Association of cystic fibrosis transmembrane conductance regulator and protein phosphatase 2C.
Cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels are rapidly deactivated by a membrane-bound phosphatase activity. The efficiency of this regulation suggests CFTR and protein phosphatases may be associated within a regulatory complex. In this paper we test that possibility using co-immunoprecipitation and cross-linking experiments. A monoclonal anti-CFTR antibody co-precipitated type 2C protein phosphatase (PP2C) from baby hamster kidney cells stably expressing CFTR but did not co-precipitate PP1, PP2A, or PP2B. Conversely, a polyclonal anti-PP2C antibody co-precipitated CFTR from baby hamster kidney membrane extracts. Exposing baby hamster kidney cell lysates to dithiobis (sulfosuccinimidyl propionate) caused the cross-linking of histidine-tagged CFTR (CFTR(His10)) and PP2C into high molecular weight complexes that were isolated by chromatography on Ni(2+)-nitrilotriacetic acid-agarose. Chemical cross-linking was specific for PP2C, because PP1, PP2A, and PP2B did not co-purify with CFTR(His10) after dithiobis (sulfosuccinimidyl propionate) exposure. These results suggest CFTR and PP2C exist in a stable complex that facilitates regulation of the channel.
Zhu
T
T
Department of Physiology, McGill University, Montréal, Québec H3G 1Y6, Canada.
Dahan
D
D
Evagelidis
A
A
Zheng
S
S
Luo
J
J
Hanrahan
J W
JW
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D016466:CHO Cells; D002458:Cell Fractionation; D002478:Cells, Cultured; D006224:Cricetinae; D003432:Cross-Linking Reagents; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000032:analysis / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests; D054648:Protein Phosphatase 2; D000071636:Protein Phosphatase 2C; D029701:Saccharomyces cerevisiae Proteins; D013388:Succinimides
D000911:Antibodies, Monoclonal; D003432:Cross-Linking Reagents; D029701:Saccharomyces cerevisiae Proteins; D013388:Succinimides; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; C035759:3,3'-dithiobis(sulfosuccinimidyl propionate); C087358:PTC1 protein, S cerevisiae; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2; D000071636:Protein Phosphatase 2C