1、细胞周期调控与细胞周期调控与p53系列研究系列研究张令强张令强军事医学科学院军事医学科学院放射与辐射医学研究所放射与辐射医学研究所蛋白质组学国家重点实验室蛋白质组学国家重点实验室ProteomeProteomeSKYSKY 1979年，被克隆发现年，被克隆发现 1993年，被年，被Science（科学）杂志评为年度明星分子（科学）杂志评为年度明星分子(Molecule of the Year 1993)人体一半以上的肿瘤有人体一半以上的肿瘤有p53基因的突变基因的突变/变异，另外一半的肿瘤有变异，另外一半的肿瘤有p53相关通路分子的异常相关通路分子的异常 是迄今为止已发现的与人类肿瘤发生相关性
2、最高的抑癌基因是迄今为止已发现的与人类肿瘤发生相关性最高的抑癌基因 被称为被称为“基因组守护神基因组守护神”、“分子警察分子警察”全世界有近全世界有近5万篇万篇p53相关的研究论文发表，平均相关的研究论文发表，平均3篇篇/天天 科学家、制药公司、临床医生关注的热点分子科学家、制药公司、临床医生关注的热点分子p53：与癌症发生、发展最为密切的抑癌基因：与癌症发生、发展最为密切的抑癌基因发展中国家发展中国家发达国家发达国家肺癌肺癌直肠癌直肠癌 乳腺癌乳腺癌胃癌胃癌 肝癌肝癌 前列腺癌前列腺癌 宫颈癌宫颈癌 头颈癌头颈癌 食管癌食管癌 白血病白血病 淋巴瘤淋巴瘤 卵巢癌卵巢癌 膀胱癌膀胱癌 肺癌肺癌
3、胃癌胃癌 肝癌肝癌恶性肿瘤中恶性肿瘤中p53基因发生突变、失去基因发生突变、失去“警察警察”功能功能 Thirty years of p53 studyArnold J.Levine and Moshe Oren.Nat.Rev.Cancer(2009)9,749Functional domains of p53Vogelstein B et al.Nature,2000,408:307p53：DNA损伤反应网络的核心调节分子损伤反应网络的核心调节分子p53的翻译后修饰的翻译后修饰Kruse and Gu.Cell,2009,137:609-622.磷酸化、泛素化、乙酰化、甲基化、磷酸化、泛素
4、化、乙酰化、甲基化、SUMO化等化等p53分子调控网络分子调控网络Reported InteractionsNovel InteractionsTP53OthersInteresting Preys71 proteins85 interactionsClassic model of p53 activationJan-Philipp Kruse and Wei Gu.Cell(2009)137,609Jan-Philipp Kruse and Wei Gu.Cell(2009)137,609Modified model of p53 activationModified model of p5
5、3 activationJan-Philipp Kruse and Wei Gu.Cell(2009)137,609ApakSetd2SmurfPACTp53 contributes to multiple normal processes and disease pathologiesKaren H.Vousden and Carol Prives.Cell(2009)137,413鉴定了一系列鉴定了一系列p53调控分子及其功能调控分子及其功能p53ATMPACTApakUbPAcSmurfSET2Mep53p53MDM2ARFPNAS(2007)JBC(2010)NCB (2009)MCB
6、i(2010)FEBS Lett(2010)Cell Signal(2008)PACT binds to Rb and p53RbG0/G1SNormal cellp53Tumor cellPACTPMLMDM2NPMGankyrinleukemiaMany cancersHCCPACT:p53 associated cellular protein testis derivedalso known as RBBP6:Rb binding protein 6Human PACT-1,1659aaMouse PACT,1560aa 831 1005 1300 1411 1594-1649 ZFR
7、INGRb bindingp53 binding1 27 42 122 162 425-475 549-593 SRPEK518-552 RINGRb bindingp53 binding1 57 97 360-410 450-496 732 906 1201 1312 1495-1550 SRPKQuestions:1,The physiological function of PACT remains unknown;2,The significance of PACT in p53 regulation is unclear.Establishment of PACT knock-out
8、 mice modelCollaboration with Dr.Xiao YangPACT knock-out:embryonic lethal at E7.5WTKOTUNEL assay to indicate apoptosisWTKODevelopment delayPACT KO mice display increased apoptosiswhich might be caused by upregulated p53 activityp53 null mutation rescue PACT KO mice from E7.5 to E11.5Although less ad
9、vanced than their Pact+/+p53-/-littermates(Left),Pact-/-p53-/-embryosdeveloped an anterior-posterior pattern with a head,trunk,and tail region(Right).E10.5PACT promotes p53 ubiquitination and degradation mediated by Mdm2PACT inhibits the transcriptional activity of p53Inactivation of PACT stabilizes
10、 p53 and induces p53-dependent apoptosis or growth retardationConclusions1.Genetic evidence indicated that PACT is a critical regulator of p53 during embryogenesis.PACT promotes Mdm2-mediated p53 degradation.鉴定了一系列鉴定了一系列p53调控分子及其功能调控分子及其功能p53ATMPACTApakUbPAcSmurfSET2Mep53p53MDM2ARFPNAS(2007)JBC(2010
11、)NCB (2009)MCBi(2010)FEBS Lett(2010)Cell Signal(2008)Mdm2 structure and its interacting proteinsp53 bindingZn1 19 102305 322438 478 491RINGNESNLSNoLS181-185 191-205 464-471Ubc940-59Smurf181-120 p300102-222 ARF210-244 acidic221 274L5/L11/L26284-374 RB273-321 Gankyrin412-437 Mdm2/MdmX438-491 Mdm2*AktS
12、166/S186ATMS395Question:How Mdm2 stability is regulated is not fully understood.Smurf belong to the HECT-type,Nedd4 family of ubiquitin ligase(E3)Smurf:Smad ubiquitination regulatory factorCa2+&p-lipid bindingSubcellular localizationSubstrate bindingRecognize PYE2 binding(N-lobe)Ubiquitin binding(C-
13、lobe)C2HECTWWSmurf1Smurf stabilizes MDM2 and inhibits its autoubiquitinationStabilizationDegradation rateUbiquitinationSmurf negatively regulates p53 dependent of Mdm2Transcriptional activityUbiquitinationUbiquitinationSmurf interacts with MDM2 in vitro and in vivoMDM2 N-terminal 75-114 interacts wi
14、th Smurf1Smurf1-WW2 but not WW1 domain interacts with MDM2Smurf enhances MDM2-MDMX interaction and inhibits MDM2 oligomerizationMDM2SmurfMDM2MDM2MDMXLower cis-E3 activityStabilization of MDM2 Higher trans-E3 activityHigh degradation of p53High cis-E3 activityAutodegradation Low trans-E3 activityLow
15、degradation of p53HECT domain-dependentE3 activity-independentAmong the Nedd4 family,four members(Smurf1/2,NEDL1/2)interact with and stabilize MDM2Why these four members?Identification of a SRF motif in the last WW domain of Smurf1/2&NEDL1/2,but not other fiveSRF motif is required for Smurf1 interac
16、ting with and stabilizing MDM2SRF motif is required for Smurf1 regulating MDM2/MDMX heterodimerizationSmurf1 regulates p53 activity dependent of the MDM2-interacting SRF motifSmurf and NEDL coordinate to regulate apoptosisConclusions1.Genetic evidence indicated that PACT is a critical regulator of p
17、53 during embryogenesis.PACT promotes Mdm2-mediated p53 degradation.2.Nedd4 family E3 ligases(Smurf1/2 and NEDL1/2)plays a role in Mdm2 stability control in their E3 activity-independent manner.鉴定了一系列鉴定了一系列p53调控分子及其功能调控分子及其功能p53ATMPACTApakUbPAcSmurfSET2Mep53p53MDM2ARFPNAS(2007)JBC(2010)NCB (2009)MCB
18、i(2010)FEBS Lett(2010)Cell Signal(2008)选择性激活选择性激活p53诱导的诱导的凋亡凋亡对于肿瘤治疗具有重要意义对于肿瘤治疗具有重要意义Karen H.Vousden and Carol Prives.Cell(2009)137,413OsterixInhibition of bone formationp53p53选择性调控是选择性调控是p53p53领域极为重要领域极为重要却还很不清楚的问题却还很不清楚的问题Apak,a novel p53 interacting protein,belongs to KRAB-type zinc finger famil
19、yKZNF:KRAB type zinc finger protein Apak：ATM&p53 associated KZNF protein688667139665ApakZinc Finger Repeats28 SQ/TQ motifs,potential ATM/ATR phosphorylation sitesp53Yeast two-hybridApak,a novel p53 interacting protein,belongs to KRAB-type zinc finger familyKZNF family is the largest family of transc
20、riptional regulators in mammals 423 members in human arranged in clusters(e.g.19q,3p,9p etc.)81 clusters(70%genes)Embryonic development Regulation of cell cycle Cell differentiation and proliferationStuart Huntley et al,Genome Research 2006,16:669Little is known about the role of KZNF family in p53
21、regulationApak represses transcriptional activity of p53 and negatively regulates p53-mediated apoptosisApak overexpressionApak knockdownApak selectively regulates the proapoptotic p53 target genesproapoptoticproarrestU2OSH1299Apak selectively regulates the proapoptotic p53 target genesApak overexpr
22、essionApak knockdownApak interacts with p53 both in vitro and in vivoMapping the interacting regionsBoth the N-terminal KRAB domain and the interacting zinc fingers are required for Apak to repress p53KRAB domain of Apak recruits KAP-1-HDAC1 complex to regulate p53 acetylationRaul Urrutia,Genome Bio
23、logy 2003,4:231Inhibitor of HDAC I III Apak represses p53 activity through regulating p53 acetylationKAP-1 is required for Apak to recruit HDAC1Apak could inhibit p53 in various cell lines but not in ATM-mutated ATS4 cellsApak requires ATM to repress p53Apak requires ATM to repress p53Surprising rep
24、ression role of ATM on p53 regulationApak requires ATM to repress p53Apak interacts with ATM(requires KAP-1)Apak forms a complex with KAP1,ATM,HDAC1 and p53 in vivoIn response to DNA damage,Apak is negatively regulated dependent of ATMIn response to DNA damage,Apak dissociates from p53&ATM(dependent
25、 of ATM)but retains the interaction with KAP-1 and HDAC1Apak dissociates from p53 dependent of ATM kinaseApak is phosphorylated by ATM kinaseApak is phosphorylated by ATM kinase on Ser68Phosphorylation of Apak is critical for p53-induced apoptosisuntreatedMMS 4 hPhosphorylation of Apak is critical f
26、or p53-induced apoptosisATMPumaBaxnucleusApakp53Ap53AIP1 NoxaKAP-1HDAC1DNA damagePPPPModel for Apak function and regulation in unstressed and stressed cellsapoptosisApak与与ATM协同协同选择性选择性调控调控p53介导的细胞介导的细胞凋亡凋亡Tian C et al.,Nature Cell Biology 688667139665Apak:ATM and p53 associated KZNF proteinATM-ApakA
27、TM-p53非应激非应激 应激应激 phosphorylationp53ApakDNA damageATM发现了发现了ATM在在p53调控调控中的中的“两面性两面性”apoptosisPAcCell.2009 May 15;137(4):609-622.Apak选择性调控选择性调控p53活性的活性的NCB文章被国际顶级文章被国际顶级刊物刊物Cell的的p53发现发现30周年纪念性综述引用周年纪念性综述引用p53ApakDNA damageKinases(ATM)Normal cellTumor cellOncogenic insultsARFMMS,etoposide,doxorubicin,
28、camptothecin,cisplatin Actinomycin D,5-fluorouracil,-lipoic acidp53ApakDNA damageKinases(ATM)Normal cellTumor cellOncogenic insultsARFMMS,etoposide,doxorubicin,camptothecin,cisplatin Actinomycin D,5-fluorouracil,-lipoic acidc-MYC,E2F1,Ras-G12VEctopic expressionApak is negatively regulated in respons
29、e to oncogenic stressDNA damage oncogene stressDNA damage oncogene stressDNA damage oncogene stressApak dissociates from p53 independent of ATM-mediated phosphorylationApak dissociates from p53 dependent of ARFApak interacts with ARF both in vitro and in vivoMapping the interacting regions:ARF(46-64
30、)&Apak-ZFThe interaction is required for ARF to regulate ApakOncogenic stress-induced ARF competes with p53 to interact with Apak and disrupts the Apak-p53 interactionATMPumaBaxnucleusApakp53Ap53AIP1 NoxaKAP-1HDAC1OncogenicstressapoptosisARFARFARFDNA损伤和癌基因激活条件下损伤和癌基因激活条件下Apak分别以分别以ATM和和ARF依依赖的方式被负调控
31、，从而使得赖的方式被负调控，从而使得p53激活激活Apak研究首次揭示研究首次揭示KRAB家族锌指蛋白在家族锌指蛋白在p53选择性调控中具有重要功能选择性调控中具有重要功能 人类基因组中多达人类基因组中多达423个成员个成员 在染色体上成簇分布在染色体上成簇分布(19q,3p,9p)参与细胞分化、增殖、周期调控参与细胞分化、增殖、周期调控 组织器官分布各异组织器官分布各异ApakKZNFbKZNFaKZNFc庞大的庞大的KRAB家族锌指蛋白（家族锌指蛋白（KZNF）家族）家族在在p53调控中可能发挥重要作用？调控中可能发挥重要作用？p53靶基因靶基因细胞学效应细胞学效应PumaBaxp53AI
32、P1NoxafasPIGsSiah-1scotinSCO2DRAMSestrinsPTENLIF细胞凋亡细胞凋亡周期阻滞周期阻滞细胞代谢细胞代谢衰老衰老/生殖生殖发育发育14-3-3 Gadd45p21B99TIGARGAMTKAIPAIBAI-1TSP1血管生成与转血管生成与转移抑制移抑制KZNFbKZNF家族家族调控分子调控分子ApakKZNFaKZNFdKZNFcKZNF家族选择性负调控家族选择性负调控p53介导的细胞学效应介导的细胞学效应KRAB家族锌指蛋白中家族锌指蛋白中p53调控分子的筛选调控分子的筛选调取所有调取所有KZNFKZNF成员序列信息成员序列信息鉴定能与鉴定能与p53p
33、53和和ATMATM相互作用的成员相互作用的成员筛选应激条件下对筛选应激条件下对p53p53抑制作用解除的成员抑制作用解除的成员 这些分子依次命名为这些分子依次命名为Apak2Apak2、Apak3 Apak3 构建表达载体构建表达载体分析含有分析含有ATMATM磷酸化位点磷酸化位点SQ/TQSQ/TQ的成员的成员明确对明确对p53p53调控的选择性调控的选择性423423个个筛选可调控筛选可调控p53p53活性分子活性分子4848个个3939个个9 9个个9 9个个（部分完成）（部分完成）鉴定到多个新的鉴定到多个新的p53选择性调控分子选择性调控分子0 0202040406060808010
34、0100120120Myc-ZNF -420 333 382 248 566 8 475 543 195 Relative p53 activity(%)DNA修复修复 凋亡、自噬凋亡、自噬细胞代谢细胞代谢KRAB型锌指蛋白家族在型锌指蛋白家族在p53调控中功能的发现调控中功能的发现为肿瘤研究、药物研发打开了一座巨大的为肿瘤研究、药物研发打开了一座巨大的“宝库宝库”Conclusions1.Genetic evidence indicated that PACT is a critical regulator of p53 during embryogenesis.PACT promotes
35、Mdm2-mediated p53 degradation.2.Nedd4 family E3 ligases(Smurf1/2 and NEDL1/2)plays a role in Mdm2 stability control in their E3 activity-independent manner.3.Identify Apak as a specific and negative regulator of p53-mediated apoptosis.In response to DNA damage and oncogenic stresses,Apak dissociates
36、 from p53 dependent of ATM and ARF,respectively.4.Display the dual regulation role of ATM on p53 and Apak.5.Hypothesize that the large KRAB type zinc finger(KZNF)family might contribute to spatial and temporal regulation of p53 activity.ApakApak如何选择性调控如何选择性调控p53p53凋亡相关靶基因的表达凋亡相关靶基因的表达Apak利用利用CASTCAS
37、T技术筛选技术筛选ApakApak结合的结合的DNADNA保守序列保守序列保守序列：保守序列：TCTTN2-30TTGT 利用利用CASTCAST技术筛选技术筛选ApakApak结合的结合的DNADNA保守序列保守序列P53 binding Consensus PuPuPu-C(A/T)(A/T)G-PyPyPyApoptosis p53AIP1CCTCCTCTCTTGCCCGGGCTTGTCGAGATG p53AIP1CCTCCTCTCTTGCCCGGGCTTGTCGAGATG BaxGGGCTCACAAGTTAGAGACAAGCCTGGGCG PumaCGCGCCTGCAAGTCCTGAC
38、TTGTCCGCGGCCell-cycle arrest GADD45 GTACAGAACATGTCTAAGCATGCTGGGGAC p21 GTCAGGAACATGTCCCAACATGTTGAGCTC p53 p53靶基因中靶基因中p53AIP1p53AIP1的内含子特异地含有的内含子特异地含有ApakApak结合元件且与结合元件且与p53p53结合元件部分重叠结合元件部分重叠Free probeSuper shift Apak/p53-BS+Myc-Apak+Apak-Ab-+-Competitor-+Apak-DNAEMSA123BApak/p53-BS+His-Apak-+Compet
39、itor -+123Apak-DNAFree probeEMSAAIPC10%InputNormal IgGApak123p53AIP1D Apak Apak结合结合p53AIP1p53AIP1的内含子的内含子在细胞内在细胞内Apak参与参与p53AIP1 基因的结合基因的结合体外实验表明体外实验表明Apak直接与直接与p53AIP1 基因基因p53应激元件结合应激元件结合Apak/p53-BS+-Mut4-+-Mut5-+-Mut6-+His-Apak+EFree probeApak-DNAEMSA1234Apak/p53-BS+-Mut4-+-Mut5-+-Mut6-+GST+-GST-p
40、53-+pAb421+Fp53DNAcomplexesFree probe12345 Apak Apak结合结合p53p53蛋白与蛋白与DNADNA对锌指的需求不同对锌指的需求不同AB 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 Relative Luciferase activityApak/p53-BS Lucp53-+Apak-+p53Apak-actinApak/p53-BS+-Mut7-+GST-+GST-p53+-pAb421+Dp53DNAcomplexFree probe1234Apak/p53-BS+-Mut7-+His-Apak-+-+CFree pro
41、beApak-DNA123EMSA0.0 1.0 2.0 3.0 4.0 5.0 Relative Luciferase activityApak/p53-BS-Mut7 LucEp53-+Apak-+p53F+-actinApakApakApak利用其利用其DNADNA结合能力抑制结合能力抑制p53AIP1p53AIP1转转录录Apak抑制抑制p53激活的激活的p53AIP1 基因的转录基因的转录不能参与不能参与DNA结合的结合的Apak丧失抑制功能丧失抑制功能Mutant 7丧失丧失Apak的结合能力，但仍结合的结合能力，但仍结合p53ApakApak与与p53p53直接竞争结合直接竞争结
42、合p53AIP1p53AIP1内含子内含子ApakApak特异结合特异结合p53AIP1p53AIP1内含子内含子ApakApak对对p53AIP1p53AIP1基因的调控效应显著高于其他基因基因的调控效应显著高于其他基因DNADNA损伤条损伤条件下，件下，ApakApak与与p53AIP1p53AIP1基因解基因解离离DNADNA损伤条损伤条件下，件下，ApakApak从核质向核仁移位从核质向核仁移位p53ATMApakAcPumaBaxp53AIP1 Noxap53ATMApakp53AIP1ApakApak选择性调控选择性调控p53p53下游凋亡基因的机制下游凋亡基因的机制 p53靶基因
43、靶基因细胞学效应细胞学效应PumaBaxp53AIP1NoxafasPIGsSiah-1scotinSCO2DRAMSestrinsPTENLIF细胞凋亡细胞凋亡周期阻滞周期阻滞细胞代谢细胞代谢衰老衰老/生殖生殖发育发育14-3-3 Gadd45p21B99TIGARGAMTKAIPAIBAI-1TSP1血管生成与转血管生成与转移抑制移抑制KZNFbKZNF家族家族调控分子调控分子ApakKZNFaKZNFdKZNFcKZNFKZNF家族选择性负调控家族选择性负调控p53p53介导的细胞学效应介导的细胞学效应Modified model of p53 activationJan-Philipp Kruse and Wei Gu.Cell(2009)137,609ApakSetd2SmurfPACTp53ATMPACTApakUbPAcSmurfSET2Mep53p53MDM2ARFAcknowledgementsLingqiang Zhang group Dahu Li Jing Nie Chunyan Tian Lin Yuan Shan Wang Ping Xie
