The stress-responsive kinases MAPKAPK2/MAPKAPK3 activate starvation-induced autophagy through Beclin 1 phosphorylation
- UT Southwestern Medical Center, United States
- Howard Hughes Medical Institute, UT Southwestern Medical Center, United States
- North Dakota State University, United States
- Hannover Medical School, Germany
Figures
Figure 1
Beclin 1 S90 is phosphorylated in response to nutrient starvation.
(A) Detection of Beclin 1 S90 phosphorylation in HeLa cells transfected with indicated Flag-Beclin 1 construct by radiolabeling, immunoprecipitation with an anti-Flag antibody and autoradiography …
Figure 2 with 3 supplements
The Beclin 1 S90 phosphorylation site is required for autophagy induction in MCF7 and U2OS cells.
(A) Western blot results of MCF7 cells transiently transfected with empty vector, and Flag epitope-tagged wild-type Beclin 1, Beclin 1 S90A, or Beclin 1 S90E. The cells were grown in normal medium …
Figure 2—figure supplement 1
Doxycycline reduces Beclin 1 expression and starvation-induced autophagy in U2OS cells that express doxycycline-inducible beclin 1 shRNA.
Detection of Beclin 1 and autophagy levels (as measured by p62 and LC3 immunoblots) in U2OS that express doxycycline-inducible beclin 1 shRNA. Cells were cultured in the presence or absence of 1 …
Figure 2—figure supplement 2
Bafilomycin A1-induced Beclin 1 S90 phosphorylation is reversed by the ROS scavenger, N-acetyl-L-cysteine.
HeLa cells were either untreated, pre-treated or not pre-treated with 5 mM N-acetyl-L-cysteine for 1 hr, followed by culture either in the presence or absence 100 nM Bafilomycin A1 for 3 hr. Baf A1, …
Figure 2—figure supplement 3
Effects of Beclin 1 S90 phosphorylation o on subcellular localization of the Beclin 1/VPS34/ATG14 complex.
Subcellular localization of Beclin 1, VPS34, and ATG14 in normal media or after starvation for 2 hr in HBSS in beclin 1 shRNA U2OS cells following treatment with 1 μg/ml doxycycline for 4 days and …
Figure 3
The Beclin 1 S90 phosphorylation site is required for tumor suppression function in MCF7 cells.
(A) Western blot detection of Beclin 1 p-S90, Flag-Beclin 1, p62, and LC3 in MCF7 cells stably transduced with retroviruses expressing indicated Beclin 1 construct. (B) Xenograft growth of cells in …
Figure 4 with 1 supplement
MK2 and MK3 mediate phosphorylation of Beclin 1 S90.
(A) In vitro kinase activity of MK3 using indicated Beclin 1 peptides as a substrate. (B) In vitro kinase activity of MK2, MK3, and MK5 using the wild-type (WT) Beclin 1 peptide shown in A as a …
Figure 4—figure supplement 1
Results of in vitro kinase screen using the Beclin 1 83–97 peptide as a substrate.
Kinases included in screen are listed in alphabetical order on the x-axis. See Supplementary file 1 for details.
Figure 5 with 1 supplement
MK2 positively regulates autophagy.
(A) Effects of wild-type, dominant negative (K/R) and constitutively active (EE) MK2 on endogenous Beclin 1 S90 phosphorylation, MK2/MK3 activation (levels of p-HSP27) and autophagy (levels of p62 …
Figure 5—figure supplement 1
Dominant p38α inhibits starvation-induced MK2 activation and Beclin 1 S90 phosphorylation.
(A) Effects of dominant-negative (DN) JNK1 and DN p38α on Beclin 1 S90 phosphorylation, MK2 activation (MK2 band shift and p-HSP27) and autophagy (p62 degradation and LC3-II conversion) in HeLa …
Figure 6
MK2 positively regulates autophagy through the Beclin 1 S90 phosphorylation site.
(A) Western blot detection of Beclin 1 S90 phosphorylation and autophagy (p62 degradation and LC3-II conversion) in MK2−/−/MK3−/− MEFs retrovirally transduced with indicated expression constructs …
Figure 7
BCL2 inhibits MK2-dependent Beclin 1 S90 phosphorylation.
(A) In vitro kinase activity of MK2 using Flag-Beclin 1 purified from HEK293 cells as a substrate in the presence of the indicated amount of GST-BCL2. (B) Structural model showing that steric …
Figure 8
Model of convergent functions of MAPK signaling molecules in amino acid starvation-induced autophagy and regulation of Beclin 1 S90 phosphorylation.
In response to nutrient starvation, JNK1 results in multi-site phosphorylation of BCL2 and disruption of BCL2/Beclin 1 binding (Wei et al., 2008). This permits active MK2/3 to phosphorylate Beclin 1 …
Additional files
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Supplementary file 1
Raw data of in vitro kinase screen with 190 kinases using the Beclin 1 peptide spanning from amino acids 83–97 as a substrate.
- https://doi.org/10.7554/eLife.05289.016
