病毒感染与PDK1 SUMO化修饰的互作研究

发布时间：2018-02-25 06:30

本文关键词： 3-磷酸肌醇依赖性蛋白激酶1 蛋白激酶B 小泛素样蛋白 Ⅲ型磷酸肌醇3-激酶　出处：《浙江大学》2016年博士论文　论文类型：学位论文

【摘要】：3-磷酸肌醇依赖性蛋白激酶(3-phosphoinositide-dependent protein kinase-1,PDPK1orPDK1)属于AGC蛋白激酶家族(cAMP和cGMP依赖性的蛋白激酶C)的一员,它能磷酸化下游的许多蛋白,如Akt、p90RSK、p70S6K、SGK和PKC等。因此PDK1的活性对下游激酶以及各细胞功能有着重要的影响,但是病毒感染是否影响PDK1的活性,尚不可知。为了解病毒感染是如何影响PDK1的活性,我们检测了不同病毒感染刺激对PDK1活性的影响。发现禽流感病毒、禽双RNA病毒、猪圆环病毒2型和猪伪狂犬病毒分别感染293T,293T/DF-1和PK-15细胞,均能诱导一条分子量为210kDa左右的PDK1修饰条带,进一步的实验证实,此条带为SUMO化修饰的PDK1。过表达FLAGPDK1和HASUMO1后,感染IBDV,并于感染后1、3、6、12h收样进行IP,通过FLAG抗体来钓,并用抗HA抗体来检测,可以在210KD左右的位置检测到条带,进一步证明PDK1的SUMO化修饰。通过网站预测到PDK1的SUMO化修饰位点分别为第207位、第296位和第495位赖氨酸,将这三个位点的赖氨酸同时突变为精氨酸后,PDK1的SUMO化修饰水平大大下降。同时还发现PDK1点突变后能够抑制下游AKT和S6K的磷酸化。此外,转染VP3的细胞中,AKT和S6K的磷酸化水平均明显上调。因此,本研究内容显示,病毒感染能够诱导PDK1的SUMO化修饰,并且PDK1的SUMO化修饰对下游AKT和S6K的磷酸化活性有着重要的影响。既然PDK1的SUMO化修饰在胞内有着重要的作用,那么胞内调控PDK1 SUMO化的机制就尤为重要。用PDK1作为诱饵蛋白来钓取胞内蛋白,发现PDK1能钓下110KD左右的条带,经过质谱鉴定发现这个条带为VPS34。我们采用CO-IP技术和PULL DOWN技术验证了 PDK1和VPS34的互作为直接互作。进一步实验证明过表达VPS34能够抑制PDK1的SUMO1修饰。同时我们发现VPS34能够破坏PDK1与UBC9的互作,从而抑制UBC9催化PDK1的SUMO化修饰。因此,研究表明VPS34能够负调控PDK1的SUMO化修饰。SUMO化对于PDK1蛋白功能的发挥具有重要作用,但是PDK1的SUMO化修饰对于调控其底物AKT活性的机制目前尚不清楚。超高分辨结果显示,PDK1的SUMO化修饰能够促进PDK1转移至膜周,并且和AKT定位在一块。免疫共沉淀结果进一步确认了 SUMO化能够促进PDK1和AKT的互作。而PDK1和AKT的互作对于AKT的磷酸化是必须的。另外,研究表明IBDV通过VP3与VPS34互作,从而破坏了 VPS34和PDK1的互作,并因此促进了 PDK1的SUMO化。对VP3的功能域进行分析,发现VP3通过CC3结构域与VPS34发生互作。而VP3通过CC1结构域与Beclin-1发生互作。但是VP3是通过CC3来调节PDK1修饰条带和AKT的活性。因此可以判定VP3是通过VPS34,而非Beclin-1来调节AKT的活性。同时研究还表明,VP3是通过AKT通路而非Beclin-1来抑制细胞自噬的。因此本章研究表明病毒感染可通过VPS34和PDK1的互作来调节PDK1的SUMO化状态,并进一步调节AKT的活性,从而调控病毒的感染。
[Abstract]:3-phosphoinositide-dependent protein kinase-1 (PDPK1 or PDK1) is a member of the AGC protein kinase family, camp and cGMP dependent protein kinase C, which phosphorylates many proteins downstream. Therefore, the activity of PDK1 has an important effect on downstream kinase and cell function, but whether virus infection affects the activity of PDK1 is not known. In order to understand how viral infection affects the activity of PDK1, We examined the effects of different viral stimuli on PDK1 activity. We found that avian influenza virus, avian double RNA virus, porcine circovirus type 2 and porcine pseudorabies virus were infected with 293Tm23T / DF-1 and PK-15 cells, respectively. All of them could induce a modified PDK1 band with molecular weight of 210kDa. Further experiments confirmed that the band was SUMO modified PDK1.After overexpression of FLAGPDK1 and HASUMO1, the band was infected with IBDV, and then collected at 1h after infection, and then was caught by FLAG antibody. Using anti-HA antibody to detect, the bands can be detected at 210KD, which further proves the SUMO modification of PDK1. The SUMO modification sites of PDK1 were predicted to be 207th, 296th and 495th, respectively. The SUMO modification level of PDK1 was significantly decreased after the lysine was mutated into arginine at the same time. It was also found that the point mutation of PDK1 could inhibit the phosphorylation of downstream AKT and S6K. The phosphorylation levels of AK T and S6K in VP3 transfected cells were significantly up-regulated. Therefore, this study showed that viral infection could induce SUMO modification of PDK1. And the SUMO modification of PDK1 has an important effect on the phosphorylation activity of downstream AKT and S6K. Since the SUMO modification of PDK1 plays an important role in the cell, The mechanism of intracellular regulation of PDK1 SUMO is especially important. Using PDK1 as bait protein to catch intracellular protein, we find that PDK1 can catch about 110 KD bands. The band was identified as VPS34.The direct interaction between PDK1 and VPS34 was verified by CO-IP and PULL DOWN techniques. Further experiments proved that overexpression of VPS34 could inhibit SUMO1 modification of PDK1. At the same time, we found that VPS34 could inhibit SUMO1 modification of PDK1. Can break the interaction between PDK1 and UBC9, Thus inhibiting the SUMO modification of PDK1 catalyzed by UBC9. Therefore, it has been shown that VPS34 can negatively regulate the SUMO modification of PDK1. SUMO plays an important role in the function of PDK1 protein. However, the mechanism of SUMO modification in regulating the activity of PDK1 substrate AKT is still unclear. The ultrahigh resolution results show that the SUMO modification of PDK1 can promote the transfer of PDK1 to the perimembrane. The results of immunoprecipitation further confirmed that SUMO could promote the interaction between PDK1 and AKT, and that the interaction between PDK1 and AKT was necessary for the phosphorylation of AKT. In addition, the study showed that IBDV interacted with VPS34 through VP3. Thus, the interaction between VPS34 and PDK1 is broken, and the SUMO of PDK1 is promoted. The functional domain of VP3 is analyzed. It is found that VP3 interacts with VPS34 through CC3 domain, while VP3 interacts with Beclin-1 through CC1 domain. But VP3 regulates the activity of PDK1 modified band and AKT through CC3. Therefore, it can be determined that VP3 regulates AKT by VPS34, not Beclin-1. Studies also show that VP3 inhibits autophagy through the AKT pathway rather than Beclin-1. Therefore, this chapter suggests that viral infection can regulate the SUMO state of PDK1 through the interaction of VPS34 and PDK1. And further regulate the activity of AKT, thereby regulating the infection of the virus.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S852.65

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