REGULATION OF UBIQUITIN LIGASE NEDD4-2 BY PHOSPHORYLATION

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 在代谢应激条件下，包括上皮钠通道在内的许多膜转运蛋白的表达和活性受到抑制，从而限制了离子梯度的耗散，并保留了维持离子梯度所需的细胞能量。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。 作者先前已经证明ENaC被AMP激活的蛋白激酶(AMPK，一种普遍存在的丝氨酸/茶氨酸激酶，在许多系统中作为代谢感受器和代谢调节器参与)抑制。AMPK不直接磷酸化ENaC，但可能与ENaC介导的Na+转运调节因子如泛素连接酶Nedd4-2相互作用。初步实验表明，当使用不能与Nedd4-2相互作用的ENaC亚基的变体时，AMPK不能抑制ENaC介导的钠电流。此外，AMPK在体外使Nedd4-2磷酸化。这一证据支持以下假设：Ned4-2可能是AMPK的直接靶标，AMPK可能使Nedd4-2磷酸化，从而增强其与ENaC的结合能力或其泛素连接酶活性。 为了验证这一假说，作者需要确定AMPK是否在体内使Nedd4-2磷酸化，以及这种磷酸化发生在什么位置。我们将对培养细胞中的AMPK活性进行调节，并纯化Nedd4-2进行分析。我们将用强阳离子交换层析分析Nedd4-2的AMPK磷酸化位点。Nedd4-2的胰酶解分馏后，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仪。

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.