IN VIVO PHOSPHORYLATION SITES IN CYSTIC FIBROSIS TRANSMEMB CONDUCTANCE REGULATO

囊性纤维化跨膜电导调节中的体内磷酸化位点

• 批准号: 7355045
• 负责人: DAVID C GADSBY
• 金额: $ 0.37万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-03-01 至 2007-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7355045
• 关键词: VIVO; PHOSPHORYLATION; SITES; CYSTIC; FIBROSIS

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. Inherited mutations in the chloride channel known as the cystic fibrosis transmembrane conductance regulator (CFTR) lead to the symptoms of cystic fibrosis. To become open, the channel must be phosphorylated by cAMP-dependent protein kinase at sites within its regulatory domain. Previous mass spectrometric experiments have identified numerous serine residues in the regulatory domain that are phosphorylated in vitro, and have allowed determination of the relative ease of in vitro phosphorylation at the various sites. The current work examines human CFTR expressed in Xenopus oocytes to determine the in vivo phosphorylation pattern. It was observed that Ser-768 of CFTR was essentially quantitatively phosphorylated in vivo. Other work in the Gadsby lab has shown that mutation of this residue to alanine, thus preventing its phosphorylation, results in a constitutively active ion channel. Thus phosphorylation of Ser-768 appears to be a negative regulator of channel opening. Other sites of in vivo serine phosphorylation were characterized, and examination of the in vivo phosphorylation state of CFTR containing the S768A mutation gave no evidence of a change in phosphorylation pattern at the other sites within the regulatory domain.

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。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。所列机构为中心，不一定是研究者所在机构。被称为囊性纤维化跨膜传导调节因子（CFTR）的氯离子通道的遗传突变导致囊性纤维化的症状。为了变得开放，通道必须在其调节结构域内的位点被cAMP依赖性蛋白激酶磷酸化。以前的质谱实验已经确定了许多丝氨酸残基的调节结构域中，在体外磷酸化，并允许在体外磷酸化的相对容易的测定在各个网站。目前的工作研究人类CFTR表达在非洲爪蟾卵母细胞，以确定在体内磷酸化模式。观察到CFTR的Ser-768在体内基本上定量磷酸化。加兹比实验室的其他工作表明，该残基突变为丙氨酸，从而阻止其磷酸化，导致组成型活性离子通道。因此，Ser-768的磷酸化似乎是通道开放的负调节剂。体内丝氨酸磷酸化的其他位点进行了表征，并且检查含有S768 A突变的CFTR的体内磷酸化状态，没有证据表明调节结构域内其他位点的磷酸化模式发生变化。