Title: Interaction of the protein phosphatase 2A and the AMP-activated kinase with the R domain of the CFTR protein
Other Titles: Interactie van het proteïne fosfatase 2A en het AMP-geactiveerde proteïne kinase met het regulatordomein van het CFTR-proteïne
Authors: Vastiau, Annick; M9417657
Issue Date: 25-May-2007
Abstract: Cystic Fibrosis is the most common lethal autosomal recessive disorder in the Caucasian population. The disease is caused by a defect in the ‘cystic fibrosis transmembrane conductance regulatorÂ’ gene, the CFTR gene.This gene encodes a 1480 amino acids long transmembrane protein with a symmetric multi-domain structure: a repeat composed of a transmembrane region, the membrane spanning domain and a nucleotide binding domain (NBD), linked by the R domain. CFTR is a cAMP-dependent chloride channel present in the apical membrane of exocrine glands. It is regulated by binding and hydrolysis of ATP at the NBDs and phosphorylation of the R domain. Different PKA and PKC phosphorylation sites exist in the R domain. Besides being a chloride channel, CFTR is a regulator of other ion channels. The R domain seems to be an important domain in the regulation of these interacting channels. In this work, proteins interacting with the R domain of CFTR were identified and the effect of the interaction on CFTR channel function was studied. Yeast two hybrid screenings with the R domain as a bait (Matchmaker screening) and the NBD1-R domain as a bait (Hybrigenics screening) identified 9 candidate interacting R domain proteins: PR65a, MIZIP, an eukaryotic translation initiation factor, a short peptide IRGRVDLVSSFFFFFF, the suppression of tumourigenicity protein, endofin, human bicaudal, theSNAP-associated protein and VSP39. The interaction of the R domain and PR65a was further detailed in chapter 2; the interaction with the short peptide could not be confirmed. The interaction of the NBD1-R domain with the other candidates should be confirmed by other independent techniques. The nature of the candidate interacting proteins suggests a role forthe R domain in the maturation, degradation, trafficking and endo/exocytosis of CFTR. The second part of this thesis describes the interaction of PR65a, a subunit of the protein phosphatase 2A (PP2A), with the R domain of CFTR. PR65a and the R domain could bind directly; the interaction was mediated by the first ten HEAT repeats of PR65a. PP2A dephosphorylated the phosphorylated R domain in vitro. Functional experiments indicated that PP2A could dephosphorylate CFTR in vivo. First, addition of a PP2A inhibitor resulted in a delay of deactivation of the CFTR channel. Second, overexpression of the first ten HEAT repeats of PR65a strongly impaired CFTR channel function, probably as a result of competition of this shortened form with the endogenous full length PR65 in the formation of a functional PP2A complex. In conclusion, PP2A could bind to and dephosphorylate the R domain, resulting in the deactivation of the CFTR chloride channel.AMPKa1 could be pulled down with the R domain. The AMPK enzyme could phosphorylate residue S768 in the R domain in vitro. Activation of AMPK resulted in phosphorylation of CFTR at residue S768, leading to the deactivation of the CFTR chloride channel. The S768A mutation completely abolished channel deactivation by AMPK. This is a first indication that a kinase can phosphorylate CFTR and that this phosphorylation can deactivate its channel. In this thesis, we described the interaction of the R domain with PP2A and AMPK, two proteins that interact with the R domain of CFTR and deactivate the CFTR channel. Besides regulation of the CFTR channel itself, these interactions might interfere with the role of CFTR in ion channel regulation and endo/exocytosis of CFTR. In addition, we found new proteins interacting with the NBD1-R domain, suggesting a possible role for theR domain in CFTR maturation, degradation, trafficking and endo/exocytosis. Insight in the regulation and the function of the R domain might lead to better insight in CFTR function, resulting in more adequate CF treatment.
Publication status: published
KU Leuven publication type: TH
Appears in Collections:Human Mutations and Polymorphisms Section (-)

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