IN VIVO PHOSPHORYLATION SITES IN CYSTIC FIBROSIS TRANSMEMB CONDUCTANCE REGULATO

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。被称为囊性纤维化跨膜传导调节剂（CFTR）的氯离子通道的遗传突变可导致囊性纤维化的症状。为了开放，通道必须在其调节区域内被camp依赖性蛋白激酶磷酸化。先前的质谱实验已经在体外磷酸化的调控域中鉴定了许多丝氨酸残基，并且已经允许确定在不同位点的体外磷酸化的相对容易程度。目前的工作是检测人CFTR在爪蟾卵母细胞中的表达，以确定体内磷酸化模式。观察到CFTR的Ser-768在体内基本上被定量磷酸化。加兹比实验室的其他工作表明，这种残基突变为丙氨酸，从而阻止了丙氨酸的磷酸化，从而产生了一个构成活性的离子通道。因此，Ser-768的磷酸化似乎是通道打开的负调节因子。对体内丝氨酸磷酸化的其他位点进行了表征，对含有S768A突变的CFTR的体内磷酸化状态的检查没有发现调节域内其他位点磷酸化模式发生变化的证据。

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.