Syntaxin 6 and CAL mediate the degradation of the cystic fibrosis transmembrane conductance regulator

Abstract

The PDZ domain-containing protein CAL mediates lysosomal trafficking and degradation of CFTR. Here we demonstrate the involvement of a CAL-binding SNARE protein syntaxin 6 (STX6) in this process. Overexpression of STX6, which colocalizes and coimmunoprecipitates with CAL, dramatically reduces the steady-state level and stability of CFTR. Conversely, overexpression of a STX6 dominant-negative mutant increases CFTR. Silencing endogenous STX6 increases CFTR but has no effect on DeltaTRL-CFTR, which cannot bind to CAL. Silencing CAL eliminates the effect of STX6 on CFTR. Both results suggest a dependence of CAL on STX6 function. Consistent with its Golgi localization, STX6 does not bind to ER-localized DeltaF508-CFTR. Silencing STX6 has no effect on DeltaF508-CFTR expression. However, overexpression of STX6 coimmunoprecipitates with and reduces temperature-rescued DeltaF508-CFTR that escapes ER degradation. Conversely, silencing STX6 enhances the effect of low temperature in rescuing DeltaF508-CFTR. Finally, in human bronchial epithelial cells, silencing endogenous STX6 leads to increases in protein levels and Cl(-) currents of both wild-type and temperature-rescued CFTR. We have identified STX6 as a new component of the CAL complex that regulates the abundance and function of CFTR at the post-ER level. Our results suggest a therapeutic role of STX6 in enhancing rescued DeltaF508-CFTR.

Figure 1.

STX6 interacts with CAL and promotes CFTR degradation. (A) HEK293 cells were cotransfected with 3 μg myc-CAL and 3 μg GFP-CFTR with or without 6 μg HA-STX6 as indicated. Forty-eight hours after transfection, cell lysates were harvested and immunoprecipitated by HA-affinity matrix. HA-STX6 interacted with both myc-CAL and GFP-CFTR. (B) HEK293 cells were cotransfected with 3 μg GFP-CFTR and increasing amount of HA-STX6 (3, 6, and 9 μg as indicated). Forty-eight hours after transfection, cell lysates were harvested and subjected to Western blotting analysis. HA-STX6 in a dose-dependent manner reduced GFP-CFTR protein expression. (C) HEK293 cells were cotransfected with 3 μg GFP-CFTR and HA-Vti1b (3, 6, and 9 μg as indicated). Forty-eight hours after transfection, cell lysates were harvested and subjected to Western blotting analysis. HA-Vti1b had no effect on GFP-CFTR protein expression. (D) HEK293 cells were cotransfected with 3 μg GFP-CFTR and STX16 (3, 6, and 9 μg as indicated). At 48 h after transfection, STX16 had no effect on GFP-CFTR protein expression. In these and all subsequent experiments, data shown are representative of at least three independent experiments.

Figure 2.

Dominant-negative interaction and silencing of endogenous SXT6 increases GFP-CFTR (A) HEK293 cells were cotransfected with 3 μg GFP-CFTR and increasing amount of HA-STX6ΔC (3, 6, and 9 μg as indicated). At 48 h after transfection, cell lysates were harvested and subjected to Western blotting analysis. HA-STX6ΔC dose-dependently increases GFP-CFTR protein expression. (B) HEK293 cells were sequentially transfected with 20 nM STX6 siRNA (Hs_STX6_6_HP) and 3 μg GFP-CFTR plasmid (see Materials and Methods). Forty-eight hours after GFP-CFTR transfection, cell lysates were harvested and subjected to Western blotting analysis. Silencing of STX6 increased GFP-CFTR protein expression. (C) HEK293 cells were sequentially transfected with 20 nM STX16 siRNA and 3 μg GFP-CFTR. Forty-eight hours after GFP-CFTR transfection, cell lysates were subjected to Western blotting analysis. Silencing of STX16 had no effect on GFP-CFTR protein expression. (D) HEK293 cells grown on coverslips were sequentially transfected with 20 nM STX6 siRNA and 3 μg GFP-CFTR as in B. Forty-eight hours after GFP-CFTR transfection, cells were fixed and subjected to indirect fluorescence immunocytochemical staining with a STX6 mAb and a CAL polyclonal antibody. CAL is pseudocolored in green, and STX6 is pseudocolored in red (E), as described in D except CFTR was detected with a mouse mAb.

Figure 3.

STX6-mediated CFTR degradation (A) HEK293 cells were transfected with 3 μg GFP-CFTR with or without 6 μg HA-STX6. Twenty-four hours after transfection, the cell culture media was replaced with serum-free media supplemented with 100 μg/ml cycloheximide. Cell lysates were collected at indicated times and subjected to Western blot analysis. (B) Percent of remaining GFP-CFTR relative to the time immediately before cycloheximide treatment. Data are the average of three independent experiments. Error bars, SDs. (C) Cells were transfected as in (A) and treated with 100 ng/ml bafilomycin A1 (BAF) and 100 μg/ml cycloheximide as indicated for 3 h. Cell lysates were collected and subjected to Western blot analysis. (D) Cells were transfected as in (A) and treated with 10 μg/ml E-64 as indicated for 16 h. Cell lysates were collected and subjected to Western blot analysis.

Figure 4.

The dependence of STX6 activity on the carboxyl PDZ biding motif and CAL. (A) HEK293 cells were sequentially transfected with 20 nM STX6 siRNA and 3 μg GFP-CFTR or 3 μg GFP-CFTRΔTRL as indicated. Forty-eight hours after the second transfection, cell lysates were collected and subjected to Western blot analysis. (B) HEK293 cells were sequentially transfected with 20 nM CAL siRNA and 3 μg GFP-CFTR with or without 6 μg HA-STX6 as indicated. Forty-eight hours after the second transfection, cell lysates were collected and subjected to SDS-PAGE and Western blotting analysis. (C) HEK293 cells were cotransfected with 3 μg GFP-CFTRΔTRL with or without 3 μg HA-STX6 as indicated. Forty-eight hours after transfection, cell lysates were harvested and immunoprecipitated by HA-affinity matrix. HA-STX6 interacted with GFP-CFTRΔTRL.

Figure 5.

The independence of STX6 on CAL in binding to CFTR. (A) HEK293 cells were sequentially transfected with 20 nM CAL siRNA and 3 μg GFP-CFTR as indicated. Forty-eight hours after the second transfection, cell lysates were collected and subjected to Western blot analysis. (B) Cell lysates in A were subjected to immunoprecipitation with HA-affinity matrix. HA-STX6 interacted with GFP-CFTR when CAL was silenced with siRNA. (C) HEK293 cells were cotransfected with 3 μg GFP-CFTR with or without 3 μg HA-STX6 as indicated. Forty-eight hours after transfection, cell lysates were harvested and immunoprecipitated by CAL antibody and protein A/G beads. The supernatant after CAL immunoprecipitation was subjected to Western blot analysis to show the complete depletion of CAL. (D) CAL-depleted lysate in C was subjected to immunoprecipitation with HA-affinity matrix. HA-STX6 interacted with GFP-CFTR even when CAL was depleted.

Figure 6.

Silencing of STX6 in CF epithelial cell line CFBE-CFTR. (A) CFBE-CFTR cells were transfected with 20 nM STX6 siRNA as indicated. Seventy-two hours after the transfection, cell lysates were collected and subjected to Western blot analysis by indicated antibodies; GAPDH was used as the loading control. (B) CFBE-CFTR cells were transfected with 20 nM STX6 siRNA or 20 nM control siRNA. Seventy-two hours after the cells were fixed and subjected to indirect fluorescence immunocytochemical staining with a CFTR mAb (left panels) or STX6 mAb (right panels). (C) CFBE-CFTR cells grown on permeable supports were transfected with 1, 10, or 20 nM STX6 siRNA or 20 nM control siRNA as indicated. Seventy-two hours after transfection, cells were mounted on the Ussing chamber setup where the short-circuit current was measured as described in Materials and Methods. In the graph, 10 μM forskolin was added at the first arrow (black), and 1 μM CFTR_inh-172 was added at the second arrow (red).

Figure 7.

STX6 interacts with CFTR in post-ER subcellular compartments. (A) HEK293 cells were transfected with 20 nM STX6 siRNA and 3 μg GFP-CFTR or 3 μg GFP-ΔF508CFTR. Forty-eight hours after the second transfection, cells lysates were collected and subjected to Western blot analysis; CHC was used as a loading control and as an immunoprecipitation control. (B) Hela-ΔF508CFTR cells were transfected with HA-STX6 as indicated and, the next day, cells were either moved to a 27°C incubator or kept at 37°C as indicated. Forty-eight hours after transfection, cell lysates were collected and immunoprecipitated with the CFTR mAb and protein A/G beads. The immunoprecipitated material and cell lysates were subjected Western blot analysis; CHC was used as a loading control and as an immunoprecipitation control. Arrows with solid line point to C bands. Arrows with dashed line point to B bands.

Figure 8.

Silencing of STX6 in CF epithelial cell line CFBE-ΔF508CFTR. (A) CFBE-ΔF508CFTR cells were transfected with 20 nM STX6 siRNA or 20 nM control siRNA as indicated. Seventy-two hours after the transfection, cell lysates were collected and subjected to Western blot analysis by indicated antibodies. (B) CFBE-CFTR cells and CFBE-ΔF508CFTR cells grown on permeable supports were transfected with 20 nM STX6 siRNA or 20 nM control siRNA as indicated. Seventy-two hours after transfection, cells were mounted on the Ussing chamber set-up and the short-circuit current was measured as described in Materials and Methods. In the graph, 50 μM genistein was added at the first arrow (black), and 1 μM CFTR_inh-172 was added at the second arrow (red). Brown line, CFBE-ΔF508CFTR grown at 37°C; yellow line, CFBE-ΔF508CFTR grown at 27°C; light blue line, CFBE-ΔF508CFTR grown at 27°C and transfected with STX6 siRNA; dark blue line, CFBE-CFTR grown at 37°C; and pink line, CFBE-CFTR grown at 37°C and transfected with STX6 siRNA.