Antagonistic Regulation of Cystic Fibrosis Transmembrane Conductance Regulator Cell Surface Expression by Protein Kinases WNK4 and Spleen Tyrosine Kinase

ABSTRACT The cystic fibrosis transmembrane conductance regulator (CFTR) protein is an epithelial receptor mediating the translocation of Salmonella enterica serovar Typhi to the gastric submucosa. Since the level of cell surface CFTR is directly related to the efficiency of serovar Typhi translocation, the goal of this study was to measure CFTR expression by the intestinal epithelium during infection. CFTR protein initially present in the epithelial cell cytoplasm was rapidly trafficked to the plasma membrane following exposure to live serovar Typhi or bacterial extracts. CFTR-dependent bacterial uptake by epithelial cells increased (>100-fold) following CFTR redistribution. The bacterial factor which triggers CFTR redistribution is heat and protease sensitive. These data suggest that serovar Typhi induces intestinal epithelial cells to increase membrane CFTR levels, leading to enhanced bacterial ingestion and submucosal translocation. This could be a key, early step in the infectious process leading to typhoid fever.

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Regulation of the cystic fibrosis transmembrane conductance regulator Cl- channel by specific protein kinases and protein phosphatases.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)53959-4 ◽

1993 ◽

Vol 268 (3) ◽

pp. 2037-2047 ◽

Cited By ~ 2

Author(s):

H.A. Berger ◽

S.M. Travis ◽

M.J. Welsh

Keyword(s):

Cystic Fibrosis ◽

Protein Kinases ◽

Protein Phosphatases ◽

Specific Protein ◽

Transmembrane Conductance Regulator ◽

Transmembrane Conductance ◽

Cl Channel ◽

Cystic Fibrosis Transmembrane

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Dynasore inhibits removal of wild-type and ΔF508 cystic fibrosis transmembrane conductance regulator (CFTR) from the plasma membrane

Biochemical Journal ◽

10.1042/bj20090389 ◽

2009 ◽

Vol 421 (3) ◽

pp. 377-385 ◽

Cited By ~ 15

Author(s):

Andrew Young ◽

Martina Gentzsch ◽

Cynthia Y. Abban ◽

Ying Jia ◽

Patricio I. Meneses ◽

...

Keyword(s):

Cystic Fibrosis ◽

Cell Surface ◽

Small Molecule ◽

Short Exposure ◽

Growth Media ◽

Transmembrane Conductance Regulator ◽

Wild Type ◽

Transmembrane Conductance ◽

Δf508 Cftr ◽

Cystic Fibrosis Transmembrane

Dynasore, a small molecule inhibitor of dynamin, was used to probe the role of dynamin in the endocytosis of wild-type and mutant CFTR (cystic fibrosis transmembrane conductance regulator). Internalization of both wild-type and ‘temperature-corrected’ ΔF508 CFTR was markedly inhibited by a short exposure to dynasore, implicating dynamin as a key element in the endocytic internalization of both wild-type and mutant CFTR. The inhibitory effect of dynasore was readily reversible upon washout of dynasore from the growth media. Corr-4 ({2-(5-chloro-2-methoxy-phenylamino)-4′-methyl-[4,5′]-bithiazolyl-2′-yl}-phenyl-methanonone), a pharmacological corrector of ΔF508 CFTR biosynthesis, caused a marked increase in the cell surface expression of mutant CFTR. Co-incubation of ΔF508 CFTR expressing cells with Corr-4 and dynasore caused a significantly greater level of cell surface CFTR than that observed in the presence of Corr-4 alone. These results argue that inhibiting the endocytic internalization of mutant CFTR provides a novel therapeutic target for augmenting the benefits of small molecule correctors of mutant CFTR biosynthesis.

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The tyrosine kinase p60c-src regulates the fast gate of the cystic fibrosis transmembrane conductance regulator chloride channel

Biophysical Journal ◽

10.1016/s0006-3495(96)79501-2 ◽

1996 ◽

Vol 71 (6) ◽

pp. 3073-3082 ◽

Cited By ~ 44

Author(s):

H. Fischer ◽

T.E. Machen

Keyword(s):

Cystic Fibrosis ◽

Tyrosine Kinase ◽

Chloride Channel ◽

Transmembrane Conductance Regulator ◽

Transmembrane Conductance ◽

Cystic Fibrosis Transmembrane

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Ablation of Internalization Signals in the Carboxyl-terminal Tail of the Cystic Fibrosis Transmembrane Conductance Regulator Enhances Cell Surface Expression

Journal of Biological Chemistry ◽

10.1074/jbc.m209275200 ◽

2002 ◽

Vol 277 (51) ◽

pp. 49952-49957 ◽

Cited By ~ 25

Author(s):

Krisztina Peter ◽

Karoly Varga ◽

Zsuzsa Bebok ◽

Carmel M. McNicholas-Bevensee ◽

Lisa Schwiebert ◽

...

Keyword(s):

Cystic Fibrosis ◽

Chloride Channel ◽

Surface Expression ◽

Transmembrane Conductance Regulator ◽

Channel Activity ◽

Transmembrane Conductance ◽

Coated Pits ◽

Double Mutation ◽

Carboxyl Terminal ◽

Cystic Fibrosis Transmembrane

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that undergoes endocytosis through clathrin-coated pits. Previously, we demonstrated that Y1424A is important for CFTR endocytosis (Prince, L. S., Peter, K., Hatton, S. R., Zaliauskiene, L., Cotlin, L. F., Clancy, J. P., Marchase, R. B., and Collawn, J. F. (1999)J. Biol. Chem.274, 3602–3609). Here we show that a second substitution in the carboxyl-terminal tail of CFTR, I1427A, on Y1424A background more than doubles CFTR surface expression as monitored by surface biotinylation. Internalization assays indicate that enhanced surface expression of Y1424A,I1427A CFTR is caused by a 76% inhibition of endocytosis. Patch clamp recording of chloride channel activity revealed that there was a corresponding increase in chloride channel activity of Y1424A,I1427A CFTR, consistent with the elevated surface expression, and no change in CFTR channel properties. Y14124A showed an intermediate phenotype compared with the double mutation, both in terms of surface expression and chloride channel activity. Metabolic pulse-chase experiments demonstrated that the two mutations did not affect maturation efficiency or protein half-life. Taken together, our data show that there is an internalization signal in the COOH terminus of CFTR that consists of Tyr1424-X-X-Ile1427where both the tyrosine and the isoleucine are essential residues. This signal regulates CFTR surface expression but not CFTR biogenesis, degradation, or chloride channel function.

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Dab2 is a key regulator of endocytosis and post-endocytic trafficking of the cystic fibrosis transmembrane conductance regulator

Biochemical Journal ◽

10.1042/bj20111566 ◽

2011 ◽

Vol 441 (2) ◽

pp. 633-643 ◽

Cited By ~ 19

Author(s):

Lianwu Fu ◽

Andras Rab ◽

Li Ping Tang ◽

Steven M. Rowe ◽

Zsuzsa Bebok ◽

...

Keyword(s):

Cystic Fibrosis ◽

Epithelial Cells ◽

Cell Surface ◽

Half Life ◽

Adaptor Proteins ◽

Airway Epithelial Cells ◽

Transmembrane Conductance Regulator ◽

Transmembrane Conductance ◽

E3 Ligases ◽

Cystic Fibrosis Transmembrane

CFTR (cystic fibrosis transmembrane conductance regulator) is expressed in the apical membrane of epithelial cells. Cell-surface CFTR levels are regulated by endocytosis and recycling. A number of adaptor proteins including AP-2 (μ2 subunit) and Dab2 (Disabled-2) have been proposed to modulate CFTR internalization. In the present study we have used siRNA (small interfering RNA)-mediated silencing of these adaptors to test their roles in the regulation of CFTR cell-surface trafficking and stability in human airway epithelial cells. The results indicate that μ2 and Dab2 performed partially overlapping, but divergent, functions. While μ2 depletion dramatically decreased CFTR endocytosis with little effect on the half-life of the CFTR protein, Dab2 depletion increased the CFTR half-life ~3-fold, in addition to inhibiting CFTR endocytosis. Furthermore, Dab2 depletion inhibited CFTR trafficking from the sorting endosome to the recycling compartment, as well as delivery of CFTR to the late endosome, thus providing a mechanistic explanation for increased CFTR expression and half-life. To test whether two E3 ligases were required for the endocytosis and/or down-regulation of surface CFTR, we siRNA-depleted CHIP [C-terminus of the Hsc (heat-shock cognate) 70-interacting protein] and c-Cbl (casitas B-lineage lymphoma). We demonstrate that CHIP and c-Cbl depletion have no effect on CFTR endocytosis, but c-Cbl depletion modestly enhanced the half-life of CFTR. The results of the present study define a significant role for Dab2 both in the endocytosis and post-endocytic fate of CFTR.

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Enhanced cell-surface stability of rescued ΔF508 cystic fibrosis transmembrane conductance regulator (CFTR) by pharmacological chaperones

Biochemical Journal ◽

10.1042/bj20071420 ◽

2008 ◽

Vol 410 (3) ◽

pp. 555-564 ◽

Cited By ~ 72

Author(s):

Karoly Varga ◽

Rebecca F. Goldstein ◽

Asta Jurkuvenaite ◽

Lan Chen ◽

Sadis Matalon ◽

...

Keyword(s):

Cystic Fibrosis ◽

Cell Surface ◽

Half Life ◽

Permissive Temperature ◽

Transmembrane Conductance Regulator ◽

Transmembrane Conductance ◽

Surface Stability ◽

Pharmacological Chaperones ◽

Δf508 Cftr ◽

Cystic Fibrosis Transmembrane

Misfolded proteins destined for the cell surface are recognized and degraded by the ERAD [ER (endoplasmic reticulum) associated degradation] pathway. TS (temperature-sensitive) mutants at the permissive temperature escape ERAD and reach the cell surface. In this present paper, we examined a TS mutant of the CFTR [CF (cystic fibrosis) transmembrane conductance regulator], CFTR ΔF508, and analysed its cell-surface trafficking after rescue [rΔF508 (rescued ΔF508) CFTR]. We show that rΔF508 CFTR endocytosis is 6-fold more rapid (∼30% per 2.5 min) than WT (wild-type, ∼5% per 2.5 min) CFTR at 37 °C in polarized airway epithelial cells (CFBE41o−). We also investigated rΔF508 CFTR endocytosis under two further conditions: in culture at the permissive temperature (27 °C) and following treatment with pharmacological chaperones. At low temperature, rΔF508 CFTR endocytosis slowed to WT rates (20% per 10 min), indicating that the cell-surface trafficking defect of rΔF508 CFTR is TS. Furthermore, rΔF508 CFTR is stabilized at the lower temperature; its half-life increases from <2 h at 37 °C to >8 h at 27 °C. Pharmacological chaperone treatment at 37 °C corrected the rΔF508 CFTR internalization defect, slowing endocytosis from ∼30% per 2.5 min to ∼5% per 2.5 min, and doubled ΔF508 surface half-life from 2 to 4 h. These effects are ΔF508 CFTR-specific, as pharmacological chaperones did not affect WT CFTR or transferrin receptor internalization rates. The results indicate that small molecular correctors may reproduce the effect of incubation at the permissive temperature, not only by rescuing ΔF508 CFTR from ERAD, but also by enhancing its cell-surface stability.

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Epitope tagging permits cell surface detection of functional CFTR

AJP Cell Physiology ◽

10.1152/ajpcell.1995.269.6.c1565 ◽

1995 ◽

Vol 269 (6) ◽

pp. C1565-C1576 ◽

Cited By ~ 34

Author(s):

M. Howard ◽

M. D. DuVall ◽

D. C. Devor ◽

J. Y. Dong ◽

K. Henze ◽

...

Keyword(s):

Cell Surface ◽

Surface Membrane ◽

Surface Expression ◽

Transmembrane Conductance Regulator ◽

Wild Type ◽

Transmembrane Conductance ◽

External Loop ◽

Glycosylation Sites ◽

Apical Membranes ◽

Cystic Fibrosis Transmembrane

The cystic fibrosis transmembrane conductance regulator (CFTR) is a phosphorylation-activated Cl channel responsible for adenosine 3',5'-cyclic monophosphate (cAMP)-induced Cl secretion across the apical membranes of epithelial cells. To optimize its detection for membrane localization studies, we tagged CFTR with epitope sequences at the carboxy terminus or in the fourth external loop. When epitopes were added to the fourth external loop, the N-linked glycosylation sites in that loop were either preserved or they were mutated to produce a deglycosylated CFTR (dgCFTR). Tagged CFTRs were expressed in HeLa cells, and their cAMP-sensitive Cl permeability was assayed using the halide-sensitive fluorophore SPQ. CFTRs containing the M2 epitope showed halide permeability responses to cAMP, whereas cells expressing CFTR with the hemagglutinin (HA) tag showed little or no cAMP response. Xenopus oocytes expressing dgCFTR, with or without the M2 epitope, showed Cl conductance responses that were 20% of the wild-type response, whereas M2-tagged constructs retaining the glycosylation sites responded like wild-type CFTR. External M2-tagged CFTR was detected in the surface membrane of nonpermeabilized cells. The surface expression of the mutant M2-tagged CFTRs correlated with processing of these mutants (Gregory et al. Mol. Cell. Biol. 11:3886-3893, 1991). M2-901/CFTR is a useful reporter for the trafficking of wild-type and mutant CFTRs to the cell surface.

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