IN VIVO PHOSPHORYLATION SITES IN CYSTIC FIBROSIS TRANSMEMB CONDUCTANCE REGULATO

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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.