von Hippel-Lindau protein binds hyperphosphorylated large subunit of RNA polymerase II through a proline hydroxylation motif and targets it for ubiquitination

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von Hippel-Lindau protein binds hyperphosphorylated large subunit of RNA polymerase II through a proline hydroxylation motif and targets it for ubiquitination
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  von Hippel–Lindau protein binds hyperphosphorylatedlarge subunit of RNA polymerase II through a prolinehydroxylation motif and targets it for ubiquitination Anna V. Kuznetsova* † , Jaroslaw Meller †‡§ , Phillip O. Schnell*, James A. Nash*, Monika L. Ignacak*, Yolanda Sanchez ¶ ,Joan W. Conaway  **, Ronald C. Conaway  **, and Maria F. Czyzyk-Krzeska* †† *Department of Molecular and Cellular Physiology,  ‡ Pediatric Informatics, Children’s Hospital Research Foundation,  ¶ Department of Molecular Genetics,University of Cincinnati College of Medicine, Cincinnati, OH 45267-0576;  § Department of Informatics, Nicholas Copernicus University, 87-100, Torun,Poland;   Stowers Institute for Medical Research, Kansas City, MO 64110; and **Department of Biochemistry and Molecular Biology, University ofKansas Medical Center, Kansas City, KS 66160Edited by Robert G. Roeder, The Rockefeller University, New York, NY, and approved January 7, 2003 (received for review October 7, 2002) The transition from transcription initiation to elongation involvesphosphorylation of the large subunit (Rpb1) of RNA polymerase IIon the repetitive carboxyl-terminal domain. The elongating hyper-phosphorylated Rpb1 is subject to ubiquitination, particularly inresponse to UV radiation and DNA-damaging agents. By usingcomputer modeling, we identified regions of Rpb1 and the adja-centsubunit6ofRNApolymeraseII(Rpb6)thatsharesequenceandstructural similarity with the domain of hypoxia-inducible tran-scription factor 1   (HIF-1  ) that binds von Hippel–Lindau tumorsuppressor protein (pVHL). pVHL confers substrate specificity tothe E3 ligase complex, which ubiquitinates HIF-  and targets it forproteasomal degradation. In agreement with the computationalmodel, we show biochemical evidence that pVHL specificallybinds the hyperphosphorylated Rpb1 in a proline-hydroxylation-dependentmanner,targetingitforubiquitination.Thisinteractionis regulated by UV radiation. T he von Hippel–Lindau tumor suppressor protein (pVHL)-associated complex, which contains elongin B, elongin C,cullin-2, and Rbx-1 (1–3) is a primary ubiquitin ligase forubiquitination of the    subunits of the hypoxia-inducible tran-scription factors (HIFs) (4–6). During normoxia, translatedHIF-  s are hydroxylated on conserved proline residues located within L(XY)LAP motifs by the O 2 , Fe(II), and 2-oxoglutarate-regulated Egl-9 family of prolyl hydroxylases (7, 8), resulting intheir ubiquitination and degradation. During hypoxia, prolinehydroxylation is inhibited; HIF-  s are not ubiquitinated, andthey accumulate and regulate transcription of the HIF-responsive genes (4–6, 9–12). Loss of pVHL function in VHL disease leads to the accumulation of HIF-  s during normoxicconditions, causing constitutive induction of HIF-responsivegenes, including angiogenic vascular endothelial growth factor(VEGF) (13, 14). This functioning, in turn, contributes to theformation of highly vascular tumors such as hemangioblastomas,angiomas, and renal clear cell carcinomas (RCCs) (15). von Hippel–Lindau disease is also associated with pheochro-mocytomas, nonmalignant tumors of adrenal medulla chromaf-fin cells, which synthesize and release large quantities of cat-echolamines and produce cardiovascular pathologies (16, 17).The molecular mechanism of the augmented catecholamineproduction is unknown. Recently, we presented evidence thatpVHL regulates expression of the rate-limiting enzyme in cat-echolamine biosynthesis, tyrosine hydroxylase (TH), and inpheochromocytoma-derived (PC12) cells (18, 19). Low levels of pVHL, resulting from expression of   VHL  antisense RNA, cor-relate with more efficient transcription of the full-length  TH  transcripts (19). In contrast, high levels of overexpressed pVHL blocktranscriptelongationbetweenexons6and8ofthe TH  gene(18). The presence of the elongation arrest site within this regionof the  TH   gene has been confirmed by using  in vitro  transcrip-tional analysis (20).Processive elongation of the initiated transcripts involvesreversible hyperphosphorylation of tandemly repeated hep-tapeptides on the carboxyl-terminal domain (CTD) of subunit 1of RNA polymerase II (Rpb1) within the RNA polymerase IIcomplex (21). This elongation-competent, hyperphosphorylatedRpb1 is ubiquitinated in a transcription-dependent manner (22,23). In particular, ubiquitination of the hyperphosphorylatedRpb1 is induced by UV radiation and DNA damage (24–26),suggesting that Rpb1 ubiquitination may play a role in thetranscription-coupled repair (27). In yeast, ubiquitination ismediated by a HECT-class Rsp5 ubiquitin ligase (28); however,the nature of the E3 ligase in mammalian cells is unknown. Wehypothesized that the hyperphosphorylated Rpb1 may be asubstrate for pVHL-associated E3 ubiquitin-ligase activity.Here,weidentifyaregionoftheRpb1  Rpb6subunitsofRNA polymerase II that shares sequence and structural similarity withthe pVHL binding domain of HIF-1  , and show that thepVHL-associated complex interacts specifically with the hyper-phosphorylated Rpb1, leading to its ubiquitination. Materials and Methods Cell Cultures and Reagents.  PC12 cell clones (18, 19) and 786-ORCC cells were described (1), and were used at the cell densityof 1.5–2.5    10 5 per cm 2 . UV irradiations (15 J  m 2 ) wereperformed in a UV Crosslinker (FB-UVXL-1000, Fisher Bio-tech, Pittsburgh).  N  -Cbz- L  -Leu- L  -Leu- L  -norvalinal (CbzLLn; 10  M) was added 30 min before UV irradiation. This medium wasremoved immediately before the irradiation and the same me-dium was returned after irradiation.Iron chloride, cobalt chloride, zinc chloride, desferrioxamine,2,2  -dipyridyl, ascorbic acid, 2-oxoglutarate, and CbzLLn werepurchased from Sigma. Reagents used in ubiquitination reaction were purchased from Boston Biochem (Boston) or AffinitiResearch Products (Hamhead, Exeter, Devon, U.K.). Thefollowing antibodies were used as follows: H14 (ResearchDiagnostics, Flanders, NJ); C21 and anti-cullin-2 (Santa CruzBiotechnology); Ig32 anti-pVHL (PharMingen); 12CA5 anti-hemagglutinin (HA) (Roche Molecular Biochemicals);anti-Rbx1 (Zymed); anti-elongin C (Signal Transduction, Lex-ington, KY); anti-elongin B polyclonal (custom made by Alpha This paper was submitted directly (Track II) to the PNAS office.Abbreviations: pVHL, von Hippel–Lindau protein; TH, tyrosine hydroxylase; PC12,phoechromocytoma cell line; CTD, carboxyl-terminal domain; Rpb1 or Rpb6, subunit 1or 6 of RNA polymerase II; HA, hemagglutinin; CbzLLn,  N  -Cbz- L -Leu- L -Leu- L -norvalinal;HIF, hypoxia-inducible factor; RCC, renal cell carcinoma; ODDD, oxygen-dependentdegradation domain. † A.V.K. and J.M. contributed equally to this work. †† Towhomcorrespondenceshouldbeaddressedat:DepartmentofMolecularandCellularPhysiology, University of Cincinnati College of Medicine, P.O. Box 670576, Cincinnati,OH 45267-0576. E-mail: Maria.Czyzykkrzeska@uc.edu. 2706–2711    PNAS    March 4, 2003    vol. 100    no. 5 www.pnas.org  cgi  doi  10.1073  pnas.0436037100  Diagnostic, San Antonio, TX); anti-HIF-2   (Novus Biologicals,Littleton, CO); anti-ubiquitin (StressGen Biotechnologies, Vic-toria, Canada); and mouse anti-rabbit IgG (clone RG-96, Sig-ma). Anti-mouse secondary antibody-bound agarose was fromSigma. Synthetic biotinylated peptides were made by AlphaDiagnostic. In Vitro   pVHL-Peptide Binding Reaction.  Ten micrograms of biotin- ylated peptide was incubated with streptavidin-coated Dyna-beads (M-280, Dynal, Great Neck, NY) in a buffer (25   l)containing 20 mM Tris at pH 8, 100 mM NaCl, 0.5% NonidetP-40, and 1 mM EDTA for 1 h at room temperature. Washedbeads were incubated with WT [pRC-cytomegalovirus (CMV)expression vector; Invitrogen] or mutated pVHL (pCI-neo-CMV expression vector; Promega), translated  in vitro  byusing [ 35 S]methionine and TNT reticulocyte lysate (Promega).Binding reaction products were washed extensively in the samebuffer and analyzed for bound [ 35 S]pVHL by using SDS  PAGE.Forthepeptidehydroxylationstep,immobilizedpeptidewasfirstincubated in the hypotonically prepared cellular extract fromPC12 cells as described below in the presence of 100  M each of FeCl 2 , ascorbic acid, and 2-oxoglutarate for 1 – 2 h at 30 or 37 ° C. Preparation of Extracts.  Intact nuclei were isolated as described(18, 19). The nuclei were resuspended in a half-pellet volume of low-salt buffer (20 mM Hepes, pH 7.9  20 mM NaCl  1 mMEDTA   20% glycerol), to which a half-pellet volume of high-saltbuffer (20 mM Hepes, pH 7.9  1 M NaCl  1 mM EDTA   20%glycerol) was added. Proteins were extracted for 30 min at 4 ° C,followed by digestion of genomic DNA and RNA with DNaseand micrococcal nuclease (15 and 88 units, respectively, per 100  l of nuclear pellet volume) for 60 min, to release DNA-boundRNA polymerase II complexes. The digestion produced DNA fragments of   600 bp, as estimated by ethidium bromide stain-ing on agarose gel. Extracts were centrifuged twice at 21,000   g   for 30 min at 4 ° C and dialyzed.Total cellular extracts were prepared by using hypotonic lysis(20 mM Tris, pH 7.5  5 mM KCl  1.5 mM MgCl 2  1 mM DTT andstandard protease inhibitors) at 4 ° C and were homogenized byusing 40 strokes of a tight-pestle Dounce homogenizer. Thelysates were digested with DNase and micrococcal nuclease asdescribed above, and centrifuged twice at 21,000    g  .Denatured lysates were obtained by boiling pellets in 3 vol of SDSlysisbuffer(1%SDS  50mMTris,pH7.5  0.5mMEDTA   1mM DTT) for 10 min. The lysates were then diluted withimmunoprecipitation buffer (see below) and centrifuged at21,000    g   for 30 min. Immunoprecipitations.  For all immunoprecipitation reactions, theagarose beads were precoated with BSA, and the primaryantibodies were preconjugated with the secondary antibodies.Reactions were performed in buffer containing 50 mM Hepes atpH 7.8, 150 mM NaCl, 5 mM MgCl 2 , 20% (vol   vol) glycerol, and0.1% Triton X-100 (immunoprecipitation buffer) and washed inthe same buffer containing in addition 0.5% Triton, 0.5% Igepal,and 0.5% sodium deoxycholate, or a buffer containing 0.5%Igepal and NaCl from 150 to 900 mM. Immunoprecipitatedproteins were eluted by boiling in SDS sample buffer, resolvedby electrophoresis in SDS  4 – 22% polyacrylamide gradient gels,and detected by immunoblotting.For hydroxylation of endogenous Rpb1, 150   g of totalprotein extract was incubated in the presence of prolyl hydrox- ylase cofactors or inhibitors for 15 – 30 min at 30 ° C. The extracts were then processed for the immunoprecipitation reaction withanti-HA antibody. For elutions of Rpb1 from pVHL-associatedcomplexes, proteins coimmunoprecipitated with anti-HA anti-bodyfromPC12VHL(WT)nuclearextractswereincubatedin40mM Tris buffer in the presence of the respective peptides for1.5 h. The eluates were analyzed by SDS  PAGE. To dephos-phorylateRpb1,extractswereincubatedwith25unitsofalkalinephosphatase (Roche Molecular Biochemicals) with or without10 mM NaF for 30 or 60 min at room temperature. Dephos-phorylated extracts were used for immunoprecipitations withanti-HA antibodies. In Vitro   Ubiquitination.  Four-hundred micrograms of total cellularextract was immunoprecipitated with anti-HA or H14 antibody,followed by four washes with high detergent immunoprecipita-tion buffer and two washes in buffer containing 50 mM Tris atpH 8 and 3 mM DTT. The immunoprecipitated complexes wereresuspended in a final reaction volume of 50   l containing ATP-regenerating buffer, 5   g   l  1 ubiquitin, 100 ng   l  1 ubiq-uitin aldehyde, CbzLLn, and 16   l of purifed rabbit reticulocytefraction II, incubated for 2 h at 37 ° C, washed in 50 mM Tris, pH8.0/3 mM DTT buffer, and analyzed by SDS  PAGE. Computational Analysis.  The PROSITE server (29) was used toidentify proteins containing proline hydroxylation motifs.HIF-1   secondary structures were predicted by using the Psi-PRED (30) server. The sequence of the human Rpb1 subunit(GenBank accession no. NP    000928) was first aligned optimally with the yeast Rpb1 structure (PDB ID code 1I50, chain A, 60%identical with mostly conservative substitutions), and then usedto build the optimal structurally biased sequence alignment withthe sequences of the human HIF-   factor (GenBank accessionno. BAB70608). HIF-1  residues 530 – 577 were aligned with the yeast Rpb1 (structure 1I50, chain A) by using the  LOOPP  programand structurally biased sequence alignment (refs. 31 – 33;  LOOPP is available at www.tc.cornell.edu  CBIO  loopp). Residues 571 – 679 of HIF-1   were aligned with the structure of the humanRpb6 subunit (structure 1qkl, chain A) by using the 3D-PSSMserver (34). Results Similarity of Rpb1  Rpb6 Subunits and HIF-1   Oxygen-DependentDegradation Domain (ODDD).  The human, murine, and yeast Rpb1subunits contain an analogous, L(XY)LAP, motif located ami-no-terminal to the binding site for Rpb6 and to the beginning of the unstructured CTD (Fig. 1; ref. 35). Comparing the sequenceof the HIF-1   ODDD with representative libraries of proteinstructures identified a region with similarities to a fragment of Rpb1 and the adjacent Rpb6 subunit. The  50-aa Rpb1 coun-terpart is 30% identical and contains the L(XY)LAP motif,including P1465 as a counterpart of the HIF-1   P564 residue.The HIF-1   secondary structures, as predicted by the  PSI - PRED method (30), are also consistent with those of Rpb1 and Rpb6.The plausible pVHL binding pocket between Rpb1 and Rpb6 with the critical pVHL-binding motif (Fig. 1  B ) is located on thesurface of the RNA polymerase II complex (see the legend toFig.7,whichispublishedassupportinginformationonthePNAS web site, www.pnas.org). The estimated statistical significance of individual alignments of the HIF-1  sequence into the Rpb1 andRpb6 structures is low (Fig. 7). However, two weak matches intoadjacent structural domains of the RNA polymerase II complex make the overall prediction stronger than suggested by theindividual estimates of significance. This prediction is furtherstrengthened by the recently published partial structure of theODDD peptide and pVHL complex (36, 37), which suggests thatODDD exists in an extended conformation and reveals that theadjacent DLQL motif stabilizes the pVHL binding. A closelyrelated motif (DLLL) is found in the predicted Rpb1 counter-part of ODDD. pVHL Binds Hyperphosphorylated Rpb1 in a Proline Hydroxylation-Dependent Manner.  The immobilized Rpb1 peptide (amino acids1440 – 1475) containing the hydroxylated proline binds to WT Kuznetsova  et al.  PNAS    March 4, 2003    vol. 100    no. 5    2707      M     E     D     I     C     A     L     S     C     I     E     N     C     E     S  [ 35 S]pVHL (Fig. 2  A ), but not to pVHL with deletions of exon 3or exon 2 or with point mutations within the    (C162T) or   (Y98N) domain (Fig. 2  B ). The nonhydroxylated peptide doesnot bind pVHL, but it acquires pVHL-binding properties afterincubation with PC12 cell extracts in the presence of Fe(II),ascorbic acid, and 2-oxoglutarate (Fig. 2 C ). The hydroxylated,but not the nonhydroxylated, peptide competes with full-length[ 35 S]HIF-2   for [ 35 S]pVHL binding (Fig. 2  D ). [ 35 S]pVHL andRpb6 do not interact under these conditions.Coimmunoprecipitation experiments using anti-pVHL anti-bodies in nuclear extracts from PC12 cells overexpressing HA-tagged human pVHL, or in control vector-transfected PC12 cells(18), reveal that both anti-VHL and anti-HA antibodies are ableto coimmunoprecipitate hyperphosphorylated Rpb1 as detectedby the H14 antibody, which is specific for phosphoserine-5 withinthe CTD repeats (refs. 38 – 40; Fig. 3  A ). In contrast, pVHL failsto coimmunoprecipitate the nonphosphorylated Rpb1 as de-tected by the C21 antibody (Fig. 3  A  and  B ), which is specific forthe nonphosphorylated peptide sequence from the CTD. ThepVHL  – Rpb1 complex is stable in high salt (up to 900 mM NaCl washes), consistent with other pVHL-binding proteins (1 – 3)(Fig. 3  B ). Dephosphorylation of extracts with alkaline phospha-tase greatly attenuates binding of pVHL to the hyperphosphor- ylated Rpb1, and does not induce binding of the hypophosphor- ylated Rpb1 (Fig. 3 C ). pVHL  – Rpb1 complex is also formed inextracts derived from RCC cells, either expressing endogenoustruncated and nonfunctional pVHL or stably transfected withHA-tagged pVHL (ref. 1; Fig. 3  D ).Incubation of cellular lysates in the presence of Fe(II), 2-oxoglutarate, and ascorbic acid substantially increases the for-mation of the pVHL  – Rpb1 complex as determined by coimmu-noprecipitation reactions (Fig. 4  A  and  B ), whereas this complex is inhibited in the presence of iron chelators, desferrioxamine,and 2,2  -dipyridyl (41), or by the addition of ZnCl 2 , a potentdivalent inhibitor of collagen prolyl hydroxylases (ref. 42;Fig. 4  A Right ). These treatments do not affect the total amountof hyperphosphorylated Rpb1 in the extracts (Fig. 4  A Left ).Pretreatment of lysates with cofactors of prolyl hydroxylasesaugments both the association of pVHL with Rpb1 and theformation of the pVHL  – HIF-2   complex (Fig. 4  B ). However,these treatments do not affect the formation of the pVHL  – elongin BC, cullin-2, and Rbx-1 complex (Fig. 4  B ), and they donot induce binding of pVHL to the hypophosphorylated Rpb1(data not shown). The synthetic 36-aa P1465-hydroxylated Rpb1peptide elutes Rpb1 and HIF-2   from the anti-HA immuno-precipitated complex, whereas the nonhydroxylated peptide isonly marginally effective (Fig. 4 C ). These data indicate that,similar to HIF-  , hyperphosphorylated Rpb1 binds pVHL in aproline hydroxylation-dependent manner. pVHL Regulates Ubiquitination and Accumulation of Hyperphosphor-ylated Rpb1.  The amount of hyperphosphorylated, but not of hypophosphorylated, Rpb1 in PC12 cells correlates inversely Fig. 1.  Computational prediction of the pVHL-binding pocket in the RNApolymerase II complex. (  A ) Sequence-to-structure alignments of the HIF-1  ODDD fragment into the carboxyl-terminal fragments of human Rpb1 andRpb6 subunits. The Rpb1 and Rpb6 secondary structures are indicated below,and the predicted HIF-1   secondary structures are shown above their se-quences.  H  ,    (and other)-helices;  E  , extended   -strands. HIF-1   motifs thatmake contact with the pVHL complex (including the Pro-564 residue) areshadedand,ifconservedinRpb1,bold.ThecriticalHIF-1  residues(L559,L562,P564, and D571) are conserved in the Rpb1 structure. The K532 residue,ubiquitinatedonHIF-1  ,isboxed.ThehumanandyeastRpb6structures(PDBID codes 1QKL and 1I50, chain F, respectively) are different by an additional  -strand occurring only on the human Rpb6 structure (boxed fragment). ( B )ThepredictedpVHL-bindingpocket(Rpb1,purple;Rpb6,red;otherfragmentsincontactwiththebindingpocketaregreen).Thecriticalprolineresidueandthe fl anking amino acids are indicated by using ball and stick models of theirsidechains.ThenumberingofresiduesisaccordingtotheyeastRpb1structurewith the yeast Leu-1430, Pro-1435, and Ile-1445 residues corresponding toLeu-1460, Pro-1465, and Leu-1475 of the human Rpb1, respectively. Fig. 2.  pVHL binds to the Rpb1 synthetic 36-aa peptide with hydroxylatedP1465. (  A ) Binding of [ 35 S]pVHL to the Rpb1 peptide hydroxylated (lane 4), ornonhydroxylated (lane 3), on P1465. ( B ) Binding of the  in vitro -translatedmutated forms of pVHL to the hydroxylated peptide. To ensure that theamountsofthelabeledmutantproteinsusedinthepeptide-bindingreactionswere the same as for the WT pVHL, the amounts of the lysate with radioac-tivelylabeledmutantproteinsusedinthebindingreactionswerenormalizedaccordinglybyusingthePhosphorImagerquanti fi cation.( C  )Hydroxylationofthe Rpb1 peptide in extract from PC12 cells. HAVHL, HA-tagged pVHL. ( D )Competition experiment of [ 35 S]HIF-2  – [ 35 S]pVHL binding by hydroxylated(lanes 3 and 4) or nonhydroxylated (lane 5) peptide. 2708    www.pnas.org  cgi  doi  10.1073  pnas.0436037100 Kuznetsova  et al.   with the levels of pVHL (Fig. 5  A ). Cells overexpressing pVHL exhibit low levels of constitutively accumulated hyperphospho-rylated Rpb1, whereas cells expressing reduced levels of pVHL (19) exhibit high levels of Rpb1 detected with H14 (Fig. 5  A  and C ).AftertreatmentwiththeproteasomalinhibitorCbzLLn,cellsexpressing high levels of pVHL accumulate the more slowlymigrating forms of Rpb1, whereas cells expressing low levels of pVHL do not. In contrast, steady-state levels of the hypophos-phorylated form of Rpb1 are not affected by CbzLLn treatment,and are independent of pVHL levels (Fig. 5  A ). Formation of thepVHL-Rpb1 complex is proportional to the concentration of pVHL and increases in cells treated with CbzLLn (Fig. 5  B ).Consistent with these data,  in vivo  ubiquitination of the hyper-phosphorylated Rpb1 correlates with the levels of pVHL in aCbzLLn-dependent manner (Fig. 5 C ).To further investigate whether the pVHL complex directlyubiquitinates hyperphosphorylated Rpb1, protein complexes were coimmunoprecipitated with either anti-HA or H14 anti-body, washed stringently, and subjected to  in vitro  ubiquitination(Fig.5  D ).ThemoreslowlymigratingformsofRpb1aredetectedonly with the full ubiquitination-reaction-containing complexescoimmunoprecipitated with anti-HA, but not with H14 (Fig. 5  D ,lanes 2 and 5). The ubiquitinated forms of Rpb1 are not detectedifATP-regeneratingbufferorubiquitinisomitted(Fig.5  D ,lanes3 and 4). These data show that pVHL targets hyperphosphory-lated Rpb1 for ubiquitination. UV Irradiation Induces pVHL–Rpb1 Interaction.  In cells overexpress-ing pVHL, UV irradiation induces an early and transient in-crease in the accumulation of hyperphosphorylated Rpb1 de-tected with H14, which decreases during 8 h of recovery (Fig.6  A ). In contrast, in cells with reduced levels of pVHL, hyper-phosphorylated Rpb1 does not increase after UV irradiation,but declines with a delay beginning after 6 h of recovery (Fig.6  A ). The disappearance of Rpb1 in pVHL-overexpressing cellsis prevented by proteasomal inhibitors, indicating that the loss of Rpb1 results from proteasomal degradation (Fig. 6  A ). Levels of the hypophosphorylated Rpb1 are not affected by UV exposureand do not depend on the concentration of pVHL. UV irradi-ation increases the amount of Rpb1 coimmunoprecipitated withpVHL, in the presence and absence of proteasomal inhibitors(Fig. 6  B ). The UV stimulus clearly increases ubiquitination of hyperphosphorylated Rpb1 in cells overexpressing pVHL, butfails to induce its ubiquitination in cells expressing low levels of pVHL (Fig. 6 C ). These data indicate that pVHL contributes tothe processing of the RNA polymerase complex in response toUV stress. Discussion Our findings extend the role of the pVHL complex. They showthat, in addition to its role as an E3 ubiquitin ligase, whichregulates the accumulation of HIF-  protein (9 – 12), and thereby Fig. 3.  pVHL speci fi cally interacts with the hyperphosphorylated Rpb1 innuclear extracts from PC12 and RCC cells. Coimmunoprecipitations (IP) usingmonoclonal antibodies against HA or pVHL or mouse anti-rabbit IgG (RG-96)in nuclear extracts from PC12 cells overexpressing human HA-tagged pVHL[PC12 VHL (WT)] (  A  and  B ), or control PC12 cells stably transfected with anempty vector (  A ). ( C  ) Dephosphorylation of Rpb1 in PC12 cellular extracts fortheindicatedtimesbytreatingtheextractswithalkalinephosphatase(AP)inthe absence (lanes 3, 4, 7, and 8) or presence (lanes 6 and 9) of NaF. Treatedextracts were subjected to immunoprecipitations using anti-HA antibodies.( D ) Anti-HA immunoprecipitations in nuclear extracts from RCC 786-O cellslacking pVHL function (lanes 1 and 2) or from cells stably transfected withHA-pVHL(1)(lanes3and4).PC12andindicatedRCCcellswerepretreatedwith10   M CbzLLn for 6 h to increase accumulation of the hyperphosphorylatedRpb1. The immunoprecipitates were washed with high-detergent immuno-precipitationbuffer(  A , C  ,and D ),orimmunoprecipitationbufferscontainingupto900mMNaCland0.5%Igepal( B ).Blotswereprobedwiththeindicatedantibodies, human (h)pVHL and rat (r)pVHL, respectively. Fig. 4.  pVHL binds Rpb1 in a proline hydroxylation-dependent manner. (  A )Preincubation of PC12 cellular extracts with FeCl 2 , ascorbic acid, and 2-oxoglutarate (100   M each) (lane 3), or with 100   M each of iron chelators:desferrioxamine (DF, lane 4) and 2,2  -dipyridyl (DPy, lane 5), or ZnCl 2  (lane 6),followed by Western blot analysis ( Left  ) or coimmunoprecipitations (IP) withanti-HA antibodies ( Right  ). IB, immunoblotting antibody. ( B ) Coimmunopre-cipitation of the components of pVHL-associated complex by using anti-HAantibody in cellular lysates (lane 1) or lysates treated under hydroxylatingconditions with Fe(II), ascorbate, and 2-oxoglutarate, as in  A . Immunoblotswere probed with the indicated antibodies. ( C  ) Elution of hyperphosphory-latedRpb1andHIF-2  withahydroxylated36-aaRpb1peptide.Rdescribestheratio of the signal detected with H14 antibody to the signal detected withanti-HA antibody, as quanti fi ed by using optical density measurements. Kuznetsova  et al.  PNAS    March 4, 2003    vol. 100    no. 5    2709      M     E     D     I     C     A     L     S     C     I     E     N     C     E     S  expression of hypoxia-inducible genes, the pVHL complex canfunction as an E3 ligase, which targets the hyperphosphorylatedRpb1 for ubiquitination and degradation. Importantly, bindingof pVHL to the full-size Rpb1 requires hydroxylation of proline-1465 within Rpb1 and phosphorylation of the CTD. To date, theproteins that pVHL targets for ubiquitination include, in addi-tion to HIF-  s, a subfamily of deubiquitinating enzymes (43, 44)and activated atypical protein kinase C (45).The exact role that ubiquitination of hyperphosphorylatedRpb1 by pVHL plays in the function of the RNA polymerasecomplex remains to be determined. Because ubiquitination of Rpb1 occurs in a transcription-dependent manner (22, 23), andbecauseourearlierobservationsindicatethatpVHLlevelsaffect in vivo  elongation of TH transcripts (18, 19), we anticipate thatubiquitination of Rpb1 by pVHL complex is likely to regulateefficient transcript elongation through elongation-pause and-arrest sites of specific genes. In particular, it may be involved inthe regulation of TH transcript elongation (18 – 20). Such po-tential role of the pVHL  – Rpb1 interaction is supported by thefact that pVHL binds in the pocket between Rpb1 and Rpb6, andthat Rpb6 promotes elongation through arrest sites by binding tothe elongation factor TFIIS (46). The pVHL-binding site islocated on the surface of the elongating RNA polymerase IIcomplex, and thus is accessible for pVHL binding during tran-scription. Interaction of pVHL with the RNA polymerase IIcomplex may also locally titrate elongins B and C from elonga-tion factor SIII (elongin ABC), thereby providing anothermechanism by which pVHL could inhibit transcription elonga-tion, as proposed based on  in vitro  studies (47).We also anticipate that the pVHL  – Rpb1 interaction has amore universal role and may regulate genes other than TH. ThepVHL  – Rpb1 interaction is regulated by UV stress, thus pVHL may play a role in the regulation of transcription complexes(transcription-coupled repair) under conditions of DNA dam-age, such as UV irradiation. In this respect, pVHL-negative cellsundergo apoptosis in response to UV treatment, whereas thepVHL-positive cells do not (48). Our data also suggest amolecular mechanism by which the loss of pVHL function in vonHippel – Lindau disease may result in tumsrcenesis.Our results demonstrate that antisense cells having decreasedlevels of pVHL accumulate hyper- but not hypophosphorylatedRpb1, resulting in decreased ubiquitination of Rpb1. The mostconsistent explanation for this finding is that the antisense cellshave decreased pVHL-associated E3 ligase activity toward thehyperphosphorylated Rpb1. However, it cannot be excluded thatpVHL may affect the activity and  or expression of some kinasesorphosphatasesinvolvedinthephosphorylationofCTD.Inviewof the role of CTD phosphorylation in pVHL binding, this lastpossibility might be an attractive regulatory mechanism increas-ing the pVHL  – Rpb1 interaction under conditions of reducedamounts of pVHL.These data provide biochemical evidence that Rpb1 can bemodified by proline hydroxylation. It is unclear whether prolinehydroxylation requires CTD phosphorylation. Two major groupsof proline hydroxylases have been identified to date: the endo-plasmic reticulum collagen proline hydroxylases (42) and theEgl-9-like group of proline hydroxylases involved in O 2 -dependent regulation of HIF-   (7, 8). Both groups hydroxylateprolines in an O 2 -, Fe(II)-, and 2-oxoglutarate-dependent man-ner; howeve, hydroxylases involved in collagen maturation areless sensitive to O 2  levels and are functional even under hypoxicconditions (42). In contrast, the HIF prolyl hydroxylases appearto be strictly O 2 -sensitive, and their activities are inhibited by adecrease in pO 2  (7). At this time, it has not been possible tomeasure the O 2 -sensitivity of prolyl hydroxylation of Rpb1 andpVHL binding because hyperphosphorylated Rpb1 disappears Fig. 5.  Accumulation and ubiquitination of hyperphosphorylated Rpb1 incells with different levels of pVHL. (  A ) Western blot analysis of hyperphos-phorylated (H14) and hypophosphorylated (C21) Rpb1 in nuclear extractsfromPC12VHL(WT)ortwodifferentclonesofPC12VHLantisense(as)cells.( B )Coimmunoprecipitations using anti-pVHL antibody from nuclear extracts ofWT and antisense cells. Ex, extract. ( C  ) Immunoprecipitation of ubiquitinatedforms of hyperphosphorylated Rpb1 from denatured cellular lysates by usingH14 antibody. ( D )  In vitro  ubiquitination reactions on protein complexescoimmunoprecipitated by using anti-HA (lanes 1 – 4) or H14 (lanes 5 – 7) anti-bodies from cellular extracts from PC12VHL(WT) cells. H14 antibody does notcoimmunoprecipitate pVHL. UbA, ubiquitin aldehyde; E, puri fi ed enzymaticfraction II from reticulocyte lysate; ATP RS, ATP-regenerating solution. Thebracket marks ubiquitinated forms of Rpb1. Fig. 6.  Rpb1 – pVHL interactions in response to the UV treatment. (  A )WesternblotanalysisofRpb1andpVHLinnuclearextractsfromcellsintheabsence ( Upper  ) or presence ( Lower  ) of CbzLLn. ( B ) Coimmunoprecipita-tion of hyperphosphorylated Rpb1 with anti-HA antibody in nuclear ex-tractsfromcellstreatedwithUVfortheindicatedtimes.Blotswereprobedwith indicated antibodies. ( C  ) Ubiquitination of the hyperphosphorylatedRpb1 in response to the UV treatment in PC12VHL(WT) and antisense cells.Two hours after UV irradiations denatured cellular lysates were immuno-precipitated with H14 antibodies and the immunoblots were probed withanti-ubiquitin antibody. 2710    www.pnas.org  cgi  doi  10.1073  pnas.0436037100 Kuznetsova  et al.
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