G-protein-coupled receptors regulate autophagy by ZBTB16-mediated ubiquitination and proteasomal degradation of Atg14L
- Chinese Academy of Sciences, China
- Harvard Medical School, United States
- Fudan University, China
- Huazhong University of Science and Technology, China
- University of North Carolina at Chapel Hill, United States
- Beth Israel Deaconess Medical Center, Harvard Medical School, United States
Figures
Figure 1 with 1 supplement
ZBTB16 mediates the proteasomal degradation of Atg14L.
(A) The cytoplasmic and nuclear fractions of HeLa cells cultured in normal media were separated by using Paris Kit (Ambion) and analyzed by western blotting using indicated antibodies. (B) HeLa …
Figure 1—figure supplement 1
ZBTB16 mediates the proteasomal degradation of Atg14L.
(A) Quantification for Figure 1B. HeLa cells were transfected with control siRNA (N.T.) or siRNA targeting ZBTB16 and cultured for 72 hr. The cell lysates were analyzed by western blotting with …
Figure 2 with 1 supplement
Ubiquitination of ATG14L by ZBTB16.
(A) 293T cells were transfected with expression vectors of FLAG-ZBTB16 and Myc-Atg14 and cultured for 24 hr. The cells were treated with MG132(10 µM) for the last 4 hr before harvesting. The cell …
Figure 2—figure supplement 1
Ubiquitination of ATG14L by ZBTB16 and Cullin3.
(A) 293T cells were transfected with expression vectors of FLAG-ZBTB16 and Myc-Atg14L and cultured for 24 hr. The cells were treated with MG132 (10 µM) for the last 4 hr before harvesting and then …
Figure 3 with 2 supplements
Serum starvation regulates ZBTB16 activity through GSK3β.
(A) HeLa cells were cultured in serum-free condition for indicated periods of time, and then the cell lysates were harvested and analyzed by western blotting using indicated antibodies. (B) HeLa …
Figure 3—figure supplement 1
Serum starvation regulates ZBTB16 activity through GSK3β.
(A) HeLa cells were serum starvation for the indicated periods of time, and then the mRNA was extracted and subjected to RT-PCR. (B–E) 7721 cells (B), SK-OV-3 cells (C), H4 cells (D) and HCT116 …
Figure 3—figure supplement 2
Serum starvation regulates ZBTB16 activity through GSK3β.
(A) HeLa cells were cultured without serum for the indicated periods of time, and then the cell lysates were harvested and analyzed by western blotting using indicated antibodies. (B) HeLa cells …
Figure 4
Phosphorylation of S184/T282 of ZBTB16 by GSK3b is functionally important for regulating Atg14L under serum starvation condition to promote autophagy.
(A) The expression vectors of Flag-tagged wild type or mutants ZBTB16 and HA-Ub were transfected into HeLa cells and cultured for 24 hr. HeLa cells were treated with or without serum-deprivation …
Figure 5 with 2 supplements
Regulation of Atg14L by GPCR mediated signaling pathways.
(A) HeLa cells were cultured in the presence (no treatment) or absence of serum (serum starvation) for 24 hr and then re-stimulated by 10% FBS or boiled FBS (boiled for 30 min at 95°C) for 1 hr. The …
Figure 5—figure supplement 1
Regulation of Atg14L by ligands and agonists of GPCR.
(A) Quantification for Figure 5A. HeLa cells were cultured in the presence (no treatment) or absence of serum (serum starvation) for 24 hr, and then re-stimulated by 10% FBS or boiled FBS (boiled …
Figure 5—figure supplement 2
Regulation of Atg14L by GPCR mediated signaling pathways.
(A) Quantification for Figure 5F. HeLa cells were transfected with control siRNA, Gαq/11, GαS and cultured for 72 hr. The cell lysates were analyzed by western blotting with indicated antibodies. …
Figure 6 with 1 supplement
Up-regulation of Atg14L and autophagy by GPCR antagonist AMD3100 in vivo ameliorated the neural dysfunction of HD transgenic mice.
(A) WT mice were dosed once intraperitoneally with AMD3100 at 10 mg/kg body weight and 24 hr later, the brain tissues were collected and analyzed by western blotting using indicated antibodies. …
Figure 6—figure supplement 1
Up-regulation of Atg14L and autophagy by GPCR antagonist AMD3100 in vivo ameliorated the neural dysfunction of HD transgenic mice.
(A) HeLa cells were treated with AMD3100 at 10 µM for indicated periods of time, and then the cell lysates were harvested and analyzed by western blotting using indicated antibodies. (B) HeLa cells …
Author response image 1
A. HeLa cells were transfected with expression vectors of Flag-ZBTB16S184A/T282A and cultured for 24h. Before harvesting the sample, the cells were treated with mTOR inhibitor Torin1(500nM) in …
Author response image 2
HeLa cells were transfected with expression vectors of Myc-Atg14 and cultured for 24h. Before harvesting the sample, the cells were treated with or without 10 µM CQ for 4h. The cell lysates were …
Author response image 3
HeLa cells were cultured in serum-free condition for indicated periods of time and then the cell lysates were harvested and analyzed by western blotting using indicated antibodies.
