The HPV16 E6 binding protein Tip-1 interacts with ARHGEF16, which activates Cdc42.
The HPV16 E6 binding protein Tip-1 interacts with ARHGEF16, which activates Cdc42.
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DOI：
10.1038/sj.bjc.6606026
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发表时间：
2011-01-18
影响因子：
8.8
通讯作者：
中科院分区：
文献类型：
--
作者：
胍交换因子（GEF）催化的Rho蛋白如Cdc42的活化已被证明在细胞转化、恶性进展和侵袭中起着至关重要的作用。我们之前已经证明HPV16 E6癌蛋白与PDZ结构域蛋白税收相互作用蛋白1 （Tip-1）结合，现在我们报告了一种新的Tip-1结合GEF的鉴定和功能分析。采用酵母双杂交、体外拉下、定点诱变、半定量PCR、共免疫沉淀和western blotting等方法鉴定/确认新的Tip-1结合伙伴，并分析细胞表达水平。重组蛋白的体外动力学分析、siRNA基因沉默和细胞内测定Rho蛋白的活化。tax - interactingprotein 1通过其羧基PDZ结合基序与ARHGEF16相互作用。在表达异位hpv16e6的转化和永活细胞中，ARHGEF16的水平升高，并且在存在高危HPV E6的情况下，ARHGEF16共同免疫沉淀Cdc42。体外动力学分析证实重组ARHGEF16激活Cdc42，并通过添加重组Tip-1和E6增强了Cdc42的活性。表达hpv16e6的细胞具有更高水平的Cdc42激活，通过siRNA沉默Tip-1或ARHGEF16来降低Cdc42的激活。这些数据表明，HPV16 E6、Tip-1和ARHGEF16可能协同激活Cdc42，并支持HPV16 E6表达与Cdc42激活之间的潜在联系。
Guanidine exchange factor (GEF)-catalysed activation of Rho proteins such as Cdc42 has been shown to have a crucial role in cellular transformation, malignant progression and invasion. We have previously shown that the HPV16 E6 oncoprotein binds to the PDZ domain protein Tax-interacting-protein 1 (Tip-1) and we now report identification and functional analysis of a novel Tip-1 binding GEF. Yeast two-hybrid, in vitro pull-down, site-directed mutagenesis, semiquantitative PCR, co-immunoprecipitation and western blotting were used to identify/confirm novel Tip-1 binding partners and analyse cellular expression levels. In vitro kinetic analyses of recombinant proteins, siRNA gene silencing and in cell assays were used to measure Rho protein activation. Tax-interacting-protein 1 was shown to interact with ARHGEF16 by its carboxyl PDZ binding motif. Levels of ARHGEF16 were increased in transformed and immortalised cells expressing ectopic HPV16 E6 and Cdc42 was co-immunoprecipitated by ARHGEF16 in the presence of high-risk HPV E6. In vitro kinetic analysis confirmed that recombinant ARHGEF16 activates Cdc42 and this was increased by the addition of recombinant Tip-1 and E6. Cells expressing HPV16 E6 had higher levels of Cdc42 activation, which was decreased by siRNA silencing of either Tip-1 or ARHGEF16. These data suggest that HPV16 E6, Tip-1 and ARHGEF16 may cooperate to activate Cdc42 and support a potential link between the expression of HPV16 E6 and Cdc42 activation.
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影响因子：
8
作者：
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通讯作者：
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