【作者】 宗鸿亮；

【导师】 顾建新；

【作者基本信息】 复旦大学 ， 生物化学与分子生物学， 2006， 博士

【摘要】 CDK11相关蛋白及cyclin D3／CDK11调控雄激素受体转录活性的研究细胞周期素依赖的激酶11（p58）(CDK11^P58，p58^PITSLRE，p58^GTA，p58^clk-1)是最早被发现的CDK11家族蛋白，在β-1，4-半乳糖基转移酶1分离纯化过程中得到，能与β-1，4-半乳糖基转移酶1相互作用，并磷酸化β-1，4-半乳糖基转移酶1，对其活性起增强作用。CDK11^p58在细胞内特异性地表达于G2／M期，并且过表达CDK11^p58能导致细胞生长缓慢，形态发生变化，有丝分裂障碍，阻滞在G2／M期。CDK11^p58具有促凋亡作用，且该作用与其激酶活性密切相关。虽然CDK11^p58在细胞生长中发挥着负调控子的重要作用，但是它的上游调控因子和下游的效应分子还没有被发现。最近的研究发现CDK11家族另一主要成员CDK11^p110参与了RNAPⅡ转录复合物的形成，并与RNA剪切拼接因子RNPS1、cyclin L等相关，因此推测CDK11信号途径与转录及转录后加工有关。我们以全长的CDK11^p58为诱饵蛋白，利用LexA酵母双杂交系统，在人胎肝cDNA文库中筛选其相互作用蛋白，并发现一个MYST家族组蛋白乙酰转移酶HBO1（Histone acetyltransferase binding to ORC1）的羧基端（330～611位氨基酸）能与CDK11^p58在酵母内发生相互作用。接着我们利用GST-pull down和免疫共沉淀的方法，证明了全长的HBO1蛋白与CDK11^p58在体外的直接结合和在细胞内的相互作用。由于CDK11^p58特异表达在G2／M期，因此内源性CDK11^p58与HBO1的生理性相互作用也仅可在细胞周期的G2／M期检测到。利用免疫荧光结合激光共聚焦我们发现该两者的相互作用发生在细胞核内。HBO1是在1999年新被克隆的具有组蛋白乙酰转移酶（HAT）活性的蛋白分子，与转录、复制均有密切关系。我们进一步研究发现在体外HAT检测反应体系中直接加入原核表达的CDK11^p58纯化蛋白，能显著促进HBO1对的HAT活性；在细胞内过表达CDK11^p58后，内源性HBO1的HAT活性也有增强；作为对照，另一个著名的组蛋白乙酰转移酶p300的HAT活性在体外及细胞内均不受CDK11^p58的影响。这些结果提示CDK11^p58可能通过对HBO1的HAT活性的调控，参与特定靶基因的转录调控，从而发挥其阻遏细胞周期及促凋亡的功能。由于HBO1已证明是雄激素受体（AR）的一个转录共抑制子，而我们实验室也报道了细胞周期素D3（cyclin D3）能与甾体类激素受体——维生素D受体（VDR）、以及激活转录因子（ATF5）结合，并调节这两者的转录活性。Cyclin D3的同源蛋白cyclin D1对AR的抑制性调控已有多篇文献报道。结合上述情况，我们猜测作为HBO1和cyclin D3的相关分子，CDK11^p58可能参与了真核细胞的转录调控。我们从AR着手，发现CDK11⁵⁸能与AR在体外和细胞内发生相互作用，并且这种作用并不依赖于AR共抑制子HBO1的介导。AR在结构上可分为转录激活结构域（TAD）、DNA结合结构域（DBD）、铰链区和配体结合结构域（LBD）几个功能相对独立的区域。CDK11^p58蛋白则有一个位于中间的丝／苏氨酸激酶活性结构域和功能尚不明确的氨基端、羧基端。我们根据这两个蛋白的功能结构域，构建了一系列片段突变体，通过体内、体外结合实验证实了介导AR与CDK11^p58相互作用的结构域分别为AR转录激活结构域氨基端的转录激活单元1（TAU1）和CDK11^p58的丝／苏氨酸激酶活性结构域。雄激素和AR组成的信号通路对于男性个体生殖系统及其它组织器官的发育、分化，以及男性肿瘤如前列腺癌的发生、发展密切相关。作为核受体家族的成员之一，AR在结合了雄激素后，转位到核内，与靶基因启动子结合，发挥转录因子的活性。利用AR的报道基因MMTV-LUC，我们发现AR介导的转录能被CDK11^p58显著抑制，并有剂量-效应关系，这种效应并不因内源性HBO1受RNAi抑制表达而丧失。CDK11^p58对AR的调控是特异性的，因为其对CMV的转录并无影响，而对AR其它靶基因如PSA-LUC、ARE-LUC均有转录抑制作用。有趣的是，虽然CDK11家族两个主要成员CDK11^p58与CDK11^p110由同一mRNA编码，CDK11^p110除具有羧基端与CDK11^p58完全一致的蛋白序列外，还有一段独有的氨基端，但这两个蛋白在功能上却截然不同。和CDK11^p58对AR的抑制作用相反的是，CDK11^p110能显著促进AR介导的靶基因转录。但在细胞内并不能检测到CDK11^p110与AR的直接相互作用。为了验证CDK11^p58对AR的调控是否依赖其激酶活性，我们构建了CDK11^p58的激酶活性缺失型点突变体D224N。D224N丧失了与AR在体内体外相互作用的能力，却能显著促进AR的转录活性。过表达cyclin D3能促进CDK11^p58的激酶活性，并与CDK11^p58协同抑制AR；利用RNAi抑制cyclin D3的内源性表达后，导致CDK11^p58激酶活性和对AR调控作用的丧失。Cyclin D3也能与AR在体内外相互结合，并可能和CDK11^p58一起与AR形成三元复合物。为了研究AR受CDK11^p58调控的机制，我们从以下几个方面考虑：1)AR的表达；2)AR的核定位；3)AR与共激活子／共抑制子的结合；4)AR与反应元件的结合；5)AR内部N／羧基端功能结构域之间的相互作用；6)AR受cyclin D3／CDK11^p58的磷酸化。结果发现AR的表达、核定位、AR与共激活子p300或共抑制子HDAC1的相互作用、以及AR分子内部N／羧基端结构域相互作用均没有改变，而AR与其反应元件的结合受CDK11^p58的抑制。体外激酶活性检测发现AR的TAD能被CDK11^p58磷酸化。CDK11^p58也能在细胞内促进AR的磷酸化。利用RNAi抑制内源性cyclin D3表达后，CDK11^p58便不能增强AR的磷酸化水平。雄激素／AR信号途径也与前列腺癌细胞的凋亡密切相关。我们发现cyclin D3／CDK11^p58能通过抑制AR的转录活性，促进前列腺癌细胞的凋亡。而激酶活性缺失型CDK11^p58能对细胞的凋亡起保护作用。综上所述，我们发现cyclin D3／CDK11⁵⁸信号途径除能调控细胞周期外，也参与了真核细胞的转录调控。更多还原

【Abstract】 Cyclin-dependent kinase 11 （p58） (CDK11^p58, p58^PITSLRE, p58^GTA, p58^clk-1) is the earliest identified CDK11 family protein. It was obtained originally through purification of beta-1,4-galactosyltransferase 1 （GalT1）. CDK11^p58 interacts with, phosphorylates and enhances the glycosyltransferase activity of GalT1. CDK11^p58 is uniquely expressed in G2/M phase of the cell cycle. Overexpression of CDK11^p58 leads to the decreased cell growth, change in morphology, disorder in mitosis, and arrest in G2/M phase. CDK11^p58 also enhances cell apoptosis in a kinase-dependent manner. Although CDK11^p58 plays an important role in the regulation of cell growth and apoptosis, its upstream regulators and downstream effectors remain unknown.CDK11^p110, another member of CDK11 family, is reported to be associated with the formation of RNAP II complex, and the splicing factors including RNPS1 and cyclin L. Thus, it is suggested that CDK11 signaling may be involved in the eukaryotic transcription and posttranscriptional modification. We screened human fetal liver cDNA library using the full length of CDK11^p58 as the bait. As a result, we found that the C-terminal fragment of HBO1 （histone acetyltransferase binding to ORC1）, an MYST family histone acetyltransferase, interacted with CDK11^p58 in yeast. The interaction between CDK11^p58 and the full length of HBO1 was further confirmed in yeast, through GST-pull down assay in vitro and coimmunoprecipitation in vivo. In consistence with the unique expression of CDK11^p58 in G2/M phase, coimmunoprecipitation of endogenous CDK11^p58 and HBO1 was only detected in G2/M phase-synchronized HeLa cells. CDK11^p58 and HBO1 were colocalized in cell nucleus as sporadic particles. HBO1 was first coloned as a new interacting protein of ORC1 and was found to possess intrinsic histone acetyltransferase （HAT） activity. We found that direct addition of recombinant CDK11^p58 protein in the in vitro HAT activity assay enhanced HBO1 HAT activity significantly. Overexpression of CDK11^p58 resulted in the increased HAT activity of endogenous HBO1 in HeLa cells. As a control, the HAT activity of p300, a well-known HAT and coactivator, remained unaffected by CDK1 l^p58 in vitro and in vivo. The above data suggest that CDK11^p58 may regulate the transcription of certain target genes through enhancing HBO1 HAT activity to inhibit cell cycle or promote apoptosis.According to the fact that HBO1 is a corepressor of AR, and that cyclin D3 is also involved in the regulation of transcription mediated by vitamin D receptor （VDR） or activating transcription factor 5 （ATF5）, and that cyclin Dl is well identified as a corepressor of AR, we hypothesize that cyclin D3/CDK11^p58 signaling may also play an important role in AR-mediated transcription. We found that CDK11^p58 interacted with AR in vitro and in mammalian cells independent of HBO1. AR is structurally divided into four independent functional domains: transcription activation domain （TAD）, DNA-binding domain （DBD）, hinge region and ligand-binding domain （LBD）. CDK11^p58 contains a Ser/Thr protein kinase domain in the middle of the molecule, an N-terminal domain and a C-terminal domain with unknown function. We constructed the deletion mutants of the two proteins based on their functional domains. Coimmunoprecipitation assay showed that the interacting domains of the two proteins were mapped to the transacription activation unit 1 （TAU1） located in AR TAD, and the kinase domain of CDK11^p58.Androgen/AR signaling is essential for the development and differentiation of male productive and non-productive systems, and is also closely related to the carcinogenesis of male tumors such as prostate cancer. Similar to other nuclear receptors, upon binding to androgen, AR is translocated into nucleus, binds to the promoters, and mediates transcription of target genes. Using the AR reporter gene MMTV-LUC, we found that AR-mediated transactivation was inhibited by CDK11^p58 in a dose-dependent manner. This repressive effect of CDK11^p58 on AR is specific because CMV-LUC which is driven by a CMV promoter is not affected by CDK11^p58. Consistently, PSA-LUC and ARE-LUC, the other two AR reporter genes, are also inhibited by CDK11^p58. The repressive role of CDK11^p58 on AR is HBO1-independent. Interestingly, although CDK11^p58 is encoded by the same mRNA as CDK11^p110, and is structurally located to the C-terminal region of CDK11^p110, the two CDK11 isoforms play distinct roles in AR regulation. CDK11^p110 did not interact with AR in cells, but enhanced the transcriptional activity of AR markedly.In order to clarify whether CDK11^p58 mediated AR regulation is dependent on its kinase activity, we constructed D224N, the kinase-deficient point mutant of CDK11^p58. We found that D224N failed to bind to and repress AR, but enhanced AR-mediated transactivation remarkably. Cyclin D3 promoted the kinase activity of CDK11^p58, and repressed the transcriptional activity of AR in synergy with CDK11^p58, while the kinase activity and repressive effect of CDK11^p58 were both lost when cyclin D3-targeted RNAi was used. Cyclin D3 interacted with AR in vitro and in vivo. Cyclin D3, CDK11^p58 and AR may form a triple complex in vivo. To elucidate the molecular mechanism by which CDK11^p58 repressesAR function, the following possible patterns were considered: 1) AR expression, 2) AR nuclear translocation, 3) AR interaction with coactivators/corepressers,4) binding of AR to ARE, 5) AR N/C interaction, and 6) direct targeting of CDK11^p58 to the AR TAD phosphorylation. We found that neither the expression, nor nuclear translocation, nor the interaction of AR with coregulators, nor the interaction of AR N/C-terminal domains was altered. But the binding of AR to its responsive element was inhibited by CDK11^p58. The in vitro kinase activity assay revealed that AR TAD and TAU1 was phosphorylated by CDK11^p58. Overexpression of CDK11^p58 led to the increased phosphorylation of AR in vivo. In the presence of cyclin D3 RNAi, CDK11^p58-mediated AR phosphorylation was quenched. Androgen/AR signaling is essential for the viability of prostate cancer cells. Cyclin D/CDK11^p58 complex is capable of promote the apoptosis of prostate cancer cells both in LNCaP cells or AR-overexpressing PC-3 cells, while the kinase-deficiently mutant D224N protected cells from apoptosis.In summary, we here report that cyclin D3/CDK11^p58 signaling is involved in the transcriptional regulation in eukaryotic cells as well as the cell cycle regulation.更多还原