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

通道激酶TRPM6和TRPM7属于α-激酶家族，是一种特殊的丝氨酸-苏氨酸蛋白 与传统的真核蛋白激酶不显示序列同源性的蛋白激酶。通道激活酶 是一种不寻常的双功能分子，由融合在离子通道上的蛋白激酶结构域组成。这些 分子在调节脊椎动物体内镁的动态平衡方面起着关键作用。TRPM7频道 已经使用电生理技术进行了表征，然而，激动素的作用 仍然不为人知。我们产生了TRPM7激酶结构域被破坏的敲除小鼠，并发现 这会导致早期胚胎死亡，这表明激酶结构域具有必要的和非 冗余功能。我们最近发现膜联蛋白1是TRPM7的内源底物。 发现TRPM7激酶使Annexin 1在保守的丝氨酸残基上磷酸化 在膜联蛋白功能中起关键作用的两亲性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 提示TRPM7和TRPM6激酶利用一种不寻常的底物识别机制。 我们的目标是确定TRPM7和TRPM6激酶结构域的生理功能，以阐明 它们的激活和底物识别的机制以及在调节通道活性中的作用。 因此，具体目标是： 1)鉴定生理底物，确定TRPM7和TRPM6识别的序列基序 激活剂。探讨TRPM7和TRPM6激酶的自磷酸化机制，以MAP 自磷酸化位点，并探讨自磷酸化在通道调节中的作用 活动(与Fleig博士合作，项目1)。已鉴定的激酶底物在镁- 将研究依赖信号传递(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的假说 激酶识别其底物中的α螺旋构象。3)确定其动力学机制。 TRPM7和TRPM6激酶，阐明它们的激活机制，并探讨其在 二价金属离子对其活性的调节作用。

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.