两对人类蛋白质（MSK1与CK2，NLK与SMAD4）相互作用及其磷酸化的研究

【作者】 石彦；

【导师】 霍克克；

【作者基本信息】 复旦大学 ， 遗传学， 2008， 博士

【摘要】 本论文分为两部分,第一部分是关于CK2与MSK1的相互作用及其对后者的磷酸化研究。第二部分是关于NLK和SMAD4的相互作用及其磷酸化的研究。在论文的第一部分,我们对MSK1两个剪切本的亚细胞定位进行了研究比较,新发现了CK2和MSK1之间的磷酸化作用关系。MSK1有两个长度不同的剪切本,Trans1和7rans2。我们通过对MSK1两个剪切本在HeLa细胞中的亚细胞定位情况进行研究,不仅证实了长的剪切本Trans1定位于细胞核中,而且发现Trans2定位于细胞质中。因此我们推测MSK1的C-端包含有负责MSK1细胞定位的核定位信号序列。我们正在构建一系列缺失突变体来确认这段序列。我们以MSK1为诱饵筛选人肝cDNA文库,筛选到了猎物CK2全酶中的β亚基。我们用GST-Pull Down和细胞免疫共沉淀的实验分别在体内和体外验证了这种相互作用的特异性。因为CK2全酶是由两个α或α’和两个β亚基组成的四聚体,所以我们也用免疫共沉淀的方法验证了MSK1和CK2α亚基之间的相互作用。我们发现MSK1和CK2之间的相互作用在细胞血清饥饿培养、anisomycin刺激培养的情况下都可以发生,并且这种相互作用可以被p38 MAPK通路的特异性抑制剂SB203580所抑制。为了确定MSK1和CK2发生相互作用的区域,我们通过构建一系列的MSK1缺失突变体,发现了MSK1的N-端和C-端激酶结构域都可以与CK2β发生相互作用,而只有C-端激酶结构域和CK2α发生相互作用。为了研究MSK1和CK2之间相互作用的生物学意义,我们分析了两者之间的磷酸化关系。我们首先用体外磷酸化的实验发现了CK2可以磷酸化MSK1的C-端区域,然后又利用质谱分析发现CK2能够磷酸化MSK1的10个丝氨酸或苏氨酸位点(分别为Ser-691,Ser-694,Thr-753,Thr755,Ser-757,Ser-758,Ser-759,Ser-760,Ser-762,Thr-793),同时发现这些位点都位于C端激酶结构域下游的一个肽段中。此外,我们还利用点突变的方法,验证了Ser-757和Ser758这两个被磷酸化位点的真实性,并且发现Ser-657也是CK2的一个磷酸化位点,但这个位点在质谱实验中没有被分析出来。在这部分工作中,我们确定了MSK1 trans1剪切本的C-末端序列中包含有核定位序列。首次发现了MSK1和CK2这一对新的相互作用,这种相互作用是p38通路活性所依赖的,并且发现了CK2能够磷酸化MSK1 C-端激酶结构域和后面肽段里面的11个丝氨酸和苏氨酸位点,这些位点的磷酸化可能在空间结构上对MSK1的激活提供了帮助。在论文的第二部分,我们首先以SMAD4作为诱饵,用酵母双杂交的方法筛选到了相互作用蛋白NLK,然后通过GST-Pull Down,免疫共沉淀的方法验证了SMAD4和NLK之间的相互作用,并且发现两者之间的相互作用不依赖于NLK的激酶活性。我们通过构建SMAD4缺失突变体,发现了SMAD4的接头结构域是和NLK发生相互作用的区域。因为接头结构域和SMADs蛋白的泛素化降解相关,这个结果提示NLK可能会通过磷酸化SMAD4介导它的降解。亚细胞共定位实验发现当单独转染SMAD4时,它在细胞质和细胞核中均有分布,但是主要均匀弥散地分布在细胞质中,当与NLK共转染时,它变成了不均匀的点状分布,并且有向核内聚集的趋势,可以和NLK共定位于细胞质和核中。这说明NLK能够改变SMAD4蛋白的亚细胞定位。我们用体外磷酸化实验证明NKL可以在体外特异性地磷酸化SMAD4,通过构建缺失突变体,进一步实验证明NLK磷酸化SMAD4的位点位于MH1结构域和接头结构域中,而不是在MH2结构域中。在细胞中共转染SMAD4和NLK,随着NLK转染量的梯度增多,SMAD4的表达量明显呈梯度下降趋势。这说明过量表达NLK会降低细胞中SMAD4的蛋白量。但这种现象是通过转录水平抑制,还是通过磷酸化介导SMAD4泛素化降解还不清楚,我们正在进行深入的研究。在第二部分中,我们新发现了SMAD4和NLK这对新的相互作用,他们的相互作用是不依赖于NLK激酶活性的。并且发现NLK可以体外磷酸化SMAD4,并且改变它的亚细胞定位。当NLK过量表达时,会降低SMAD4在细胞中的蛋白量。因为SMAD4是SMADs家族蛋白中的核心成员,也是一个重要的抑癌基因,它与各种不同的SMADs蛋白协同作用是TGF-β超家族信号途径转导中的关键环节。NLK是进化中十分保守的丝/苏氨酸激酶,在组织的发育中起着重要的作用。本文的研究结果为进一步深入研究这两个蛋白质的功能及其调控机制奠定了有利的基础。更多还原

【Abstract】 This thesis is composed of two separate parts:The phosphorylation of MSK1 by CK2 and the function of it;NLK can reduce the protein level of SMAD4 through phosphorylate SMAD4.In partⅠ,we found the different subcellur localization of the two transcripts of MSK1.Identified MSK1 can be phosphorylated by CK2.MSK1 have two isoforms, trans1 and trans2.We found that the longer trans1 localized in the nuclear,but the shorter trans2 localized in the cytoplasm.This suggest that the sequence trans1 longer than shorter have the nuclear localization sequence.We are constructing series truncated mutants to identife the NLS sequence.Through yeast two-hybrid screening,we obtained CK2β.We identified the interaction between MSK1 and CK2 in vitro and in vivo.And also identified the interaction between MSK1 and CK2α.We found MSK1 can interact with CK2 under starverd and anisomycin stimulated condition. This interaction can be inhibited by SB203580,a p38 specific inhibitor.Through truncated mutants CoIP,we identified CK2βcan interact with both the N-terminal truncate and the C-terminal truncate,but CK2αcan only interact with the N-termianl truncate.Using theμRPLC-ESI-MS,we identified 10 Ser/Thr sites within the C-terminal of MSK1 can be phosphorylated by CK2:Ser-691,Ser-694,Thr-753,Thr755,Ser-757, Ser-758,Ser-759,Ser-760,Ser-762,Thr-793.We further identified Ser-657 as another site can be phorylated by CK2 using the site mutation method which is not identified by mass spectrum.Taken together,we found there is a nuclear localization sequence in the C-terminal sequence of MSK1.Identified the interection between MSK1 and CK2,the interaction is p38 MAPK signal dependent.And further identified 11 sites within the C-termianl of MSK1 can be phosphorylated by CK2.These phosphorylation sites may play an important role in the alteration of the three dimensional structure of MSK1 leading it to activation.In partⅡ,we first use yeast two-hybrid system identified the interaction between the SMAD4 and NLK.We further identified the interaction between SMAD4 and NLK in vitro and in vivo,and found the interaction is kinase activity independent.Series truncated mutants assay displayed that the linker sequence between the MH1 domain and MH2 domain of SMAD4 are sufficient and necessary for this specific interaction.The linker sequence is relevant to polyubiquitination -dependent degradation.So we suggest that NLK can induce the degradation of SMAD4.Colocalization assay indicate that the localization of SMAD4 can be altered when cotransfected with NLK.It altered from equal localization in cytoplasm and in nuclear to assemble localization in cytoplasm and a little can switch into the nuclear.We identified SMAD4 can be phosphorylated by NLK in vitro,and the phosphorylate site is within the MH1 domain and the linker sequence.When overexpress NLK in 293T cell,the protein level of SMAD4 is reduced.This reduction happened in transcription level or through polyubiquitination-dependent degradation is unknown.The mechanism need our further research.In partⅡ,we identified the interaction between SMAD4 and NLK.The interaction is kinase activity independent.We also found NLK can phosphorylate SMAD4 and can alter the localization of SMAD4.We further identified when overexpress NLK,the protein level of SMAD4 can be reduced.SMAD4 is the core member of SMADs family,is an important tumor-suppress gene.The interaction between SMAD4 and R-SMADs is important for the TGF-βsignaling transduction.NLK is one conserved Ser/Thr kinase,having important role in the tissue organization.Our research provide more advantaged information for further research of the function and control mechanism of these two proteins.更多还原