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Molecular Insights into Activation and Substrate Recognition of Protein Kinase R

蛋白激酶 R 激活和底物识别的分子洞察

基本信息

批准号：
8288483
负责人：
Madhusudan Dey
金额：
$ 33.45万
依托单位：
UNIVERSITY OF WISCONSIN MILWAUKEE
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-06-01 至 2016-05-31
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): Abstract: The active PKR kinase domain (KD) phosphorylates the Ser51 residue of translation initiation factor eIF2¿. The activation of PKR KD requires precise dimerization and autophosphorylation, including threonine-446 (T446) autophosphorylation in the activation loop. Then, the phospho-PKR (active) recognizes eIF2¿ and transfers the ¿- phosphate of ATP to the Ser51 residue of eIF2¿. We hypothesize that, in latent PKR, the activation loop collapses into the active-site cavity, and dimerization of the N-terminal double-stranded RNA binding domains of PKR would cause a precise conformational change of the C-terminal KD dimer. As a result, the activation loop residue T446 is phosphorylated by any of three mechanisms: trans-inter-dimer, cis-intra-dimer and trans- intra-dimer. We will dissect each of these mechanisms, and uncouple the role of dimerization and T446 phosphorylation, in particular, how dimerization promotes T446 autophosphorylation and how T446 autophosphorylation influences dimerization. Also, we will take an electron paramagnetic resonance (EPR) spectroscopy based approach to examine the movement of the Ser51 loop of a cysteine-less eIF2¿ in the apo-form, in the phosphorylated form, and with/without addition of PKR. PUBLIC HEALTH RELEVANCE: Project Narrative: Protein kinase R is directly involved in anti-viral defense responses and anti-proliferative effects on cancer cells, this fundamental knowledge of PKR will be useful to design anti- viral and anti-proliferative modulators.

描述（由申请人提供）：翻译后摘要：活性PKR激酶结构域（KD）磷酸化翻译起始因子eIF 2的Ser 51残基。PKR KD的活化需要精确的二聚化和自磷酸化，包括活化环中的苏氨酸-446（T446）自磷酸化。然后，磷酸化PKR（活性）识别eIF 2并将ATP的的Ser 51残基上。我们假设，在潜伏性PKR中，激活环塌陷到活性位点空腔中，PKR的N-末端双链RNA结合结构域的二聚化将导致C-末端KD二聚体的精确构象变化。因此，活化环残基T446通过三种机制中的任一种被磷酸化：反式二聚体间、顺式二聚体内和反式二聚体内。我们将剖析这些机制中的每一个，并解开二聚化和T446磷酸化的作用，特别是，二聚化如何促进T446自磷酸化和T446自磷酸化如何影响二聚化。此外，我们将采取基于电子顺磁共振（EPR）光谱的方法来检查无半胱氨酸eIF 2 <$的Ser 51环在apo形式，磷酸化形式和添加/不添加PKR的情况下的运动。公共卫生相关性：项目叙述：蛋白激酶R直接参与对癌细胞的抗病毒防御反应和抗增殖作用，PKR的这一基础知识将有助于设计抗病毒和抗增殖调节剂。