Project 2: Structure Function of TRPM6/7 Alpha Kinase Domains

项目2：TRPM6/7α激酶结构域的结构功能

• 批准号: 7285826
• 负责人: ALEXEY G. RYAZANOV
• 金额: $ 33.28万
• 依托单位: UNIV OF MED/DENT NJ-R W JOHNSON MED SCH
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-03-01 至 2012-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7285826
• 关键词: Amphipathic Alpha Helix; Annexins; Binding; Cessation of life; Collaborations; Dependence; Disruption; Embryo; Family; Fluorescence Resonance Energy Transfer; Goals; Homeostasis; Investigation; Ion Channel; Ions; Kinetics; Knockout Mice; Magnesium; Maps; Metals; Modeling; Molecular Conformation; Monitor; Neurons; Nonmuscle Myosin Type IIA; Peptide Library; Peptides; Phosphorylation; Phosphorylation Site; Phosphotransferases; Physiological; Play; Protein Kinase; Protein-Serine-Threonine Kinases; Reaction; Regulation; Research Personnel; Role; Scanning; Sequence Homology; Serine; Signal Transduction; Signaling Molecule; Site; Structure; Techniques; Testing; Vertebrates; combinatorial; divalent metal; novel; programs; research study

Channel kinases TRPM6 and TRPM7 belong to the alpha-kinase family, unusual protein serine-threonine kinases that do not display sequence homology to conventional eukaryotic protein kinases. Channel kinases are unusual bifunctional molecules consisting of a protein kinase domain fused to an ion channel. These molecules play a key role in the regulation of magnesium homeostasis in vertebrates. The TRPM7 channel has been characterized using electrophysiological techniques, however the role of the kinasedomain remains unknown. We generated knockout mouse with a disruption of the TRPM7 kinase domain and found that this results in early embryonic lethality, suggesting that the kinase domain has an essential and non- redundant function. We have recently identified annexin 1 as an endogenous substrate for TRPM7.We found that TRPM7 kinase phosphorylates annexin 1 at a conserved serine residue located within an amphipathic alpha-helix that plays a crucial role in annexin function. Our preliminary experiments suggest that TRPM7 kinase can phosphorylate myosin IIA and several other substrates. Our experiments also suggest that TRPM7 and TRPM6 kinases utilize an unusual mechanism of substrate recognition. Our goal is to determine physiological function of TRPM7 and TRPM6 kinase domains, to elucidate the mechanism of their activation and substrate recognition and the role in the regulation of channel activity. Therefore the specific aims are: 1) To identify physiological substrates and to determine sequence motif recognized by TRPM7 and TRPM6 kinases. To investigate mechanism of autophosphorylation of TRPM7 and TRPM6 kinases, to map autophosphorylation sites, and to investigate the role of autophosphorylation in the regulation of channel activity (in collaboration with Dr. Fleig, Project 1). The role of identified kinase substrates in magnesium- dependent signaling will be investigated (in collaboration with Dr. Scharenberg, Project 3). 2) To investigate mechanism of substrate recognition and to test the hypothesis that TRPM7 andTRPM6 kinases recognize alpha helical conformation in its substrates. 3) To determine the kinetic mechanismof TRPM7 and TRPM6 kinases, to elucidate the mechanism of their activation, and to investigate the role of divalent metal ions in the modulation of their activity.

通道激酶TRPM6和TRPM7属于α-激酶家族，不寻常的蛋白质丝氨酸 - 苏氨酸 不与常规真核蛋白激酶显示序列同源的激酶。通道激酶 是由与离子通道融合的蛋白激酶结构域组成的异常双功能分子。这些 分子在脊椎动物中镁稳态的调节中起关键作用。 TRPM7通道 已经使用电生理技术来表征，但是Kinasasedomain的作用 仍然未知。我们生成了敲除鼠标，并破坏了TRPM7激酶结构域并发现 这会导致早期的胚胎致死性，这表明激酶结构域具有必不可少的和非 - 冗余功能。我们最近将膜联蛋白1确定为TRPM7的内源性底物。 发现TRPM7激酶在位于一个位于一个保守的丝氨酸残基处磷酸化膜联蛋白1 在膜联蛋白功能中起着至关重要的作用的两亲性α-螺旋。我们的初步实验表明 该TRPM7激酶可以磷酸化肌球蛋白IIA和其他几种底物。我们的实验也是如此 建议TRPM7和TRPM6激酶利用底物识别的异常机制。 我们的目标是确定TRPM7和TRPM6激酶结构域的生理功能，以阐明 它们激活和底物识别的机制以及在通道活性调节中的作用。 因此，具体目的是： 1）识别生理底物并确定TRPM7和TRPM6识别的序列基序 激酶。研究TRPM7和TRPM6激酶的自磷酸化机制，以绘制 自磷酸化位点，并研究自磷酸化在通道调节中的作用 活动（与Fleig博士合作，项目1）。鉴定的激酶底物在镁中的作用 依赖信号将进行研究（与Scharenberg博士合作，项目3）。 2）研究底物识别的机制并检验TRPM7和TRPM6的假设 激酶识别其底物中的α螺旋构象。 3）确定动力学机理 TRPM7和TRPM6激酶，以阐明其激活的机制，并研究 二价金属离子在其活性调节中。