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与睾丸高表达蛋白质RNF138相互作用的蛋白激酶NLK的功能研究和睾丸特异表达蛋白质RSA-14-44的功能研究
【作者】 梁俊波；
【作者基本信息】 北京协和医学院 ， 生物化学与分子生物学， 2010， 博士
【摘要】 1.丝氨酸/苏氨酸蛋白激酶NLK的功能研究本组前期在精子发生的分子机制研究中分离了一种睾丸高表达蛋白质为RNF138[1],后经研究提示它可与一种进化上保守的丝氨酸/苏氨酸蛋白激酶NLK(Nemo-like kinase, NLK)结合[2]。Zeng等人的研究指出,果蝇的Nemo蛋白能够磷酸化BMP信号的效应分子MAD并促进其出核,从而拮抗BMP信号通路[3]。最近的研究显示,Smad4能够结合NLK并被其磷酸化[4]。这些结果提示,NLK可能参与TGF-p信号通路调节,TGF-p信号能够调节细胞增殖、分化、迁移、凋亡等一系列细胞反应；而该信号的异常调节则可能导致各种疾病的发生,如纤维化、恶性肿瘤、自身免疫疾病和心血管疾病等。因此进一步明确NLK在TGF-β信号通路中的功能,将有助于进一步揭示TGF-β信号通路的调节机制,从而深化对TGF-β信号失调和相关疾病发生过程的认识。鉴于揭示NLK在TGF-β信号通路中的调节作用已成为当前研究的热点,本文也由此作了深入的探讨。我们首先利用免疫共沉淀实验,发现NLK与Smad3之间存在相互作用,并且两者的结合依赖于Smad3的MH2结构域。免疫荧光实验显示,在TGF-p刺激条件下NLK与Smad3在U20S细胞中共定位。在此基础上,通过双荧光素酶报告基因系统分析NLK对Smad3介导的转录反应的影响,我们发现,在不同的细胞模型中,瞬时表达NLK均可以抑制Smad3介导的转录反应。同时,我们利用RNA干扰技术,降低HaCaT、HeLa、HepG2和HCT-116细胞中内源性NLK的表达,检测Smad3及其下游靶基因PAI-1(plasminogen activator inhibitor-1, PAI-1)、CTGF (connective tissue growth factor, CTGF)、FN (fibronectin, FN)、p21、p15的变化。结果显示,降低细胞内NLK的表达水平,磷酸化Smad3以及Smad3总的蛋白质水平均显著上调,而Smad3下游靶基因的蛋白水平也有不同程度的升高。在此基础上,我们制备了NLK基因敲除的HCT-116细胞株,即NLK+/-和NLK-/-。首先检测NLK基因敲除的HCT-116细胞中细胞凋亡的变化,结果显示该类细胞并未发生显著的凋亡。随后,利用NLK基因敲除的HCT-116细胞,通过CCK-8法、平板克隆形成实验和软琼脂实验观察NLK基因缺失对HCT-116细胞增殖的影响。结果显示,NLK基因缺失抑制细胞增殖过程。2.睾丸特异表达GTPase RSA-14-44蛋白的功能研究哺乳动物的精子发生是一个典型的发育过程,其中涉及生精干细胞遗传信息的编译、重组、分配以及生精细胞形态的显著变化。精子发生过程的一个突出特征是具有阶段特异性,即在特定的发育阶段,出现特异的细胞或组织结构,以执行相应的特殊功能,而这些特异性结构绝大部分都是精子发生过程中阶段特异性基因表达的结果。因此,寻找并研究精子发生过程中差异表达的基因有助于我们深刻理解精子发生的独特机制。在前期工作中,我们鉴定了一个睾丸特异表达的Rho GTPase,并将其命名为RSA-14-44。进一步的实验证实RSA-14-44与20S蛋白酶体的催化亚基PSMB5之间存在相互作用,并且两者的结合依赖于RSA-14-44C末端190位的半胱氨酸。此外,RSA-14-44主要与PSMB5的前体相互作用,对细胞蛋白酶体活性水平的影响甚微。本研究主要集中于两个问题,(1)RSA-14-44与PSMB5亚细胞定位与其相互作用的具体关联,(2)RSA-14-44对PSMB5前体加工过程的影响。我们在MCF-7细胞瞬时表达Flag-RSA-14-44和Myc-PSMB5,观察两者在细胞中的定位情况。结果显示,RSA-14-44分布于细胞浆,并集中分布于细胞核周；而PSMB5在胞浆胞核均有分布,两者部分共定位于细胞核周。值得注意的是,将RSA-14-44190位的半胱氨酸突变为丝氨酸后,其定位从细胞核周扩展至细胞核内；蛋白质组分分离及分析的结果也证实了这一结果。因此,RSA-14-44第190位半胱氨酸的突变会导致其错误的亚细胞定位进而影响RSA-14-44与PSMB5之间的结合。我们在HEK293T细胞中共表达PSMB5和不同剂量的RSA-14-44。结果显示,伴随着RSA-14-44表达水平的提高,PSMB5前体的蛋白质水平显著下降,而PSMB5前体加工后所产生的PSMB5的蛋白质水平基本保持稳定。在此基础上,我们利用CHX (cycloheximide)阻断蛋白质合成,观察RSA-14-44表达上调对PSMB5前体的稳定性的影响。结果表明,RSA-14-44表达水平的上升可以显著降低PSMB5前体蛋白的稳定性,而加工成熟的PSMB5的稳定性不受影响；当190位半胱氨酸发生突变后,RSA-14-44对PSMB5前体蛋白稳定性的影响被消除,表明RSA-14-44通过结合PSMB5,参与负调节PSMB5前体蛋白质的稳定性,进而调节蛋白酶体的合成。
【Abstract】 1. Exploring the function of NLK, a conserved serine/threonine protein kinaseThe transforming growth factor-β(TGF-β) family members including TGF-βisoforms, activins, bone morphogenetic proteins (BMPs), and growth and differentiation factors (PDFs), are the secreted cytokines that stimulate a broad array of cellular responses such as cell proliferation, differentiation, migration and apoptosis. Misregulation of these signaling involve in various diseases including fibrosis, malignant tumors, auto-immune diseases and vascular disorders. Thus, tightly controlling TGF-βsignaling is essential for maintaining normal cellular responses and tissue homeostasis.Nemo-like kinase (NLK) is an evolutionally conserved serine/threonine protein kinase. It was demonstrated that Nemo could antagonize BMP signaling through phosphorylating MAD, the BMP effector in Drosophila, which promote exporting MAD from the nucleus to the cytoplasm. Recent study also showed that Smad4 was phosphorylated by NLK, suggesting that NLK might be involved in regulating TGF-P signaling. Thus, we certainly get new insight into the mechanism of TGF-P signaling pathway by exploring the roles of NLK in the phosphoryation of Smads.NLK associates with Smad3 in vivo, which was confirmed by the co-immunoprecipitation assay. Detailed analyses further showed that the MH2 domain, located in the C terminal of Smad3, was essential for its association with NLK. Endogenous Smad3 co-localizes with transiently expressed NLK in U2OS cells in response to TGF-βstimuli.Based on these information, we investigated the possible effects of NLK on Smad3-mediated transcription by a dual-luciferase reporter assay. NLK suppresses Smad3-mediated transcription in both HEK293T and HeLa cells. Using the RNAi technology, we knocked down the expression of NLK in HaCaT, HeLa, HepG2 and HCT-116 cells and confirm the inhibition of Smad3-mediated transcription by NLK. As expected, the protein level of p21, a classical target gene of Smad3, is elevated obviously.Down-regulated cell proliferation was further observed in the NLK knockout HCT-116 cells (NLK+/- and NLK-/-), which was confirmed by the results of plate colony formation assay and the soft agar assay. However, no significant change of apoptosis was observed in the NLK+/- and NLK-/- HCT-116 cells.2. Investigating the function of RSA-14-44, a testis-specific Rho GTPaseMammalian spermatogenesis is a paradigm for development in which the genetic information from male germ stem cells is re-edited, re-organized and finally distributed into spermatozoa while a dramatic cellular metamorphosis occurs in germ cells. This intricate process is built upon a system that features distinctive patterns of structure and regulation, most of which are the results of a unique and well-coordinated program of gene expression. Thus, identification and characterization of these differentially expressed genes has provided us additional insight into the unique mechanisms of spermatogenesis.In the previous study, we identified a testis-specific Rho GTPase named RSA-14-44, which associates with PSMB5, a catalytic subunit of the 20S proteasome during spermatogenesis. Detailed analyses showed that only the precursors of PSMB5 associate with RSA-14-44, which was not a direct regulator of proteasome activity. Furthermore, RSA-14-44 C190S resulted in the disruption of association between RSA-14-44 and PSMB5.In the present study, we clarified the effect of the C190S mutant on the location of RSA-14-44 and the possible roles of RSA-14-44 in the autocleavage of PSMB5.First, we found that in transfected MCF-7 cells, RSA-14-44 partially co-localize with PSMB5 in a perinuclear region. Furthermore, we compared the localization of the RSA-14-44C190S mutant with wide-type protein by immunofluorescence. In contrast to the perinuclear centered pattern of the wild-type protein, RSA-14-44c190S is dispersed in both nuclear and cytoplasmic regions. This dramatic difference resulting from the C190S mutation was further confirmed by the immunoblot analysis of subcellular fractions.To specify the possible effects of RSA-14-44 on the autocleavage of PSMB5, we co-expressed PSMB5 with various amounts of RSA-14-44. Surprisingly, the levels of PSMB5 precursors decrease significantly as a result of the increase in the doses of RSA-14-44; nevertheless, no disturbance was observed in the level of mature PSMB5. Following this clue, we measured the stability of PSMB5 precursor under transient expression of RSA-14-44 to determine the precise role of RSA-14-44 in down-regulating its protein level. Actually, the PSMB5 precursor is unstable and its stability is down-regulated dramatically by co-expression of RSA-14-44. In contrast, mature PSMB5 has a much longer life time and is less sensitive to the expression of RSA-14-44. Disrupting the association of RSA-14-44 and PSMB5 completely reversed the effect of RSA-14-44 on the stability of the PSMB5 precursor, which suggests this association is a prerequisite for RSA-14-44 regulation of proteasome biogenesis.