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)

Hung Caohuy,Qingfeng Yang,Yvonne Eudy,Thien-An Ha,Andrew E Xu,Matthew Glover,Raymond A Frizzell,Catherine Jozwik,Harvey B Pollard

doi:10.1074/jbc.m114.598649

Abstract

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.

Highlights

The discovery of small molecules to correct the trafficking defect of the mutant ⌬F508-CFTR protein has been challenging
serum/glucocorticoid-induced protein kinase 1 (SGK1)[Ser(P)422] and C4-CER coincidently bind phosphoinositidedependent kinase 1 (PDK1) and permit PDK1 to autophosphorylate at Ser241
We show that C4-CER functionally rescues ⌬F508-CFTR in cultured Cystic fibrosis (CF) pancreas and lung cells as well as in primary cultures of homozygous ⌬F508-CFTR human bronchial epithelial cells

Summary

Background

The discovery of small molecules to correct the trafficking defect of the mutant ⌬F508-CFTR protein has been challenging. C4-CER-mediated activation of both PDK1 and SGK1 is independent of the PI3K/Akt/mammalian target of rapamycin signaling pathway. We previously reported that the glucocorticoid dexamethasone (DEX) functionally rescues the ⌬F508-CFTR trafficking defect in CF lung and pancreas epithelial cells [10] Such rescue depends upon the coincident activation of 3-phosphoinositidedependent kinase 1 (PDK1) by phosphoinositide-3-kinase (PI3K) and of serum/glucocorticoid-induced protein kinase 1 (SGK1) by DEX. C4-CER-mediated rescue requires both PDK1 and SGK1 but is totally independent of the PI3K/Akt/mammalian target of rapamycin (mTOR) pathway. These data suggest that C4-CER may be a candidate therapeutic for CF, by rescuing the most common mutation, ⌬F508-CFTR, from proteasomal destruction

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION