ABCMdb

PMID: 25384981

Caohuy H, Yang Q, Eudy Y, Ha TA, Xu AE, Glover M, Frizzell RA, Jozwik C, Pollard HB
Activation of 3-phosphoinositide-dependent kinase 1 (PDK1) and serum- and glucocorticoid-induced protein kinase 1 (SGK1) by short-chain sphingolipid C4-ceramide rescues the trafficking defect of DeltaF508-cystic fibrosis transmembrane conductance regulator (DeltaF508-CFTR).
J Biol Chem. 2014 Dec 26;289(52):35953-68. doi: 10.1074/jbc.M114.598649. Epub 2014 Nov 10., [PubMed]

Sentences

No. Mutations Sentence Comment

82 ABCC7 p.Ser422Asp In Vitro Phosphorylation of CFTR-Wild-type and èc;F508-CFTR immunoprecipitates from untreated repaired and CFPAC-1 cell lysates, respectively, were washed with phosphorylation buffer (50 mM Tris, pH 7.5, 0.1 mM EGTA, 0.1% 2-mercaptoethanol) and then resuspended in 30 òe;l of the same buffer containing 100 ng of purified recombinant human S422D-SGK1 mutant protein (Millipore) and 1afb; Mg2af9; -ATP mixture (Millipore). Login to comment 336 ABCC7 p.Ser422Asp The in vitro kinase reaction mixture consists of active PDK1, mutant S422D-SGK1, and substrate CFTR that was immunoprecipitated from either untreated CFPAC-1 or repaired CFPAC-1 pLJ6 cells. Login to comment 415 ABCC7 p.Ser422Asp Phosphorylation of immunoprecipitated CFTR or phosphorylation at Thr256 of S422D-SGK1 in the whole reaction mixture(WRM)wasdetectedbyWesternblotting.D,effectofC4-CERonCFTRubiquitination.ThecelllysatespreparedasdescribedinBweresubjectedtoCFTR immunoprecipitation, followed by Western blot analysis for total CFTR expression or CFTR ubiquitination. Login to comment 425 ABCC7 p.Ser422Asp Furthermore, the importance of active SGK kinase for the addition of CFTR to the plasma membrane in Xenopus oocytes (50) is consistent with our observation that PDK1-activated S422D-SGK1 phosphorylates both wild-type and mutant CFTR. Login to comment 427 ABCC7 p.Val510Asp For instance, the introduction of the suppressor mutation V510D to èc;F508-CFTR mediates rescue of èc;F508-CFTR and increases the half-life of the membrane-bound èc;F508-CFTR (39). Login to comment