The HTLV-I Tax protein binding domain of cyclin-dependent kinase 4 (CDK4) includes the regulatory PSTAIRE helix -: art. no. 54
The HTLV-I Tax protein binding domain of cyclin-dependent kinase 4 (CDK4) includes the regulatory PSTAIRE helix -: art. no. 54
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DOI：
10.1186/1742-4690-2-54
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发表时间：
2005-09-15
期刊：
影响因子：
3.3
通讯作者：
Grassmann, R
中科院分区：
文献类型：
--
作者：
Fraedrich, K;Müller, B;Grassmann, R
背景：人类T细胞白血病病毒1型(HTLV-1)的Tax癌蛋白在转基因小鼠中具有致白血病作用，并能在体外诱导T细胞的永久性生长。在成人T细胞白血病培养物中发现有活性的CDK全酶复合体，并通过激活细胞周期蛋白依赖性激酶(CDK)CDK4来刺激从G1到S的转变。Tax蛋白直接和特异性地与CDK4和细胞周期蛋白D2相互作用，需要结合才能增强CDK4的活性。TRAX与细胞周期蛋白D/CDK复合体的蛋白质-蛋白质接触增加了CDK4与其正调控亚单位细胞周期蛋白D的结合，使复合体抵抗p21(CIP)抑制。结果：通过体外结合、下拉杂交和哺乳动物双杂交分析，分析了TAX的N末端是否具有结合CDK4的能力。这些实验表明，40个氨基酸的片段足以与CDK4和细胞周期蛋白D2相互作用。为了定义一个税收结合结构域并分析税收如何影响激酶活性，我们测试了一系列CDK4缺失突变体。不同的分析揭示了两个区域，它们在缺失时一致地导致结合活性降低。这些细胞被分离出来，并进行哺乳动物双杂交分析，以测试它们与Tax N末端相互作用的可能性。这些实验同时揭示了CDK4的N-末端和C-末端结合。结论：由于CDK4的N-端和C-端在预测的蛋白质三维结构中是相邻的，它们可能包含一个单一的结合域，与Tax的N-端相互作用。
Background: The Tax oncoprotein of human T-cell leukemia virus type 1 (HTLV-1) is leukemogenic in transgenic mice and induces permanent T-cell growth in vitro. It is found in active CDK holoenzyme complexes from adult T-cell leukemia-derived cultures and stimulates the G1-to-S phase transition by activating the cyclin-dependent kinase ( CDK) CDK4. The Tax protein directly and specifically interacts with CDK4 and cyclin D2 and binding is required for enhanced CDK4 kinase activity. The protein-protein contact between Tax and the components of the cyclin D/CDK complexes increases the association of CDK4 and its positive regulatory subunit cyclin D and renders the complex resistant to p21(CIP) inhibition. Tax mutants affecting the N-terminus cannot bind cyclin D and CDK4.Results: To analyze, whether the N-terminus of Tax is capable of CDK4-binding, in vitro binding , pull down -, and mammalian two-hybrid analyses were performed. These experiments revealed that a segment of 40 amino acids is sufficient to interact with CDK4 and cyclin D2. To define a Tax-binding domain and analyze how Tax influences the kinase activity, a series of CDK4 deletion mutants was tested. Different assays revealed two regions which upon deletion consistently result in reduced binding activity. These were isolated and subjected to mammalian two-hybrid analysis to test their potential to interact with the Tax N-terminus. These experiments concurrently revealed binding at the N- and C-terminus of CDK4. The N- terminal segment contains the PSTAIRE helix, which is known to control the access of substrate to the active cleft of CDK4 and thus the kinase activity.Conclusion: Since the N- and C-terminus of CDK4 are neighboring in the predicted three-dimensional protein structure, it is conceivable that they comprise a single binding domain, which interacts with the Tax N- terminus.