Cystic Fibrosis transmembrane conductance regulator (CFTR) Cl - channels are rapidly and efficiently deactivated by a type 2C serine/threonine phosphatase (PP2C). The efficiency of this regulation suggests that PP2C and CFTR are associated within a regulatory complex. Previous work has shown that PP2C and CFTR can be co-immunoprecipitated and reversibly coupled by exposing cell lysates to the hydrophilic cross-linker dithiobis [sulfosuccinimidyl proprionate]. While it is clear that PP2C can associate with CFTR, the nature of the interaction between them has not been defined. The goal of this study was to identify specific region(s) within CFTR that mediate this interaction. The interaction between CFTR and PP2C was strong enough to be reproduced in the absence of cross-linking agents, supporting its physiological relevance. We then translated CFTR in vitro and incubated it with cell lysates containing endogenous PP2C, however interaction between the proteins was not observed under these conditions. Since the association seemed to require some factor present in cells, a distal fragment of CFTR was expressed in vivo and used for pull down assays. PP2C was co-purified under these conditions, suggesting an interaction between PP2C and the C-Tail of CFTR. To further narrow down the region of interaction, three residues within the C-Tail (Y1424, LL1430/1431) that had been shown previously to interact with the AMP-activated protein kinase (AMPK, a known PP2C substrate and possible targeting protein) were mutated to alanines. When this mutant was used in a pull-down assay, no association with PP2C was observed, consistent with a role for AMPK-binding residues and AMPK in the association of PP2C with CFTR.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.29482 |
| Date | January 2002 |
| Creators | Tryansky, Jonathan N. |
| Contributors | Hanrahan, John (advisor) |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Master of Science (Department of Physiology.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 001941013, proquestno: MQ85832, Theses scanned by UMI/ProQuest. |
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