【作者】 谢琳娜；

【导师】 韩家淮；

【作者基本信息】 厦门大学 ， 生化与分子生物学， 2006， 硕士

【摘要】 细胞内的信号转导途径调节细胞对于来自胞内外刺激的反应。丝裂原活化蛋白激酶信号转导途径是真核生物中普遍存在的信号转导机制之一,三级激酶级联反应是其通路的核心成份。然而2002年Ge等发现TAB1所介导的p38α自我磷酸化反应打破了传统的MAPK家族只能通过三级激酶的磷酸化途径来激活的观念,这一直接激活机制,成为MAPK激活机制的一个有力补充。此外,TAB1与p38α的相互作用在p38家族中是十分特异的。因而确定TAB1和p38α相互作用必需区域氨基酸的序列将有助于对这一令人感兴趣的蛋白?蛋白相互作用的分子机制的理解。本论文分析了TAB1的缺失突变体和点突变体与p38α的相互作用,发现其羧基末端的脯氨酸残基(Pro412)是与p38α结合的必要位点,并进一步发现Pro412氨基末端存在一个貌似“D-结构域”的结合位点,而Pro412正好处于结合位点的ΦB+3的位置。通过突变体分析,我们发现以前报道的p38α中的疏水结合槽也与该相互作用有关,而CD结构域和ED位点却与之无关。同时,用不与TAB1相互作用的p38β和p38α构建嵌合拼接体,发现Thr218和Ile275是p38α特异性结合于TAB1的重要位点。并且将其中的任何一个残基置换为p38β的相应氨基酸都会阻止p38α和TAB1结合。当p38α的底物和激活物与p38α结合时,结合位点(p38α的疏水结合槽)发生了较大的构象改变,而Thr218和Ile275恰在这个位置附近,这提示我们TAB1诱导p38α自我磷酸化也可能是由于p38α发生了类似于p38α与其底物或激酶相互作用时引起的独特的构象变化。更多还原

【Abstract】 Intracellular signal transduction pathways regulate cellular responses to physiological and nonphysiological stimuli. The mitogen-activated protein kinases (MAPKs) play an important role in a variety of biological processes. MAPK kinases are in turn activated by phosphorylation by the MAPK kinase kinases (MAPKKKs). Ge et al.(2002) showed that there is another way to activate the so-called stress-activated MAPK known as p38α. They have revealed that TAB1 interacts with p38αand induces p38αautophosphorylation. Therefore, the determination of binding sequence of TAB1 and p38αis critical for us to know the interesting protein-protein interaction between them.Here, we examined the sequence requirements in TAB1 and p38αthat drive their interaction. Deletion and point mutations in TAB1 reveal that a proline residue in the C terminus of TAB1 (Pro412) is necessary for its interaction with p38α. Furthermore, a cryptic D-domain like docking site was identified adjacent to the N terminus of Pro412, putting Pro412 in theΦB+3 position of the docking site. Through mutational analysis, we found that the previously identified hydrophobic docking groove in p38αis involved in this interaction, whereas the CD domain and ED site are not. Furthermore, chimeric analysis using TAB1 non-binding p38β, revealed a previously unidentified locus of p38αcomprising Thr218 and Ile275 that is essential for specific binding of p38αto TAB1. Converting either of these residues to the corresponding amino acid of p38βabolishes p38αinteraction with TAB1. Adjacent to Thr218 and Ile275 is a site where large conformational changes occur in the presence of docking-site peptides derived from p38αsubstrates and activators. The results suggest that TAB1-induced autophosphorylation of p38αresults from conformational changes that are similar but unique to those seen in p38αinteractions with its substrates and activating kinases.更多还原