REGULATION OF UBIQUITIN LIGASE NEDD4-2 BY PHOSPHORYLATION

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 在代谢应激的条件下，许多膜转运蛋白（包括上皮钠通道ENaC）的表达和活性受到抑制，从而限制了离子梯度的消散并保留了维持它们所需的细胞能量。 将膜运输与能量产生和代谢状态联系起来的细胞机制才刚刚开始被揭示。 作者先前已经表明，ENaC被AMP激活的蛋白激酶（AMPK，一种普遍存在的丝氨酸/苏氨酸激酶，在许多系统中作为代谢传感器和代谢调节剂参与）抑制。AMPK不直接磷酸化ENaC，但可能与ENaC介导的Na+转运的调节剂如泛素连接酶Nedd 4 -2相互作用。 初步实验表明，AMPK是不能抑制ENaC介导的Na+电流时，使用的ENaC亚基不能与Nedd 4 -2相互作用的变体。此外，AMPK在体外磷酸化Nedd 4 -2。 这一系列证据表明以下假设：Nedd 4 -2可能作为AMPK的直接靶点，AMPK可能磷酸化Nedd 4 -2，从而增强其与ENaC结合的能力或其泛素连接酶活性。 为了验证这一假设，作者需要确定AMPK是否在体内磷酸化Nedd 4 -2以及这种磷酸化发生在什么位点。 我们将在培养的细胞中调节AMPK活性并纯化Nedd 4 -2用于分析。 我们将通过Nedd 4 - 2的胰蛋白酶消化物的强阳离子交换色谱分离，然后通过非反相液相色谱结合电喷雾电离-四极杆飞行时间串联质谱分析Nedd 4 -2的AMPK磷酸化位点。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Under conditions of metabolic stress, the expression and activity of many membrane transport proteins including the epithelial sodium channel, ENaC, are inhibited, thereby limiting the dissipation of ionic gradients and preserving the cellular energy required to maintain them. The cellular mechanisms that link membrane transport to energy production and metabolic status are only beginning to be revealed. The authors have previously shown that ENaC is inhibited by AMP-activated protein kinase (AMPK, a ubiquitous serine/theonine kinase that participates as a metabolic sensor and metabolic regulator in many systems). AMPK does not directly phosphorylate ENaC, but may interact with regulators of ENaC-mediated Na+ transport such as the ubiquitin ligase, Nedd4-2. Preliminary experiments indicate that AMPK is unable to inhibit ENaC-mediated Na+ current when using variants of ENaC subunits unable to interact with Nedd4-2. Additionally, AMPK phosphorylates Nedd4-2 in vitro. This line of evidence suggests the following hypothesis: Nedd4-2 may serve as a direct target of AMPK, and AMPK may phosphorylate Nedd4-2 and therefore augment its ability to bind to ENaC or its ubiquitin ligase activity. In order to test this hypothesis, the authors need to establish whether AMPK phosphorylates Nedd4-2 in vivo and at what sites this phosphorylation occurs. We will modulate AMPK activity in cultured cells and purify Nedd4-2 for analysis. We will analyze sites of AMPK phosphorylation of Nedd4-2 by strong cation exchange chromatography fractionation of a tryptic digest of Nedd4-2 followed by non-reverse phase liquid chromatography coupled with electrospray ionization-quadrupole time of flight tandem mass spectrometry.