly B.D. Ye S.Q. Garcia J.G. Semenza G.L. Transcriptional regulation of vascular endothelial cell responses to hypoxia by HIF-1.Blood. 2005; 105: 659-669Crossref PubMed Scopus (814) Google Scholar, Elvidge et al., 2006Elvidge G.P. Glenny L. Appelhoff R.J. Ratcliffe P.J. Ragoussis J. Gleadle J.M. Concordant regulation of gene expression by hypoxia and 2-oxoglutarate-dependent dioxygenase inhibition: the role of HIF-1α, HIF-2α, and other pathways.J. Biol. Chem. 2006; 281: 15215-15226Crossref PubMed Scopus (334) Google Scholar). HIF-1 is required for embryonic development in mice (Iyer et al., 1998Iyer N.V. Kotch L.E. Agani F. Leung S.W. Laughner E. Wenger R.H. Gassmann M. Gearhart J.D. Lawler A.M. Yu A.Y. Semenza G.L. Cellular and developmental control of O2 homeostasis by hypoxia-inducible factor 1α.Genes Dev. 1998; 12: 149-162Crossref PubMed Scopus (1931) Google Scholar, Ryan et al., 1998Ryan H.E. Lo J. Johnson R.S. HIF-1α is required for solid tumor formation and embryonic vascularization.EMBO J. 1998; 17: 3005-3015Crossref PubMed Scopus (1285) Google Scholar) and plays key roles in ischemic cardiovascular disease, stroke, and cancer (Melillo, 2004Melillo G. HIF-1: a target for cancer, ischemia and inflammation–too good to be true?.Cell Cycle. 2004; 3: 154-155Crossref PubMed Google Scholar, Ran et al., 2005Ran R. Xu H. Lu A. Bernaudin M. Sharp F.R. Hypoxia preconditioning in the brain.Dev. Neurosci. 2005; 27: 87-92Crossref PubMed Scopus (127) Google Scholar, Moeller and Dewhirst, 2006Moeller B.J. Dewhirst M.W. HIF-1 and tumor radiosensitivity.Br. J. Cancer. 2006; 95: 1-5Crossref PubMed Scopus (143) Google Scholar). Delineation of the mechanisms that regulate HIF-1 activity in these contexts has become a major challenge of contemporary molecular and cell biology. HIF-1 is a heterodimeric transcription factor that consists of HIF-1α and HIF-1β subunits (Wang et al., 1995Wang G.L. Jiang B.H. Rue E.A. Semenza G.L. Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension.Proc. Natl. Acad. Sci. USA. 1995; 92: 5510-5514Crossref PubMed Scopus (4695) Google Scholar). The amino-terminal half of each subunit consists of basic-helix-loop-helix (bHLH) and PAS domains that mediate dimerization and DNA binding (Jiang et al., 1996aJiang B.H. Rue E. Wang G.L. Roe R. Semenza G.L. Dimerization, DNA binding, and transactivation properties of hypoxia-inducible factor 1.J. Biol. Chem. 1996; 271: 17771-17778Crossref PubMed Scopus (854) Google Scholar). HIF-1α protein is rapidly accumulated during hypoxia and is degraded under nonhypoxic conditions (Wang et al., 1995Wang G.L. Jiang B.H. Rue E.A. Semenza G.L. Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension.Proc. Natl. Acad. Sci. USA. 1995; 92: 5510-5514Crossref PubMed Scopus (4695) Google Scholar, Jiang et al., 1996bJiang B.H. Semenza G.L. Bauer C. Marti H.H. Hypoxia-inducible factor 1 levels vary exponentially over a physiologically relevant range of O2 tension.Am. J. Physiol. 1996; 271: C1172-C1180PubMed Google Scholar, Salceda and Caro, 1997Salceda S. Caro J. Hypoxia-inducible factor 1α (HIF-1α) protein is rapidly degraded by the ubiquitin-proteasome system under normoxic conditions. Its stabilization by hypoxia depends on redox-induced changes.J. Biol. Chem. 1997; 272: 22642-22647Crossref PubMed Scopus (1334) Google Scholar, Huang et al., 1998Huang L.E. Gu J. Schau M. Bunn H.F. Regulation of hypoxia-inducible factor 1α is mediated by an O2-dependent degradation domain via the ubiquitin-proteasome pathway.Proc. Natl. Acad. Sci. USA. 1998; 95: 7987-7992Crossref PubMed Scopus (1769) Google Scholar). The von Hippel-Lindau (VHL) protein binds both to HIF-1α and to Elongin-C, which, in turn, recruits Elongin-B and other subunits of an E3 ubiquitin ligase, thus targeting HIF-1α for ubiquitination and degradation by the 26S proteasome (Salceda and Caro, 1997Salceda S. Caro J. Hypoxia-inducible factor 1α (HIF-1α) protein is rapidly degraded by the ubiquitin-proteasome system under normoxic conditions. Its stabilization by hypoxia depends on redox-induced changes.J. Biol. Chem. 1997; 272: 22642-22647Crossref PubMed Scopus (1334) Google Scholar, Maxwell et al., 1999Maxwell P.H. Wiesener M.S. Chang G.W. Clifford S.C. Vaux E.C. Cockman M.E. Wykoff C.C. Pugh C.W. Maher E.R. Ratcliffe P.J. The tumor suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis.Nature. 1999; 399: 271-275Crossref PubMed Scopus (3858) Google Scholar). Hydroxylation of the 826-amino-acid human HIF-1α protein at proline residue 402 and/or 564 is required for VHL binding and subsequent degradation (Ivan et al., 2001Ivan M. Kondo K. Yang H. Kim W. Valiando J. Ohh M. Salic A. Asara J.M. Lane W.S. Kaelin Jr., W.G. HIFα targeted for VHL-mediated destruction by proline hydroxylation: implications for O2 sensing.Science. 2001; 292: 464-468Crossref PubMed Scopus (3596) Google Scholar, Jaakkola et al., 2001Jaakkola P. Mole D.R. Tian Y.M. Wilson M.I. Gielbert J. Gaskell S.J. Kriegsheim A. Hebestreit H.F. Mukherji M. Schofield C.J. et al.Targeting of HIF-α to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation.Science. 2001; 292: 468-472Crossref PubMed Scopus (4125) Google Scholar, Yu et al., 2001Yu F. White S.B. Zhao Q. Lee F.S. HIF-1α binding to VHL is regulated by stimulus-sensitive proline hydroxylation.Proc. Natl. Acad. Sci. USA. 2001; 98: 9630-9635Crossref PubMed Scopus (614) Google Scholar). Three prolyl hydroxylases (PHD1-3) were identified in mammalian cells and shown to utilize O2 and α-ketoglutarate as substrates to generate 4-hydroxyproline at P402 and/or P564 of HIF-1α (Bruick and McKnight, 2001Bruick R.K. McKnight S.L. A conserved family of prolyl-4-hydroxylases that modify HIF.Science. 2001; 294: 1337-1340Crossref PubMed Scopus (1989) Google Scholar, Epstein et al., 2001Epstein A.C. Gleadle J.M. McNeill L.A. Hewitson K.S. O'Rourke J. Mole D.R. Mukherji M. Metzen E. Wilson M.I. Dhanda A. et al.C. elegans EGL-9 and mammalian homologs define a family of dioxygenases that regulate HIF by prolyl hydroxylation.Cell. 2001; 107: 43-54Abstract Full Text Full Text PDF PubMed Scopus (2432) Google Scholar). The HIF-1α transactivation domain is regulated by the binding of FIH-1 (factor inhibiting HIF-1 [Mahon et al., 2001Mahon P.C. Hirota K. Semenza G.L. FIH-1: a novel protein that interacts with HIF-1α and VHL to mediate repression of HIF-1 transcriptional activity.Genes Dev. 2001; 15: 2675-2686Crossref PubMed Scopus (1036) Google Scholar]), which hydroxylates asparagine 803 to block binding of the coactivators CBP and p300 (Lando et al., 2002Lando D. Peet D.J. Gorman J.J. Whelan D.A. Whitelaw M.L. Bruick R.K. FIH-1 is an asparaginyl hydroxylase enzyme that regulates the transcriptional activity of hypoxia-inducible factor.Genes Dev. 2002; 16: 1466-1471Crossref PubMed Scopus (1115) Google Scholar). HIF-2α, which is expressed in a more restricted distribution of cell types than HIF-1α, also heterodimerizes with HIF-1β and is regulated by O2-dependent asparagine and proline hydroxylation. The levels of HIF-1α protein in normoxic tissues vary widely (Stroka et al., 2001Stroka D.M. Burkhardt T. Desbaillets I. Wenger R.H. Neil D.A. Bauer C. Gassmann M. Candinas D. HIF-1 is expressed in normoxic tissue and displays an organ-specific regulation under systemic hypoxia.FASEB J. 2001; 15: 2445-2453Crossref PubMed Scopus (523) Google Scholar), but the molecular basis for this regulation is unknown. HIF-1α degradation is regulated in an O2-independent manner by heat-shock protein 90 (HSP90), a molecular chaperone that protects client proteins from misfolding and degradation (Neckers and Ivy, 2003Neckers L. Ivy S.P. Heat shock protein 90.Curr. Opin. Oncol. 2003; 15: 419-424Crossref PubMed Scopus (356) Google Scholar, Whitesell and Lindquist, 2005Whitesell L. Lindquist S.L. HSP90 and the chaperoning of cancer.Nat. Rev. Cancer. 2005; 5: 761-772Crossref PubMed Scopus (1832) Google Scholar). HSP90 binds to the HIF-1α PAS domain, and the HSP90 inhibitors geldanamycin and 17-allylaminogeldanamycin (17-AAG) induce proteasomal degradation of HIF-1α even in renal carcinoma cells that lack functional VHL (Gradin et al., 1996Gradin K. McGuire J. Wenger R.H. Kvietkova I. Whitelaw M.L. Toftgard R. Tora L. Gassmann M. Poellinger L. Functional interference between hypoxia and dioxin signal transduction pathways: competition for recruitment of the Arnt transcription factor.Mol. Cell. Biol. 1996; 16: 5221-5231Crossref PubMed Scopus (364) Google Scholar, Isaacs et al., 2002Isaacs J.S. Jung Y.J. Mimnaugh E.G. Martinez A. Cuttitta F. Neckers L.M. Hsp90 regulates a von Hippel Lindau-independent hypoxia-inducible factor-1α-degradative pathway.J. Biol. Chem. 2002; 277: 29936-29944Crossref PubMed Scopus (572) Google Scholar, Isaacs et al., 2004Isaacs J.S. Jung Y.J. Neckers L. Aryl hydrocarbon nuclear translocator (ARNT) promotes oxygen-independent stabilization of hypoxia-inducible factor-1α by modulating an Hsp90-dependent regulatory pathway.J. Biol. Chem. 2004; 279: 16128-16135Crossref PubMed Scopus (71) Google Scholar, Mabjeesh et al., 2002Mabjeesh N.J. Post D.E. Willard M.T. Kaur B. Van Meir E.G. Simons J.W. Zhong H. Geldanamycin induces degradation of hypoxia-inducible factor 1α protein via the proteosome pathway in prostate cancer cells.Cancer Res. 2002; 62: 2478-2482PubMed Google Scholar). In the present study, we identified the receptor for activated C-kinase 1 (RACK1) as a HIF-1α-interacting protein through a proteomics-based screen. RACK1 was originally identified as an anchoring protein for activated protein kinase C (PKC) (Ron et al., 1994Ron D. Chen C.H. Caldwell J. Jamieson L. Orr E. Mochly-Rosen D. Cloning of an intracellular receptor for protein kinase C: a homolog of the β subunit of G proteins.Proc. Natl. Acad. Sci. USA. 1994; 91: 839-843Crossref PubMed Scopus (627) Google Scholar). However, RACK1 is now recognized as a multifunctional scaffold protein that plays an important role in diverse biological processes, including intracellular signal transduction (McCahill et al., 2002McCahill A. Warwicker J. Bolger G.B. Houslay M.D. Yarwood S.J. The RACK1 scaffold protein: a dynamic cog in cell response mechanisms.Mol. Pharmacol. 2002; 62: 1261-1273Crossref PubMed Scopus (322) Google Scholar) and assembly of the 80S ribosome from 40S and 60S subunits (Ceci et al., 2003Ceci M. Gaviraghi C. Gorrini C. Sala L.A. Offenhauser N. Marchisio P.C. Biffo S. Release of eIF6 (p27BBP) from the 60S subunit allows 80S ribosome assembly.Nature. 2003; 426: 579-584Crossref PubMed Scopus (298) Google Scholar). Here, we demonstrate that RACK1 competes with HSP90 for binding to HIF-1α, links HIF-1α to Elongin-C, and promotes HIF-1α degradation. We utilized a proteomics-based approach to identify proteins that bind to HIF-1α. HEK293 cell lysates were passed over a column containing Sepharose-4B covalently linked to glutathione S-transferase (GST) or a fusion protein consisting of GST and residues 531–826 of human HIF-1α (GST-HIF-1α [531–826]). Proteins that bound to GST or GST-HIF-1α (531–826) were eluted and labeled with the fluorescent dyes Cy3 and Cy5, respectively (Figure 1A). The labeled proteins were pooled and fractionated by two-dimensional SDS-PAGE. The gel was scanned, 527 fluorescent spots were identified, and their Cy5/Cy3 ratios were calculated. A Cy5/Cy3 ratio ≥ 2.5 was chosen to distinguish specific binding to HIF-1α from nonspecific binding to GST. Two spots that met this criterion were excised from the gel (Figure 1B). Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry was performed, and the data were analyzed by using a mass fingerprint search engine to query the NCBI Protein Database, which identified the two proteins as FIH-1 and RACK1 (Figure 1C). The identification of FIH-1, an established HIF-1α-interacting protein (Mahon et al., 2001Mahon P.C. Hirota K. Semenza G.L. FIH-1: a novel protein that interacts with HIF-1α and VHL to mediate repression of HIF-1 transcriptional activity.Genes Dev. 2001; 15: 2675-2686Crossref PubMed Scopus (1036) Google Scholar), validated this approach. In contrast to FIH-1, RACK1 was not previously known to interact with HIF-1α. To confirm the proteomics result, we performed in vitro binding assays. In vitro-transcribed and -translated RACK1 bound to GST-HIF-1α (531–826), but not to GST alone (Figure 1D). GST-HIF-1α (531–826) also bound to endogenous RACK1 from HEK293 cell lysates (Figure 1E). Coimmunoprecipitation (CoIP) of RACK1 and endogenous HIF-1α from desferrioxamine-treated cells (Figure 1F) demonstrated that RACK1 also interacts with HIF-1α in human cells. To investigate the functional consequences of RACK1 binding to HIF-1α, HEK293T cells were cotransfected with vectors encoding FLAG-tagged, full-length HIF-1α and T7-tagged RACK1 (T7-RACK1). FLAG-HIF-1α protein levels decreased in a dose-dependent manner as T7-RACK1 protein levels were increased (Figure 2A). The anti-RACK1 antibody (Ab) detected both T7-RACK1 and endogenous RACK1, which also served as a loading control. Transfection of the T7-RACK1 vector markedly decreased the levels of endogenous HIF-1α protein induced by exposure of HEK293T cells to 1% O2 (Figure 2B). HIF-2α protein levels were also decreased by T7-RACK1 under both 20% and 1% O2 (Figure 2C). HIF-1α is degraded in an O2-dependent manner through the activity of PHD2, which hydroxylates proline residues 402 and 564 (Bruick and McKnight, 2001Bruick R.K. McKnight S.L. A conserved family of prolyl-4-hydroxylases that modify HIF.Science. 2001; 294: 1337-1340Crossref PubMed Scopus (1989) Google Scholar, Epstein et al., 2001Epstein A.C. Gleadle J.M. McNeill L.A. Hewitson K.S. O'Rourke J. Mole D.R. Mukherji M. Metzen E. Wilson M.I. Dhanda A. et al.C. elegans EGL-9 and mammalian homologs define a family of dioxygenases that regulate HIF by prolyl hydroxylation.Cell. 2001; 107: 43-54Abstract Full Text Full Text PDF PubMed Scopus (2432) Google Scholar, Berra et al., 2003Berra E. Benizri E. Ginouves A. Volmat V. Roux D. Pouyssegur J. HIF prolyl-hydroxylase 2 is the key oxygen sensor setting low steady-state levels of HIF-1α in normoxia.EMBO J. 2003; 22: 4082-4090Crossref PubMed Scopus (1006) Google Scholar), and VHL, which binds to hydroxylated HIF-1α and promotes its ubiquitination and subsequent proteasomal degradation (Maxwell et al., 1999Maxwell P.H. Wiesener M.S. Chang G.W. Clifford S.C. Vaux E.C. Cockman M.E. Wykoff C.C. Pugh C.W. Maher E.R. Ratcliffe P.J. The tumor suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis.Nature. 1999; 399: 271-275Crossref PubMed Scopus (3858) Google Scholar, Tanimoto et al., 2000Tanimoto K. Makino Y. Pereira T. Poellinger L. Mechanism of regulation of the hypoxia-inducible factor-1α by the von Hippel-Lindau tumor suppressor protein.EMBO J. 2000; 19: 4298-4309Crossref PubMed Google Scholar). RCC4 renal carcinoma cells lack functional VHL and have high endogenous HIF-1α protein levels (Maxwell et al., 1999Maxwell P.H. Wiesener M.S. Chang G.W. Clifford S.C. Vaux E.C. Cockman M.E. Wykoff C.C. Pugh C.W. Maher E.R. Ratcliffe P.J. The tumor suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis.Nature. 1999; 399: 271-275Crossref PubMed Scopus (3858) Google Scholar, Hu et al., 2003Hu C.J. Wang L.Y. Chodosh L.A. Keith B. Simon M.C. Differential roles of hypoxia-inducible factor 1α (HIF-1α) and HIF-2α in hypoxic gene regulation.Mol. Cell. Biol. 2003; 23: 9361-9374Crossref PubMed Scopus (956) Google Scholar). In RCC4 cells that were transduced with a retrovirus encoding RACK1, HIF-1α protein levels were greatly decreased compared to levels in cells transduced with a retrovirus encoding GFP or uninfected parental cells (Figure 2D). To analyze the effect of RACK1 on HIF-1 transcriptional activity, cells were cotransfected with FLAG-HIF-1α expression vector and p2.1, a reporter plasmid that contains a hypoxia-response element (HRE) upstream of both the SV40 promoter- and firefly luciferase-coding sequences (Semenza et al., 1996Semenza G.L. Jiang B.H. Leung S.W. Passantino R. Concordet J.-P. Maire P. Giallongo A. Hypoxia response elements in the aldolase A, enolase 1, and lactate dehydrogenase A gene promoters contain essential binding sites for hypoxia-inducible factor 1.J. Biol. Chem. 1996; 271: 32529-32537Crossref PubMed Scopus (1246) Google Scholar). Transcriptional activity mediated by FLAG-HIF-1α was also inhibited in a dose-dependent manner by cotransfection of the T7-RACK1 vector (Figure 2E, middle columns). Therefore, RACK1 reduces HIF-1α protein levels and, by doing so, reduces HIF-1 transcriptional activity. Full-length HIF-1α protein containing the proline-to-alanine substitutions P402A/P564A is resistant to PHD2-mediated hydroxylation and VHL-mediated ubiquitination/degradation. Similar to its effect on wild-type FLAG-HIF-1α (Figure 2F, lanes 2 and 3), RACK1 decreased FLAG-HIF-1α (P402A/P564A) protein levels (Figure 2F, lanes 6 and 7) and transcriptional activity mediated by the double mutant protein (Figure 2E, right columns). In contrast, PHD2 inhibited transcriptional activity mediated by wild-type FLAG-HIF-1α, but not by FLAG-HIF-1α (P402A/P564A) (Figure 2E). Expression vector encoding FLAG-tagged bacterial alkaline phosphatase was also transfected into the cells, and the levels of FLAG-BAP were not affected by RACK1 (Figure 2F, middle). FLAG-BAP- and FLAG-HIF-1α-coding sequences were inserted into the same plasmid backbone to control for any potential effects of RACK1 on transcription or translation of the tagged proteins, demonstrating that RACK1 specifically induces the degradation of FLAG-HIF-1α. In support of this conclusion, RACK1-induced degradation of FLAG-HIF-1α was blocked by addition of the proteasome inhibitor MG132 (Figure 2F, compare lanes 3 and 5). Taken together, the data shown in Figure 2 demonstrate that RACK1 induces degradation of HIF-1α that is independent of prolyl hydroxylation and VHL function, but dependent on proteasome activity. To complement our studies investigating the effect of RACK1 gain of function, we also performed an analysis of RACK1 loss of function by expressing a short hairpin RNA (shRNA) directed against RACK1 or a scrambled negative control (SNC) shRNA. In HEK293T cells, shRNA-RACK1 decreased levels of RACK1 mRNA (Figures 3E and 3F) and protein (Figures 3A and 3C). shRNA-RACK1 increased FLAG-HIF-1α protein levels in a dose-dependent manner, and there was a remarkable negative correlation between the levels of RACK1 and FLAG-HIF-1α in multiple experiments (Figure 3A and data not shown). shRNA-RACK1 also increased endogenous HIF-1α protein levels at both 20% and 1% O2 (Figure 3C). Transcription of the HRE reporter gene mediated by overexpressed FLAG-HIF-1α (Figure 3B) or endogenous HIF-1α (Figure 3D) was increased in cells cotransfected with shRNA-RACK1 at both 20% and 1% O2. HIF-1α mRNA levels were not affected by shRNA-RACK1, whereas the levels of mRNAs encoded by the HIF-1 target genes VEGF and GLUT1 were increased by shRNA-RACK1, but not by shRNA-SNC or empty vector, as determined by conventional RT-PCR (Figure 3E) and quantitative real-time RT-PCR (Figure 3F). The data demonstrate that RACK1 loss of function increases endogenous HIF-1α protein levels and the expression of HIF-1 target genes. RACK1 was originally identified as a platform to bring together activated PKC and its substrates (Ron et al., 1994Ron D. Chen C.H. Caldwell J. Jamieson L. Orr E. Mochly-Rosen D. Cloning of an intracellular receptor for protein kinase C: a homolog of the β subunit of G proteins.Proc. Natl. Acad. Sci. USA. 1994; 91: 839-843Crossref PubMed Scopus (627) Google Scholar, McCahill et al., 2002McCahill A. Warwicker J. Bolger G.B. Houslay M.D. Yarwood S.J. The RACK1 scaffold protein: a dynamic cog in cell response mechanisms.Mol. Pharmacol. 2002; 62: 1261-1273Crossref PubMed Scopus (322) Google Scholar). To investigate whether PKC was involved in the RACK1-mediated inhibition of HIF-1α, we used RO-32-0432, which inhibits all known PKC isoforms with an IC50 ≤ 0.1 μM (Wilkinson et al., 1993Wilkinson S.E. Parker P.J. Nixon J.S. Isoenzyme specificity of bisindolylmaleimides, selective inhibitors of protein kinase C.Biochem. J. 1993; 294: 335-337Crossref PubMed Scopus (483) Google Scholar). Exposure of HEK293T cells to 5 μM RO-32-0432 had no effect on the RACK1-mediated degradation of HIF-1α protein (data not shown). Although PKC-ɛ was reported to interact with HIF-1α in the mouse heart (Ping et al., 2001Ping P. Zhang J. Pierce Jr., W.M. Bolli R. Functional proteomic analysis of protein kinase C ɛ signaling complexes in the normal heart and during cardioprotection.Circ. Res. 2001; 88: 59-62Crossref PubMed Scopus (181) Google Scholar), we found that expression of constitutively active PKC-ɛ had no effect on HIF-1α protein levels and HIF-1 transcriptional activity in HEK293T cells (data not shown). Exposure of cells to O2 induces PHD/VHL-dependent degradation of HIF-1α. In contrast, the HSP90 inhibitors geldanamycin and 17-AAG induce degradation of HIF-1α that is independent of PHD/VHL activity. HSP90 binding stabilizes HIF-1α, and disruption of the HSP90-HIF-1α interaction by geldanamycin or 17-AAG results in HIF-1α degradation even in cells without functional VHL (Isaacs et al., 2002Isaacs J.S. Jung Y.J. Mimnaugh E.G. Martinez A. Cuttitta F. Neckers L.M. Hsp90 regulates a von Hippel Lindau-independent hypoxia-inducible factor-1α-degradative pathway.J. Biol. Chem. 2002; 277: 29936-29944Crossref PubMed Scopus (572) Google Scholar, Isaacs et al., 2004Isaacs J.S. Jung Y.J. Neckers L. Aryl hydrocarbon nuclear translocator (ARNT) promotes oxygen-independent stabilization of hypoxia-inducible factor-1α by modulating an Hsp90-dependent regulatory pathway.J. Biol. Chem. 2004; 279: 16128-16135Crossref PubMed Scopus (71) Google Scholar, Mabjeesh et al., 2002Mabjeesh N.J. Post D.E. Willard M.T. Kaur B. Van Meir E.G. Simons J.W. Zhong H. Geldanamycin induces degradation of hypoxia-inducible factor 1α protein via the proteosome pathway in prostate cancer cells.Cancer Res. 2002; 62: 2478-2482PubMed Google Scholar). We analyzed purified GST-HIF-1α fusion proteins that contain different domains of HIF-1α for their ability to bind to endogenous RACK1 and HSP90 present in HEK293T cell lysates. GST-HIF-1α (1–329) bound strongly to both HSP90 and RACK1, whereas GST-HIF-1α (429–608) bound to neither HSP90 nor RACK1; both GST-HIF-1α (575–786) and (786–826) bound weakly to RACK1 (Figure 4A). Thus, although our initial identification of RACK1 was based on its interaction with HIF-1α residues 531–826, RACK1 shows its strongest interaction with residues 1–329. GST-RACK1 did not bind to HSP90 (Figure 4B, lane 1), but it inhibited the binding of HSP90 to GST-HIF-1α (1–329) in a dose-dependent manner (lanes 2–4). If RACK1 competes with HSP90 for binding to GST-HIF-1α (1–329), then a truncated form of HIF-1α containing only residues 1–329 may be subject to RACK1-mediated degradation. T7-RACK1 did in fact decrease FLAG-HIF-1α (1–329) protein levels in cotransfected cells (Figure 4C, compare lanes 2 and 3). The proteasome inhibitor MG132 blocked RACK1-mediated degradation of FLAG-HIF-1α (1–329) (compare lanes 3 and 5). The levels of FLAG-HIF-1α (1–329) protein were not regulated in response to changes in the cellular O2 concentration (lanes 1 and 2), which is mediated through HIF-1α residues 400–600 (Huang et al., 1998Huang L.E. Gu J. Schau M. Bunn H.F. Regulation of hypoxia-inducible factor 1α is mediated by an O2-dependent degradation domain via the ubiquitin-proteasome pathway.Proc. Natl. Acad. Sci. USA. 1998; 95: 7987-7992Crossref PubMed Scopus (1769) Google Scholar). Thus, RACK1 competes with HSP90 for binding to HIF-1α (1–329), and this competition is sufficient to destabilize HIF-1α in an O2-independent manner. To further investigate the competition between RACK1 and HSP90, we divided GST-HIF-1α (1–329) into smaller domains and studied their binding. HIF-1α residues 81–200 and 81–329 bound to both RACK1 and HSP90, whereas residues 1–80 and 201–329 bound to neither RACK1 nor HSP90 (Figure 4D). Thus, residues 81–200 represent the minimal sequence analyzed that binds to RACK1 and HSP90. GST-HIF-1α (81–200) bound to HSP90 from HEK293T cell lysates, and the addition of GST-RACK1 decreased HSP90 binding in a dose-dependent manner (Figure 4E). Reciprocally, the addition of GST-HSP90 dose dependently decreased RACK1 binding to GST-HIF-1α (81–200) (Figure 4F). These data indicate that RACK1 and HSP90 compete for binding to residues 81–200, which encompass the PAS-A subdomain of HIF-1α (Wang et al., 1995Wang G.L. Jiang B.H. Rue E.A. Semenza G.L. Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension.Proc. Natl. Acad. Sci. USA. 1995; 92: 5510-5514Crossref PubMed Scopus (4695) Google Scholar). To determine whether the competitive binding of RACK1 and HSP90 to HIF-1α was relevant to the mechanism of action of 17-AAG, we analyzed protein-protein interactions in 17-AAG-treated cells. The 17-AAG-induced degradation of FLAG-HIF-1α (P402A/P564A) was blocked by the proteasome inhibitor MG132 (Figure 5A, left). Although FLAG-HIF-1α (P402A/P564A) levels were not affected by 17-AAG in the presence of MG132, CoIP of HSP90 by anti-FLAG Ab was decreased in 17-AAG-treated cells, and CoIP of RACK1 was increased (Figure 5A, right). Decreased HSP90 and increased RACK1 binding to HIF-1α were observed when 17-AAG was added directly to cell lysates in vitro, followed by IP of endogenous HIF-1α (Figure 5B). The increased interaction of RACK1 and HIF-1α in the presence of 17-AAG may simply reflect the loss of competition by HSP90 for binding to HIF-1α. Alternatively, RACK1 binding may be required for HIF-1α degradation in 17-AAG-treated cells. To distinguish between these models, HEK293T cells were cotransfected with FLAG-HIF-1α (P402A/P564A) and shRNA-RACK1 or shRNA-SNC. 17-AAG decreased FLAG-HIF-1α (P402A/P564A), but not HIF-1β or β-actin, protein levels when shRNA-SNC was used (Figure 5C, lane 2). However, the degradation of FLAG-HIF-1α (P402A/P564A) by 17-AAG was abolished in the presence of shRNA-RACK1 (lane 4). These results indicate that the mechanism of action of 17-AAG is dependent on RACK1 activity. To delineate the mechanism of RACK1-mediated degradation of HIF-1α, we investigated the connection between RACK1 and HIF-1α E3 ubiquitin ligase. VHL is the substrate-recognition subunit that recruits HIF-1α to Elongin-C/B (Kamura et al., 2000Kamura T. Sato S. Iwai K. Czyzyk-Krzeska M. Conaway R.C. Conaway J.W. Activation of HIF-1α ubiquitination by a reconstituted von Hippel-Lindau (VHL) tumor suppressor complex.Proc. Natl. Acad. Sci. USA. 2000; 97: 10430-10435Crossref PubMed" @default.
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• W2086248882 title "RACK1 Competes with HSP90 for Binding to HIF-1α and Is Required for O2-Independent and HSP90 Inhibitor-Induced Degradation of HIF-1α" @default.
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