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 ΔF508-cystic fibrosis transmembrane conductance regulator (ΔF508-CFTR).
Cystic fibrosis (CF) is due to a folding defect in the CF transmembrane conductance regulator (CFTR) protein. The most common mutation, ΔF508, prevents CFTR from trafficking to the apical plasma membrane. Here we show that activation of the PDK1/SGK1 signaling pathway with C4-ceramide (C4-CER), a non-toxic small molecule, functionally corrects the trafficking defect in both cultured CF cells and primary epithelial cell explants from CF patients. The mechanism of C4-CER action involves a series of mutual autophosphorylation and phosphorylation events between PDK1 and SGK1. Detailed mechanistic studies indicate that C4-CER initially induces autophosphorylation of SGK1 at Ser(422). SGK1[Ser(P)(422)] and C4-CER coincidently bind PDK1 and permit PDK1 to autophosphorylate at Ser(241). Then PDK1[Ser(P)(241)] phosphorylates SGK1[Ser(P)(422)] at Thr(256) to generate fully activated SGK1[Ser(422), Thr(P)(256)]. SGK1[Ser(P)(422),Thr(P)(256)] phosphorylates and inactivates the E3 ubiquitin ligase Nedd4-2. ΔF508-CFTR is thus free to traffic to the plasma membrane. Importantly, C4-CER-mediated activation of both PDK1 and SGK1 is independent of the PI3K/Akt/mammalian target of rapamycin signaling pathway. Physiologically, C4-CER significantly increases maturation and stability of ΔF508-CFTR (t½ ∼10 h), enhances cAMP-activated chloride secretion, and suppresses hypersecretion of interleukin-8 (IL-8). We suggest that candidate drugs for CF directed against the PDK1/SGK1 signaling pathway, such as C4-CER, provide a novel therapeutic strategy for a life-limiting disorder that affects one child, on average, each day.
From the Department of Anatomy, Physiology, and Genetics, School of Medicine, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814 and hung.caohuy@usuhs.edu.
Hung
H
Caohuy
From the Department of Anatomy, Physiology, and Genetics, School of Medicine, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814 and.
Qingfeng
Q
Yang
From the Department of Anatomy, Physiology, and Genetics, School of Medicine, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814 and.
Yvonne
Y
Eudy
From the Department of Anatomy, Physiology, and Genetics, School of Medicine, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814 and.
Thien-An
TA
Ha
From the Department of Anatomy, Physiology, and Genetics, School of Medicine, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814 and.
Andrew E
AE
Xu
the Department of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15224.
Matthew
M
Glover
the Department of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15224.
Raymond A
RA
Frizzell
From the Department of Anatomy, Physiology, and Genetics, School of Medicine, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814 and.
Catherine
C
Jozwik
From the Department of Anatomy, Physiology, and Genetics, School of Medicine, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814 and harvey.pollard@usuhs.edu.
Harvey B
HB
Pollard
D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002518:Ceramides / Q000494:pharmacology*; D003550:Cystic Fibrosis / Q000188:drug therapy; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism*; D004353:Drug Evaluation, Preclinical; D056827:Endosomal Sorting Complexes Required for Transport / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D017874:Immediate-Early Proteins / Q000378:metabolism*; D016209:Interleukin-8 / Q000378:metabolism; D000075702:Nedd4 Ubiquitin Protein Ligases; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D055550:Protein Stability; D021381:Protein Transport; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D017384:Sequence Deletion; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism; D000099184:Serum-Glucocorticoid Regulated Kinases
D002518:Ceramides; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D056827:Endosomal Sorting Complexes Required for Transport; D017874:Immediate-Early Proteins; D016209:Interleukin-8; D000075702:Nedd4 Ubiquitin Protein Ligases; D019869:Phosphatidylinositol 3-Kinases; D017346:Protein Serine-Threonine Kinases; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D044767:Ubiquitin-Protein Ligases; D000099184:Serum-Glucocorticoid Regulated Kinases; C505032:CFTR protein, human; C000596772:N-butanoylsphingosine; C000631429:PDK1 protein, human; C000618483:Nedd4 protein, human; C000618485:Nedd4L protein, human
Ceramide; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator (CFTR); Inflammation; Membrane Trafficking