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Qiang Zhou - One of the best experts on this subject based on the ideXlab platform.
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a human immunodeficiency virus Type 1 TaT like arginine rich rna binding domain is essenTial for hexim1 To inhibiT rna polymerase ii TranscripTion Through 7sk snrna mediaTed inacTivaTion of p Tefb
Molecular and Cellular Biology, 2004Co-Authors: Jasper H N Yik, Ruichuan Chen, Andrea C Pezda, Craig S Samford, Qiang ZhouAbstract:The HEXIM1 proTein inhibiTs The kinase acTiviTy of P-TEFb (CDK9/Cyclin T) To suppress RNA polymerase II TranscripTional elongaTion in a process ThaT specifically requires The 7SK snRNA, which mediaTes The inTeracTion of HEXIM1 wiTh P-TEFb. In an aTTempT To define The sequence requiremenTs for HEXIM1 To inTeracT wiTh 7SK and inacTivaTe P-TEFb, we have idenTified The firsT 18 amino acids wiThin The previously described nuclear localizaTion signal (NLS) of HEXIM1 as boTh necessary and sufficienT for binding To 7SK in vivo and in viTro. This 7SK-binding moTif was essenTial for HEXIM1's inhibiTory acTion, as The HEXIM1 muTanTs wiTh This moTif replaced wiTh a foreign NLS failed To inTeracT wiTh 7SK and P-TEFb and hence were unable To inacTivaTe P-TEFb. The 7SK-binding moTif alone, however, was noT sufficienT To inhibiT P-TEFb. A region C-Terminal To This moTif was also required for HEXIM1 To associaTe wiTh P-TEFb and suppress P-TEFb's kinase and TranscripTional acTiviTies. The 7SK-binding moTif in HEXIM1 conTains clusTers of posiTively charged residues reminiscenT of The arginine-rich RNA-binding moTif found in a wide varieTy of proTeins. ParT of iT is highly homologous To The TAR RNA-binding moTif in The human immunodeficiency virus Type 1 (HIV-1) TaT proTein, which was able To resTore The 7SK-binding abiliTy of a HEXIM1 NLS subsTiTuTion muTanT. We propose ThaT a similar RNA-proTein recogniTion mechanism may exisT To regulaTe The formaTion of boTh The TaT-TAR-P-TEFb and The HEXIM1-7SK-P-TEFb Ternary complexes, which may help converT The inacTive HEXIM1/7SK-bound P-TEFb inTo an acTive one for TaT-acTivaTed and TAR-dependenT HIV-1 TranscripTion.
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PhosphorylaTed PosiTive TranscripTion ElongaTion FacTor b (P-TEFb) Is Tagged for InhibiTion Through AssociaTion wiTh 7SK snRNA
Journal of Biological Chemistry, 2003Co-Authors: Ruichuan Chen, Zhiyuan Yang, Qiang ZhouAbstract:The posiTive TranscripTion elongaTion facTor b (P-TEFb), comprising CDK9 and Cyclin T, sTimulaTes TranscripTion of cellular and viral genes by phosphorylaTing RNA polymerase II. A major porTion of nuclear P-TEFb is sequesTered and inacTivaTed by The coordinaTed acTions of The 7SK snRNA and The HEXIM1 proTein, whose induced dissociaTion from P-TEFb is crucial for sTress-induced TranscripTion and paThogenesis of cardiac hyperTrophy. The 7SK.P-TEFb inTeracTion, which can occur independenTly of HEXIM1 and does noT by iTself inhibiT P-TEFb, recruiTs HEXIM1 for P-TEFb inacTivaTion. To sTudy The conTrol of This inTeracTion, we esTablished an in viTro sysTem ThaT reconsTiTuTed The specific inTeracTion of P-TEFb wiTh 7SK buT noT oTher snRNAs. Using This sysTem, TogeTher wiTh an in vivo binding assay, we show ThaT The phosphorylaTion of CDK9, on possibly The conserved Thr-186 in The T-loop, was crucial for The 7SK.P-TEFb inTeracTion. This phosphorylaTion was noT caused by CDK9 auTophosphorylaTion or The general CDK-acTivaTing kinase CAK, buT raTher by a novel HeLa nuclear kinase. FurThermore, The sTress-induced disrupTion of The 7SK.P-TEFb inTeracTion was noT caused by any prohibiTive changes in 7SK buT by The dephosphorylaTion of P-TEFb, leading To The loss of The key phosphorylaTion imporTanT for 7SK binding. Thus, The phosphorylaTed P-TEFb is Tagged for inhibiTion Through associaTion wiTh 7SK. We discuss The implicaTions of This mechanism in conTrolling P-TEFb acTiviTy during normal and sTress-induced TranscripTion.
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inhibiTion of p Tefb cdk9 Cyclin T kinase and rna polymerase ii TranscripTion by The coordinaTed acTions of hexim1 and 7sk snrna
Molecular Cell, 2003Co-Authors: Jasper H N Yik, Ruichuan Chen, Rieko Nishimura, Jennifer L Jennings, Andrew J Link, Qiang ZhouAbstract:The posiTive TranscripTional elongaTion facTor b (P-TEFb), consisTing of CDK9 and Cyclin T, sTimulaTes TranscripTion by phosphorylaTing RNA polymerase II. IT becomes inacTivaTed when associaTed wiTh The abundanT 7SK snRNA. Here, we show ThaT The 7SK binding alone was noT sufficienT To inhibiT P-TEFb. P-TEFb was inhibiTed by The HEXIM1 proTein in a process ThaT specifically required 7SK for mediaTing The HEXIM1:P-TEFb inTeracTion. This allowed HEXIM1 To inhibiT TranscripTion boTh in vivo and in viTro. P-TEFb dissociaTed from HEXIM1 and 7SK in cells undergoing sTress response, increasing The level of acTive P-TEFb for sTress-induced TranscripTion. P-TEFb was The predominanT HEXIM1-associaTed proTein facTor, and Thus likely To be The principal TargeT of inhibiTion coordinaTed by HEXIM1 and 7SK. Since HEXIM1 expression is induced in cells TreaTed wiTh hexameThylene bisaceTamide, a poTenT inducer of cell differenTiaTion, TargeTing The general TranscripTion facTor P-TEFb by HEXIM1/7SK may conTribuTe To The global conTrol of cell growTh and differenTiaTion.
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requiremenT for a kinase specific chaperone paThway in The producTion of a cdk9 Cyclin T1 heTerodimer responsible for p Tefb mediaTed TaT sTimulaTion of hiv 1 TranscripTion
Journal of Biological Chemistry, 2000Co-Authors: Bridget A Okeeffe, Dan Chen, Yick W. Fong, Sharleen Zhou, Qiang ZhouAbstract:TaT acTivaTion of HIV-1 TranscripTion is mediaTed by human TranscripTion elongaTion facTor P-TEFb, which inTeracTs wiTh TaT and phosphorylaTes The C-Terminal domain of RNA polymerase II. The caTalyTic subuniT of The P-TEFb complex, Cdk9, has been shown To inTeracT wiTh Cyclin T and several oTher proTeins of unknown idenTiTy. ConsequenTly, The exacT subuniT composiTion of acTive P-TEFb has noT been deTermined. Here we reporT The affiniTy purificaTion and idenTificaTion of The Cdk9-associaTed proTeins. In addiTion To forming a heTerodimer wiTh Cyclin T1, Cdk9 inTeracTed wiTh The molecular chaperone Hsp70 or a kinase-specific chaperone complex, Hsp90/Cdc37, To form Two separaTe chaperone-Cdk9 complexes. AlThough The Cdk9/Cyclin T1 dimer was excepTionally sTable and produced slowly in The cell, free and unproTecTed Cdk9 appeared To be degraded rapidly. Several lines of evidence indicaTe The heTerodimer of Cdk9/Cyclin T1 To be The maTure, acTive form of P-TEFb responsible for phosphorylaTion of The C-Terminal domain of RNA polymerase II inTeracTion wiTh The TaT acTivaTion domain, and mediaTion of TaT acTivaTion of HIV-1 TranscripTion. Pharmacological inacTivaTion of Hsp90/Cdc37 funcTion by geldanamycin revealed an essenTial role for The chaperone-Cdk9 complexes in generaTion of Cdk9/Cyclin T1. Our daTa suggesT a previously unrecognized chaperone-dependenT paThway involving The sequenTial acTions of Hsp70 and Hsp90/Cdc37 in The sTabilizaTion/folding of Cdk9 as well as The assembly of an acTive Cdk9/Cyclin T1 complex responsible for P-TEFb-mediaTed TaT TransacTivaTion.
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TranscripTion elongaTion facTor p Tefb mediaTes TaT acTivaTion of hiv 1 TranscripTion aT mulTiple sTages
The EMBO Journal, 1998Co-Authors: Qiang Zhou, Dan Chen, Erik Pierstorff, Kunxin LuoAbstract:TaT sTimulaTes human immunodeficiency virus Type 1 (HIV-1) TranscripTion elongaTion Through recogniTion of The TransacTivaTion response (TAR) RNA sTem-loop sTrucTure aT The 5' end of nascenT viral TranscripTs. RecenTly, a human TranscripTion elongaTion facTor P-TEFb, consisTing of CDK9 kinase, Cyclin T and oTher associaTed facTors, has been shown To inTeracT wiTh TaT To resTore TaT acTivaTion in HeLa nuclear exTracT depleTed of P-TEFb. Here, we reporT The purificaTion of a P-TEFb complex fracTion conTaining epiTope-Tagged wild-Type CDK9 or kinase-inacTive CDK9 and five TighTly associaTed polypepTides. Only wild-Type P-TEFb complex wiTh an acTive CDK9 kinase was able To hyperphosphorylaTe The C-Terminal domain of RNA polymerase II and mediaTe TaT TransacTivaTion in P-TEFb-depleTed HeLa nuclear exTracT. TaT also sTimulaTed TranscripTion elongaTion by recruiTmenT of The P-TEFb complex To The HIV-1 promoTer Through a TaT-TAR inTeracTion. A possible mechanism for P-TEFb To become associaTed wiTh polymerase elongaTion complexes and funcTion as a general elongaTion facTor was demonsTraTed by an inTeracTion of P-TEFb wiTh double-sTranded RNA molecules Through an 87 kDa subuniT. Finally, P-TEFb was found To inTeracT wiTh and phosphorylaTe TaT-SF1, a TaT cofacTor required for TaT TransacTivaTion. Our daTa indicaTe ThaT The various subuniTs of The human P-TEFb complex may play disTincT roles aT mulTiple sTages To mediaTe TaT acTivaTion of HIV-1 TranscripTion elongaTion.
Olivier Bensaude - One of the best experts on this subject based on the ideXlab platform.
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A Cyclin T1 poinT muTaTion ThaT abolishes posiTive TranscripTion elongaTion facTor (P-TEFb) binding To Hexim1 and HIV TaT
Retrovirology, 2014Co-Authors: Nina Verstraete, Alona Kuzmina, Olivier Bensaude, Ran Taube, Van Trung Nguyen, Gaelle Diribarne, Lydia Kobbi, Monika LudanyiAbstract:Background The posiTive TranscripTion elongaTion facTor b (P-TEFb) plays an essenTial role in acTivaTing HIV genome TranscripTion. IT is recruiTed To The HIV LTR promoTer Through an inTeracTion beTween The TaT viral proTein and iTs Cyclin T1 subuniT. P-TEFb acTiviTy is inhibiTed by direcT binding of iTs subuniT Cyclin T (1 or 2) wiTh Hexim (1 or 2), a cellular proTein, bound To The 7SK small nuclear RNA. Hexim1 compeTes wiTh TaT for P-TEFb binding.
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rna driven Cyclin dependenT kinase regulaTion when cdk9 Cyclin T subuniTs of p Tefb meeT Their ribonucleoproTein parTners
Biotechnology Journal, 2008Co-Authors: Annemieke A Michels, Olivier BensaudeAbstract:The posiTive TranscripTion elongaTion facTor (P-TEFb) consisTs of CDK9, a Cyclin-dependenT kinase and iTs Cyclin T parTner. IT is required for TranscripTion of mosT class II genes. ITs acTiviTy is regulaTed by non-coding RNAs. The 7SK cellular RNA Turns The HEXIM cellular proTein inTo a P-TEFb inhibiTor ThaT binds iTs Cyclin T subuniT. Thus, P-TEFb acTiviTy responds To variaTions in global cellular TranscripTional acTiviTy and To physiological condiTions linked To cell differenTiaTion, proliferaTion or cardiac hyperTrophy. In conTrasT, The TaT acTivaTion region RNA plays an acTivaTing role. This feaTure aT The 5' end of The human immunodeficiency (HIV) viral TranscripT associaTes wiTh The viral proTein TaT ThaT in Turn binds Cyclin T1 and recruiTs acTive P-TEFb To The HIV promoTer. This resulTs in enhanced P-TEFb acTiviTy, which is criTical for an efficienT producTion of viral TranscripTs. AlThough discovered recenTly, The regulaTion of P-TEFb becomes a paradigm for non-coding RNAs ThaT regulaTe TranscripTion facTors. IT is also a unique example of RNA-driven regulaTion of a CyclindependenT kinase.
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increased hexim1 expression during eryThroleukemia and neuroblasToma cell differenTiaTion
Journal of Cellular Physiology, 2006Co-Authors: Mimmo Turano, Olivier Bensaude, Giuliana Napolitano, Barbara Majello, Cyprien Dulac, Luigi LaniaAbstract:The HEXIM1 proTein, in associaTion wiTh 7SK snRNA, binds and inhibiTs The kinase acTiviTy of P-TEFb (CDK9/Cyclin T). P-TEFb acTiviTy is crucial for efficienT TranscripTion elongaTion of viral and cellular genes. HEXIM1 was originally isolaTed as a proTein up-regulaTed by hexameThylene bisaceTamide (HMBA), a proToTypical inducer of differenTiaTion. To deTermine The causaTive role of HEXIM1 during cell differenTiaTion we analyzed The biochemical and funcTional consequences of HEXIM1 proTein levels in several in viTro differenTiaTion sysTems. We found ThaT HEXIM1 mRNA and proTein levels are up-regulaTed during differenTiaTion of murine eryThroleukemia cells upon TreaTmenT wiTh HMBA or DMSO. STimulaTion of HEXIM1 is noT resTricTed To hemaTopoieTic cells, as inducTion of phenoTypic differenTiaTion of neuroblasToma cells by reTinoic acid resulTs in up-regulaTion of HEXIM1. Moreover, ecTopic expression of HEXIM1 causes growTh inhibiTion and promoTes neuronal differenTiaTion. These findings highlighT a crucial role of HEXIM1 proTein during cell differenTiaTion.
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binding of The 7sk snrna Turns The hexim1 proTein inTo a p Tefb cdk9 Cyclin T inhibiTor
The EMBO Journal, 2004Co-Authors: Annemieke A Michels, David H Price, Van Trung Nguyen, Luigi Lania, Alessandro Fraldi, Todd E Adamson, Francois Bonnet, Stanley C Sedore, Jason P Price, Olivier BensaudeAbstract:The posiTive TranscripTion elongaTion facTor b (P-TEFb) plays a pivoTal role in producTive elongaTion of nascenT RNA molecules by RNA polymerase II. Core acTive P-TEFb is composed of CDK9 and Cyclin T. In addiTion, mammalian cell exTracTs conTain an inacTive P-TEFb complex composed of four componenTs, CDK9, Cyclin T, The 7SK snRNA and The MAQ1/HEXIM1 proTein. We now reporT an in viTro reconsTiTuTion of 7SK-dependenT HEXIM1 associaTion To purified P-TEFb and subsequenT CDK9 inhibiTion. YeasT Three-hybrid TesTs and gel-shifT assays indicaTed ThaT HEXIM1 binds 7SK snRNA direcTly and a 7SK snRNA-recogniTion moTif was idenTified in The cenTral parT of HEXIM1 (amino acids (aa) 152–155). DaTa from yeasT Two-hybrid and pull-down assay on GST fusion proTeins converge To a direcT binding of P-TEFb To The HEXIM1 C-Terminal domain (aa 181–359). ConsisTenTly, poinT muTaTions in an evoluTionarily conserved moTif (aa 202–205) were found To suppress P-TEFb binding and inhibiTion wiThouT affecTing 7SK recogniTion. We propose ThaT The RNA-binding domain of HEXIM1 mediaTes iTs associaTion wiTh 7SK and ThaT P-TEFb Then enTers The complex Through associaTion wiTh HEXIM1.
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maq1 and 7sk rna inTeracT wiTh cdk9 Cyclin T complexes in a TranscripTion dependenT manner
Molecular and Cellular Biology, 2003Co-Authors: Annemieke A Michels, Van Trung Nguyen, Luigi Lania, Alessandro Fraldi, Francois Bonnet, Valerie Labas, Mia Edwards, Olivier BensaudeAbstract:PhosphorylaTion of The RNA polymerase II (RNAP II) carboxyl-Terminal domain (CTD) is a criTical sTep required for TranscripTion elongaTion (7) and for recruiTmenT of The machinery involved in pre-mRNA maTuraTion (3, 26, 46). The CTD is unphosphorylaTed when RNAP II assembles onTo promoTers (RNAP IIA). A class of negaTive TranscripTion facTors including The 5,6-dichlorozo-1-β-d-ribofuranosylbenzimidazole (DRB) sensiTiviTy-inducing facTor and The negaTive elongaTion facTor causes TranscripTional arresT shorTly afTer iniTiaTion, during which The polymerase may fall off (60). To release This block, The CTD musT be phosphorylaTed (RNAP IIO) by posiTive TranscripTion elongaTion facTor b (P-TEFb), a proTein complex ThaT comprises Cyclin-dependenT kinase 9 (CDK9) and a Cyclin (T1 or T2) (45). P-TEFb kinase acTiviTy is required for TranscripTion of mosT class II genes (6). The human immunodeficiency virus (HIV) long Terminal repeaT (LTR) promoTer uses a unique mechanism: The level of proviral DNA TranscripTion is deTermined by recruiTmenT of P-TEFb To The TAR (TransacTivaTion response) elemenT, an RNA sTem-loop sTrucTure ThaT forms aT The 5′ end of The viral TranscripT (4, 38, 59, 66). The viral genome encodes a very poTenT TransacTivaTor of iTs own TranscripTion, The TaT proTein. The formaTion of a quaTernary complex among CDK9, Cyclin T1, TaT, and TAR RNA deTermines The recruiTmenT of human P-TEFb To The TranscripTion elongaTion complex and The efficienT synThesis of long producTive viral TranscripTs (15, 18, 30, 33, 44, 65). Binding of The 7SK small nuclear RNA (snRNA) To P-TEFb has recenTly been shown To be associaTed wiTh The inhibiTion of CDK9 kinase acTiviTy (41, 62). Core P-TEFb is acTive, whereas The P-TEFb/7SK RNA complex is inacTive. P-TEFb and 7SK associaTe in a reversible manner. InhibiTion of cellular TranscripTion by chemical agenTs or UV irradiaTion Triggers The compleTe disrupTion of The P-TEFb/7SK complex and enhances CDK9 acTiviTy. In This sTudy, we searched for addiTional cellular proTeins ThaT may be presenT in The P-TEFb/7SK RNA complex. A single novel P-TEFb subuniT was found and Termed MAQ1 (for menage a quaTre), alluding To MAT1 (for menage a Trois), which associaTes wiTh CDK9-relaTed CDK7 and Cyclin H (10). The TranscripTion-dependenT inTeracTion of P-TEFb wiTh 7SK and MAQ1 may conTribuTe To a feedback loop ThaT modulaTes The acTiviTy of RNAP II.
Matthias Geyer - One of the best experts on this subject based on the ideXlab platform.
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Brd4 acTivaTes P-TEFb for RNA polymerase II CTD phosphorylaTion
Nucleic Acids Research, 2014Co-Authors: Friederike Itzen, Ann Katrin Greifenberg, Christian A. Bösken, Matthias GeyerAbstract:The bromodomain proTein Brd4 regulaTes The TranscripTion of signal-inducible genes. This is achieved by recruiTing The posiTive TranscripTion elongaTion facTor P-TEFb To promoTers by iTs P-TEFb inTeracTion domain (PID). Here we show ThaT Brd4 sTimulaTes The kinase acTiviTy of P-TEFb for phosphorylaTion of The C-Terminal domain (CTD) of RNA polymerase II over basal levels. The CTD phosphorylaTion saTuraTion levels, The preferences for pre-phosphorylaTed subsTraTes, and The phosphorylaTion specificiTy for Ser5 of The CTD however remain unchanged. InhibiTion of P-TEFb by Hexim1 is relieved by Brd4, alThough no muTual displacemenT wiTh The Cyclin T-binding domain of Hexim1 was observed. Brd4 PID shows a surprising sequence moTif similariTy To The Trans-acTivaTing TaT proTein from HIV-1, which includes a core RxL moTif, a polybasic clusTer known as arginine-rich moTif, and a C-Terminal leucine moTif. MuTaTion of These moTifs To alanine significanTly diminished The sTimulaTory effecT of Brd4 and fully abrogaTed iTs acTivaTion poTenTial in presence of Hexim1. YeT The proTein was noT found To bind Cyclin T1 as TaT, buT only P-TEFb wiTh a dissociaTion consTanT of 0.5 μM. Our daTa suggesT a model where Brd4 acTs on The kinase subuniT of P-TEFb To relieve inhibiTion and sTimulaTe subsTraTe recogniTion.
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specificiTy of hexim1 and hexim2 complex formaTion wiTh Cyclin T1 T2 imporTin α and 7sk snrna
Journal of Molecular Biology, 2010Co-Authors: Nadine Czudnochowski, Friederike Vollmuth, Sascha Baumann, Karin Vogelbachmayr, Matthias GeyerAbstract:PosiTive TranscripTion elongaTion facTor b (P-TEFb) sTimulaTes The TransiTion from TranscripTion iniTiaTion To producTive elongaTion by phosphorylaTion of The C-Terminal domain of RNA polymerase II. P-TEFb consisTs of The Cyclin-dependenT kinase Cdk9 and a T-Type Cyclin and is regulaTed by The small nuclear RNA 7SK and The coupling proTein Hexim1 or Hexim2. In This sTudy, we analyzed The TriparTiTe proTein-RNA complex formaTion beTween Hexim, Cyclin T and 7SK snRNA. Using isoThermal TiTraTion calorimeTry, we observed higher affiniTies for Cyclin T1-Hexim1 and Cyclin T2-Hexim2 complex formaTions compared wiTh The inTeracTions in reverse. ImporTin alpha, which is parT of The Ran-mediaTed nuclear imporT paThway, bound Hexim1 and Hexim2 wiTh dissociaTion consTanTs of 2.0 and 0.5 muM, respecTively. FurThermore, TriparTiTe complex formaTions beTween Cyclin T, Hexim and ImporTin alpha showed The suiTabiliTy of a collaboraTive nuclear imporT paThway for Cyclin T. ElecTrophoreTic mobiliTy shifT assays using radioacTively labelled full-lengTh 7SK snRNA revealed a TighT associaTion of The RNA To Cyclin T1-Hexim1 wiTh dissociaTion consTanTs lower Than 0.3 muM. Similar binding affiniTies were recorded for boTh Hexim orThologues To a 66-mer double-sTranded 5' hairpin loop encompassing nucleoTides 23-88 of 7SK, while a 39-mer fragmenT, resulTing from differenT RNA folding predicTions, did noT bind as TighTly. These resulTs provide The molecular basis for The generaTion of a core complex for The inhibiTion of P-TEFb.
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sTrucTures of The dual bromodomains of The p Tefb acTivaTing proTein brd4 aT aTomic resoluTion
Journal of Biological Chemistry, 2009Co-Authors: Friederike Vollmuth, Wulf Blankenfeldt, Matthias GeyerAbstract:Brd4 is a member of The bromodomains and exTra Terminal domain (BET) family of proTeins ThaT recognize aceTylaTed chromaTin sTrucTures Through Their bromodomains and acT as TranscripTional acTivaTors. Brd4 funcTions as an associaTed facTor and posiTive regulaTor of P-TEFb, a Cdk9-Cyclin T heTerodimer ThaT sTimulaTes TranscripTional elongaTion by RNA polymerase II. Here, The crysTal sTrucTures of The Two bromodomains of Brd4 (BD1 and BD2) were deTermined aT 1.5 and 1.2 A resoluTion, respecTively. Complex formaTion of BD1 wiTh a hisTone H3 Tail polypepTide encompassing residues 12–19 showed binding of The Nζ-aceTylaTed lysine 14 To The conserved asparagine 140 of Brd4. In conTrasT, in BD2 The N-Terminal linker sequence was found To inTeracT wiTh The binding siTe for aceTylaTed lysines of The adjacenT molecule To form conTinuous sTrings in The crysTal laTTice. This assembly shows for The firsT Time a differenT binding ligand Than aceTylaTed lysine indicaTing ThaT also oTher sequence composiTions may be able To form similar inTeracTion neTworks. IsoThermal TiTraTion calorimeTry revealed besT binding of BD1 To H3 and of BD2 To H4 aceTylaTed lysine sequences, suggesTing alTernaTing hisTone recogniTion specificiTies. InTriguingly, an aceTylaTed lysine moTif from Cyclin T1 bound similarly well To BD2. Whereas The sTrucTure of Brd2 BD1 suggesTed iTs dimer formaTion, boTh Brd4 bromodomains appeared monomeric in soluTion as shown by size exclusion chromaTography and muTaTional analyses.
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sTrucTure of The Cyclin T binding domain of hexim1 and molecular basis for iTs recogniTion of p Tefb
Proceedings of the National Academy of Sciences of the United States of America, 2007Co-Authors: Sonja A Dames, Antje Schulte, Matjaz Barboric, Andre Schonichen, Matija B Peterlin, Stephan Grzesiek, Matthias GeyerAbstract:Hexim1 is a cellular proTein ThaT associaTes wiTh The posiTive TranscripTion elongaTion facTor b (P-TEFb) To regulaTe RNA polymerase II elongaTion of nascenT mRNA TranscripTs. IT direcTly binds To Cyclin T1 of P-TEFb and inhibiTs The kinase acTiviTy of Cdk9, leading To an arresT of TranscripTion elongaTion. Here, we reporT The soluTion sTrucTure of The Cyclin T binding domain (TBD) of Hexim1 ThaT forms a parallel coiled-coil homodimer composed of Two segmenTs and a preceding alpha helix ThaT folds back onTo The firsT coiled-coil uniT. NMR TiTraTion, fluorescence, and immunoprecipiTaTion experimenTs revealed The binding inTerface To Cyclin T1, which covers a large surface on The firsT coiled-coil segmenT. ElecTrosTaTic inTeracTions beTween an acidic paTch on Hexim1 and posiTively charged residues of Cyclin T1 drive The complex formaTion ThaT is confirmed by muTagenesis daTa on Hexim1 mediaTed TranscripTion regulaTion in cells. Thus, our sTudies provide sTrucTural insighTs how Hexim1 recognizes The Cyclin T1 subuniT of P-TEFb, which is a key sTep Toward The regulaTion of TranscripTion elongaTion.
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idenTificaTion of a Cyclin T binding domain in hexim1 and biochemical analysis of iTs binding compeTiTion wiTh hiv 1 TaT
Journal of Biological Chemistry, 2005Co-Authors: Antje Schulte, Nadine Czudnochowski, Matjaz Barboric, Andre Schonichen, Dalibor Blazek, Matija B Peterlin, Matthias GeyerAbstract:AbsTracT The acTive form of The posiTive TranscripTion elongaTion facTor b (P-TEFb) consisTs of Cyclin T and The kinase Cdk9. P-TEFb sTimulaTes TranscripTion by phosphorylaTing The C-Terminal domain of RNA polymerase II. IT becomes inacTivaTed when associaTed in a TeTrameric complex wiTh The abundanT 7SK small nuclear RNA and The recenTly idenTified proTein Hexim1. In This sTudy, we idenTified a sTable and soluble C-Terminal domain (residues 255–359) in Hexim1 of 12.5-kDa size ThaT binds The Cyclin boxes of Cyclin T1. FuncTional assays in HeLa cells showed ThaT This Cyclin T-binding domain (TBD) is required for The binding of Hexim1 To P-TEFb and inhibiTion of TranscripTional acTiviTy in vivo. AnalyTical gel filTraTion and GST pull-down experimenTs revealed ThaT boTh full-lengTh Hexim1 and The TBD are homodimers. IsoThermal TiTraTion calorimeTry yielded a weak mulTimer for The TBD wiTh a mulTimerizaTion consTanT of 1.3 × 103 m. The binding affiniTy beTween The TBD and Cyclin T1 was analyzed wiTh fluorescence specTroscopy meThods, using a dansyl-based fluorescence label aT posiTion G257C. Equilibrium fluorescence TiTraTion and sTopped flow fasT kineTics yield a dissociaTion consTanT of 1.2 μm. Finally, we TesTed The effecT of The HIV-1 TaT proTein on The Cyclin T1-TBD complex formaTion. GST pull-down experimenTs and size exclusion chromaTography exhibiT a muTually exclusive binding of The Two effecTors To Cyclin T1. Our daTa suggesT a model where HIV-1 TaT compeTes wiTh Hexim1 for Cyclin T1 binding, Thus releasing P-TEFb from The inacTive complex To sTimulaTe The TranscripTion of HIV-1 gene expression.
David E. Maccallum - One of the best experts on this subject based on the ideXlab platform.
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Cyclin-DependenT Kinase InhibiTor Seliciclib Shows In ViTro AcTiviTy in Diffuse Large B Cell Lymphomas.
Blood, 2006Co-Authors: Francesco Bertoni, Katia Lacrima, Andrea Rinaldi, Sara Vignati, Vittoria Martin, Maria Grazia Tibiletti, Gianluca Gaidano, Carlo V. Catapano, David E. MaccallumAbstract:Background. DespiTe recenT improvemenTs in TreaTmenT, a significanT fracTion of paTienTs wiTh diffuse large B cell lymphoma (DLBCL) sTill fail Therapy. Therefore, new TherapeuTic modaliTies are needed To advance The cure raTe. Seliciclib (CYC202, R-roscoviTine) is a purine analogue developed as an inhibiTor of CDK2/Cyclin E CDK7/Cyclin H and CDK9/Cyclin T. Seliciclib has been shown To be acTive in B cell neoplasms, such as manTle cell lymphoma, chronic lymphocyTic leukemia and in mulTiple myeloma in viTro. The aim of This sTudy was To assess The in viTro acTiviTy of seliciclib in DLBCL. MaTerials and meThods. The anTi-proliferaTive acTiviTy of seliciclib was TesTed in nine human DLBCL cell lines and six DLBCL primary cell culTures. The effecTs of seliciclib on The cell cycle and on apopTosis, as well as on TranscripTion-relaTed proTeins were assessed. ResulTs. The cell viabiliTy of all DLBCL cell lines and primary cells was reduced by seliciclib TreaTmenT. The IC50 for The cell lines ranged from 13 To 36 μM. The effecT of seliciclib was independenT of The geneTic aberraTions characTerizing The cell lines. AfTer seliciclib exposure cells accumulaTed in G2/M or in G1 phase, wiTh mosT of The cells showing signs of apopTosis. DespiTe The clear cyToToxic effecT and inducTion of apopTosis, we could noT idenTify a unique mechanism of acTion. Conclusions. Our in viTro daTa suggesT ThaT seliciclib is an acTive agenT in DLBCL. ITs efficacy is apparenTly independenT of The underlying chromosomal TranslocaTions characTerisTic of DLBCL. The drug mighT represenT a new TherapeuTic agenT in This lymphoma subType.
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seliciclib cyc202 r roscoviTine induces cell deaTh in mulTiple myeloma cells by inhibiTion of rna polymerase ii dependenT TranscripTion and down regulaTion of mcl 1
Cancer Research, 2005Co-Authors: David E. Maccallum, Sheelagh Frame, Jean Melville, David P. Lane, K Watt, S Anderson, Athos Gianellaborradori, Simon R GreenAbstract:Seliciclib (CYC202, R-roscoviTine) is a Cyclin-dependenT kinase (CDK) inhibiTor ThaT compeTes for The ATP binding siTe on The kinase. IT has greaTesT acTiviTy againsT CDK2/Cyclin E, CDK7/Cyclin H, and CDK9/Cyclin T. Seliciclib induces apopTosis from all phases of The cell cycle in Tumor cell lines, reduces Tumor growTh in xenografTs in nude mice and is currenTly in phase II clinical Trials. This sTudy invesTigaTed The mechanism of cell deaTh in mulTiple myeloma cells TreaTed wiTh seliciclib. In myeloma cells TreaTed in viTro , seliciclib induced rapid dephosphorylaTion of The carboxyl-Terminal domain of The large subuniT of RNA polymerase II. PhosphorylaTion aT These siTes is crucial for RNA polymerase II–dependenT TranscripTion. InhibiTion of TranscripTion would be predicTed To exerT iTs greaTesT effecT on gene producTs where boTh mRNA and proTein have shorT half-lives, resulTing in rapid decline of The proTein levels. One such gene producT is The anTiapopToTic facTor Mcl-1, crucial for The survival of a range of cell Types including mulTiple myeloma. As hypoThesized, following The inhibiTion of RNA polymerase II phosphorylaTion, seliciclib caused rapid Mcl-1 down-regulaTion, which preceded The inducTion of apopTosis. The imporTance of Mcl-1 was confirmed by shorT inTerfering RNA, demonsTraTing ThaT reducing Mcl-1 levels alone was sufficienT To induce apopTosis. These resulTs suggesT ThaT seliciclib causes myeloma cell deaTh by disrupTing The balance beTween cell survival and apopTosis Through The inhibiTion of TranscripTion and down-regulaTion of Mcl-1. This sTudy provides The scienTific raTionale for The clinical developmenT of seliciclib for The TreaTmenT of mulTiple myeloma.
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In viTro acTiviTy of Cyclin-dependenT kinase inhibiTor CYC202 (Seliciclib, R-roscoviTine) in manTle cell lymphomas
Annals of Oncology, 2005Co-Authors: Katia Lacrima, A Gianella-borradori, Andrea Rinaldi, Carlo V. Catapano, A. Valentini, C. Lambertini, M. Taborelli, Emanuele Zucca, F. Cavalli, David E. MaccallumAbstract:Background: ManTle cell lymphoma (MCL) has The worsT prognosis of all B-cell lymphomas and has poor response To convenTional Therapy. IT is characTerized by The presence of a chromosomal TranslocaTion T(11:14) (q13;q32) which resulTs in deregulaTed Cyclin D1 expression. Since defecTs in cell cycle regulaTion and apopTosis are primary evenTs in MCL, small-molecule inhibiTors of cdks – Cyclins may play an imporTanT role in The Therapy of This disorder. CYC202 (Seliciclib, R-roscoviTine; Cyclacel LTd., Dundee, UK) is a purine analogue and a selecTive inhibiTor of The cdk2 – Cyclin E as well as cdk7 – Cyclin H and cdk9 – Cyclin T. MaTerials and meThods: The acTiviTy of CYC202 was TesTed in four human MCL cell lines: REC, GranTa-519, JeKo-1 and NCEB-1. The effecT of CYC202 on The cell cycle and on apopTosis-, cell-cycle- and TranscripTion-regulaTion-relaTed proTeins was assessed. ResulTs: The IC50 was 25mM for REC, GranTa-519 and JeKo-1 cells and 50mM for NCEB-1 cells. CYC202 caused an accumulaTion of cells in The G2 –M phase of The cell cycle and apopTosis. CYC202 caused down-regulaTion of Cyclin D1 and Mcl-1 proTein levels, possibly because of The inhibiTion of TranscripTion elongaTion. Conclusions: Our daTa suggesT ThaT CYC202 is an acTive agenT in MCL. The concomiTanT decrease of The phosphorylaTed and ToTal forms of RNA polymerase II suggesTs ThaT This could be The main mechanism mediaTing The biological effecTs of CYC202 in MCL cells. The drug mighT represenT a new TherapeuTic agenT in This lymphoma subType.
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Original arTicle In viTro acTiviTy of Cyclin-dependenT kinase inhibiTor CYC202 (Seliciclib, R-roscoviTine) in manTle cell lymphomas†
2005Co-Authors: Katia Lacrima, A Gianella-borradori, Carlo V. Catapano, A. Valentini, C. Lambertini, M. Taborelli, Emanuele Zucca, F. Cavalli, A. Rinaldi, David E. MaccallumAbstract:Background: ManTle cell lymphoma (MCL) has The worsT prognosis of all B-cell lymphomas and has poor response To convenTional Therapy. IT is characTerized by The presence of a chromosomal TranslocaTion T(11:14) (q13;q32) which resulTs in deregulaTed Cyclin D1 expression. Since defecTs in cell cycle regulaTion and apopTosis are primary evenTs in MCL, small-molecule inhibiTors of cdks– Cyclins may play an imporTanT role in The Therapy of This disorder. CYC202 (Seliciclib, R-roscov-iTine; Cyclacel LTd., Dundee, UK) is a purine analogue and a selecTive inhibiTor of The cdk2–Cyclin E as well as cdk7–Cyclin H and cdk9–Cyclin T. MaTerials and meThods: The acTiviTy of CYC202 was TesTed in four human MCL cell lines: REC, GranTa-519, JeKo-1 and NCEB-1. The effecT of CYC202 on The cell cycle and on apopTosis-, cell-cycle- and TranscripTion-regulaTion-relaTed proTeins was assessed. ResulTs: The IC50 was 25mM for REC, GranTa-519 and JeKo-1 cells and 50mM for NCEB-1 cells. CYC202 caused an accumulaTion of cells in The G2–M phase of The cell cycle and apopTosis. CYC202 caused down-regulaTion of Cyclin D1 and Mcl-1 proTein levels, possibly because of The inhibiTion of TranscripTion elongaTion. Conclusions: Our daTa suggesT ThaT CYC202 is an acTive agenT in MCL. The concomiTanT decrease of The phosphorylaTed and ToTal forms of RNA polymerase II suggesTs ThaT This could be The main mechanism mediaTing The biological effecTs of CYC202 in MCL cells. The drug mighT represenT a new TherapeuTic agenT in This lymphoma subType. Key words: apopTosis, cdk inhibiTor, Cyclin D1, lymphom
Ruichuan Chen - One of the best experts on this subject based on the ideXlab platform.
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a human immunodeficiency virus Type 1 TaT like arginine rich rna binding domain is essenTial for hexim1 To inhibiT rna polymerase ii TranscripTion Through 7sk snrna mediaTed inacTivaTion of p Tefb
Molecular and Cellular Biology, 2004Co-Authors: Jasper H N Yik, Ruichuan Chen, Andrea C Pezda, Craig S Samford, Qiang ZhouAbstract:The HEXIM1 proTein inhibiTs The kinase acTiviTy of P-TEFb (CDK9/Cyclin T) To suppress RNA polymerase II TranscripTional elongaTion in a process ThaT specifically requires The 7SK snRNA, which mediaTes The inTeracTion of HEXIM1 wiTh P-TEFb. In an aTTempT To define The sequence requiremenTs for HEXIM1 To inTeracT wiTh 7SK and inacTivaTe P-TEFb, we have idenTified The firsT 18 amino acids wiThin The previously described nuclear localizaTion signal (NLS) of HEXIM1 as boTh necessary and sufficienT for binding To 7SK in vivo and in viTro. This 7SK-binding moTif was essenTial for HEXIM1's inhibiTory acTion, as The HEXIM1 muTanTs wiTh This moTif replaced wiTh a foreign NLS failed To inTeracT wiTh 7SK and P-TEFb and hence were unable To inacTivaTe P-TEFb. The 7SK-binding moTif alone, however, was noT sufficienT To inhibiT P-TEFb. A region C-Terminal To This moTif was also required for HEXIM1 To associaTe wiTh P-TEFb and suppress P-TEFb's kinase and TranscripTional acTiviTies. The 7SK-binding moTif in HEXIM1 conTains clusTers of posiTively charged residues reminiscenT of The arginine-rich RNA-binding moTif found in a wide varieTy of proTeins. ParT of iT is highly homologous To The TAR RNA-binding moTif in The human immunodeficiency virus Type 1 (HIV-1) TaT proTein, which was able To resTore The 7SK-binding abiliTy of a HEXIM1 NLS subsTiTuTion muTanT. We propose ThaT a similar RNA-proTein recogniTion mechanism may exisT To regulaTe The formaTion of boTh The TaT-TAR-P-TEFb and The HEXIM1-7SK-P-TEFb Ternary complexes, which may help converT The inacTive HEXIM1/7SK-bound P-TEFb inTo an acTive one for TaT-acTivaTed and TAR-dependenT HIV-1 TranscripTion.
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PhosphorylaTed PosiTive TranscripTion ElongaTion FacTor b (P-TEFb) Is Tagged for InhibiTion Through AssociaTion wiTh 7SK snRNA
Journal of Biological Chemistry, 2003Co-Authors: Ruichuan Chen, Zhiyuan Yang, Qiang ZhouAbstract:The posiTive TranscripTion elongaTion facTor b (P-TEFb), comprising CDK9 and Cyclin T, sTimulaTes TranscripTion of cellular and viral genes by phosphorylaTing RNA polymerase II. A major porTion of nuclear P-TEFb is sequesTered and inacTivaTed by The coordinaTed acTions of The 7SK snRNA and The HEXIM1 proTein, whose induced dissociaTion from P-TEFb is crucial for sTress-induced TranscripTion and paThogenesis of cardiac hyperTrophy. The 7SK.P-TEFb inTeracTion, which can occur independenTly of HEXIM1 and does noT by iTself inhibiT P-TEFb, recruiTs HEXIM1 for P-TEFb inacTivaTion. To sTudy The conTrol of This inTeracTion, we esTablished an in viTro sysTem ThaT reconsTiTuTed The specific inTeracTion of P-TEFb wiTh 7SK buT noT oTher snRNAs. Using This sysTem, TogeTher wiTh an in vivo binding assay, we show ThaT The phosphorylaTion of CDK9, on possibly The conserved Thr-186 in The T-loop, was crucial for The 7SK.P-TEFb inTeracTion. This phosphorylaTion was noT caused by CDK9 auTophosphorylaTion or The general CDK-acTivaTing kinase CAK, buT raTher by a novel HeLa nuclear kinase. FurThermore, The sTress-induced disrupTion of The 7SK.P-TEFb inTeracTion was noT caused by any prohibiTive changes in 7SK buT by The dephosphorylaTion of P-TEFb, leading To The loss of The key phosphorylaTion imporTanT for 7SK binding. Thus, The phosphorylaTed P-TEFb is Tagged for inhibiTion Through associaTion wiTh 7SK. We discuss The implicaTions of This mechanism in conTrolling P-TEFb acTiviTy during normal and sTress-induced TranscripTion.
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inhibiTion of p Tefb cdk9 Cyclin T kinase and rna polymerase ii TranscripTion by The coordinaTed acTions of hexim1 and 7sk snrna
Molecular Cell, 2003Co-Authors: Jasper H N Yik, Ruichuan Chen, Rieko Nishimura, Jennifer L Jennings, Andrew J Link, Qiang ZhouAbstract:The posiTive TranscripTional elongaTion facTor b (P-TEFb), consisTing of CDK9 and Cyclin T, sTimulaTes TranscripTion by phosphorylaTing RNA polymerase II. IT becomes inacTivaTed when associaTed wiTh The abundanT 7SK snRNA. Here, we show ThaT The 7SK binding alone was noT sufficienT To inhibiT P-TEFb. P-TEFb was inhibiTed by The HEXIM1 proTein in a process ThaT specifically required 7SK for mediaTing The HEXIM1:P-TEFb inTeracTion. This allowed HEXIM1 To inhibiT TranscripTion boTh in vivo and in viTro. P-TEFb dissociaTed from HEXIM1 and 7SK in cells undergoing sTress response, increasing The level of acTive P-TEFb for sTress-induced TranscripTion. P-TEFb was The predominanT HEXIM1-associaTed proTein facTor, and Thus likely To be The principal TargeT of inhibiTion coordinaTed by HEXIM1 and 7SK. Since HEXIM1 expression is induced in cells TreaTed wiTh hexameThylene bisaceTamide, a poTenT inducer of cell differenTiaTion, TargeTing The general TranscripTion facTor P-TEFb by HEXIM1/7SK may conTribuTe To The global conTrol of cell growTh and differenTiaTion.
