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. 2013 Nov;19(11):1478-88.
doi: 10.1038/nm.3322. Epub 2013 Oct 20.

Mst1 inhibits autophagy by promoting the interaction between Beclin1 and Bcl-2

Yasuhiro Maejima  1 , Shiori Kyoi, Peiyong Zhai, Tong Liu, Hong Li, Andreas Ivessa, Sebastiano Sciarretta, Dominic P Del Re, Daniela K Zablocki, Chiao-Po Hsu, Dae-Sik Lim, Mitsuaki Isobe, Junichi Sadoshima
Affiliations

Mst1 inhibits autophagy by promoting the interaction between Beclin1 and Bcl-2

Yasuhiro Maejima et al. Nat Med. 2013 Nov.

Abstract

Here we show that Mst1, a proapoptotic kinase, impairs protein quality control mechanisms in the heart through inhibition of autophagy. Stress-induced activation of Mst1 in cardiomyocytes promoted accumulation of p62 and aggresome formation, accompanied by the disappearance of autophagosomes. Mst1 phosphorylated the Thr108 residue in the BH3 domain of Beclin1, which enhanced the interaction between Beclin1 and Bcl-2 and/or Bcl-xL, stabilized the Beclin1 homodimer, inhibited the phosphatidylinositide 3-kinase activity of the Atg14L-Beclin1-Vps34 complex and suppressed autophagy. Furthermore, Mst1-induced sequestration of Bcl-2 and Bcl-xL by Beclin1 allows Bax to become active, thereby stimulating apoptosis. Mst1 promoted cardiac dysfunction in mice subjected to myocardial infarction by inhibiting autophagy, associated with increased levels of Thr108-phosphorylated Beclin1. Moreover, dilated cardiomyopathy in humans was associated with increased levels of Thr108-phosphorylated Beclin1 and signs of autophagic suppression. These results suggest that Mst1 coordinately regulates autophagy and apoptosis by phosphorylating Beclin1 and consequently modulating a three-way interaction among Bcl-2 proteins, Beclin1 and Bax.

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Figures

Figure 1
Figure 1. Suppression of autophagy during chronic phase of MI is detrimental for cardiac remodeling
(a) Representative immunofluorescent images of staining with p62/SQSTM1 (green), DAPI (blue), and ProteoStat® aggresome detection reagent (red) in Tg-DN-Mst1, Mst1−/− Beclin1+/− Beclin1+/−-Tg-DN-Mst1, and NTg mice 6 weeks after coronary artery ligation are shown. Results represent means from 4 independent experiments. (b) Quantitative analysis of the number of GFP-LC3 puncta 6 weeks after coronary artery ligation in Tg-DN-Mst1, Mst1−/− Beclin1 +/− Beclin1+/−-Tg-DN-Mst1, and NTg mice that were crossed with Tg-GFP-LC3 mice is shown. Representative images of GFP-LC3 puncta are shown in Supplementary Figure 1a. Data are reported as mean ± SEM. (c) Representative immunoblot images of heart homogenates from Tg-DN-Mst1, Mst1−/− Beclin1+/− Beclin1+/− -Tg-DN-Mst1, and NTg mice with phospho-Mst1 (Thr183), total Mst1, total Beclin1, p62/SQSTM1, LC3, and GAPDH antibodies after 6 weeks of MI are shown. Results represent means from 3 independent experiments. (d) Upper: Quantitative analysis of the LV scar size in Tg-DN-Mst1, Mst1−/− Beclin1+/− Beclin1+/− -Tg-DN-Mst1, and NTg mice after 6 weeks of MI is shown. Data are reported as mean ± SEM. * P < 0.05 vs NTg mice after 6-week MI. Lower: Representative images of four consecutive myocardial slices stained with Masson’s trichrome in Tg-DN-Mst1, Mst1−/− Beclin1+/− Beclin1+/− -Tg-DN-Mst1, and NTg mice after 6 weeks of MI are shown. (e) Echocardiographic analyses were conducted after 6 weeks of MI. Left: Comparison of fractional shortening (%FS) in Tg-DN-Mst1, Mst1−/−Beclin1+/−Beclin1+/− -Tg-DN-Mst1, and NTg mice. Data are reported as mean ± SEM. * P < 0.05 vs NTg mice after 6-week MI. Right: Representative images of echocardiographs of Tg-DN-Mst1, Mst1−/−Beclin1+/− Beclin1+/−-Tg-DN-Mst1, and NTg mice 6 weeks after coronary artery ligation are shown.
Figure 2
Figure 2. Mst1 promotes accumulation of protein aggresomes and p62/SQSTM1 and inhibits autophagy in cardiomyocytes
(a) Immunoblot analysis of heart homogenates with ubiquitin-specific antibody. Results represent means from 3 independent experiments. (b) Left: Representative immunofluorescent images of staining with p62/SQSTM1 (green), DAPI (blue), and ProteoStat® aggresome detection reagent (red), a marker of protein aggregation, in NTg and Tg-Mst1 hearts are shown. Right: The number of cells with aggresomes co-localized with p62/SQSTM1 in cardiomyocytes, indicated by yellow color in the merged images (arrows), was counted (n = 5 in each group). Data are reported as mean ± SEM. (c) Upper: Representative immunofluorescent images of staining with p62/SQSTM1 (green), DAPI (blue), and ProteoStat® aggresome detection reagent (red) in Ad-LacZ-transduced and Ad-Mst1-transduced cultured cardiomyocytes are shown. Lower: The number of CMs with aggresomes co-localized with p62/SQSTM1, indicated by yellow color in the merged images (arrows), was counted (n = 6 in each group). Data are reported as mean ± SEM. (d) Representative images of aggresomes detected by transmission electron microscopy in NTg and Tg-Mst1 hearts. The aggresomes (arrow), embedded in a dense meshwork of intermediate filaments (IF) adjacent to the Golgi apparatus (Go), appeared as electron dense amorphous objects. N: Nucleus, Ly: Lysosome. Results represent means from 4 independent experiments. (e) Long-lived protein degradation assay. Quantitative analysis of the degradation rate of long-lived proteins, as indicated by the ratio of the TCA-soluble medium to TCA-precipitated cell lysate 3H-l-valine radioactivity, is shown (n = 3 in each group). Data are reported as mean ± SEM. (f) Left: Representative images of autophagosomes (arrows and insets) detected by transmission electron microscopy after 48 hours of starvation in Tg-Mst1, Tg-DN-Mst1, Mst1−/−, and NTg hearts are shown. Right: Quantitative analysis of the number of autophagosomes is shown (n = 3 in each group). Data are reported as mean ± SEM. * P < 0.05 vs NTg - Baseline; # P < 0.05 vs NTg - starvation; † P < 0.05 vs Tg-Mst1 - Baseline; ¶ P < 0.05 vs Tg-DN-Mst1 - Baseline; § P < 0.05 vs Mst1−/− - Baseline. (g) Representative immunoblot images of heart homogenates with antibodies to p62/SQSTM1, LC3, Mst1, and GAPDH are shown. Results represent means from 3 independent experiments. (h) Cardiomyocytes were transduced with Ad-Mst1, Ad-shMst1, or Ad-LacZ 24 hours after Ad-mRFP-GFP-LC3 transduction and treated with glucose-free media. The mean numbers of autophagosomes, represented by yellow puncta in merged images, and autolysosomes, represented by red puncta in merged images, per cell are shown (n = 3 in each group). Data are reported as mean ± SEM. * P < 0.05 vs Ad-LacZ/GD(−); # P < 0.05 vs Ad-LacZ/GD(+). Representative images of fluorescent LC3 puncta after Ad-mRFP-GFP-LC3 transduction are shown in Supplementary Figure 2e.
Figure 3
Figure 3. Mst1 physically interacts with Beclin1 and enhances its binding with Bcl-2/Bcl-xL, thereby inhibiting the kinase activity of the Beclin1-Vps34 (Class III PI3K) complex through modulation of the Atg14L-Beclin1 interaction and Beclin1 homodimerization in CMs
(a) Cardiomyocytes were transduced with or without Ad-Mst1. Forty-eight hours after transduction, lysates were extracted for immunoprecipitation with Mst1-specific antibody or control IgG, followed by probing with ULK1, Atg4B, Atg5, Beclin1, Atg7, or LC3 antibody. Representative images are shown. Results represent means from 3 independent experiments. (b) Lipid kinase assay of Beclin1-Vps34 complex. Cardiomyocytes were transduced with Ad-Mst1 or Ad-LacZ. Left: Endogenous Vps34 complex was immunoprecipitated using Atg14L-specific antibody for the in vitro kinase assay. The resulting radioactive phosphatidylinositol 3-phosphate (PtdIns(3,4,5)P3), a reaction product, was separated by TLC. Right: Radioactive PtdIns(3,4,5)P3 was excised from thin layer plates and the radioactivity was measured in an LSC (n = 4 in each group). Data are reported as mean ± SEM. (c) Membrane-associated phosphoinositide 3-kinase (PI3K) assay in situ. Cardiomyocytes were transduced with Ad-Mst1, Ad-DN-Mst1, Ad-sh-Mst1, or Ad-LacZ 24 hours after Ad-GFP-2xFYVE transduction and then treated with or without 3-MA (5 mM) or glucose deprivation for 2 hours. Upper: Representative images of GFP-2xFYVE dots are shown. Lower: Quantitative analysis of the number of GFP-2xFYVE dots is shown (n = 6 in each group). Data are reported as mean ± SEM. * P < 0.05 vs Ad-LacZ, starvation (−). (d) Cardiomyocytes were transduced with Ad-Mst1 or Ad-LacZ. Forty-eight hours after transduction, lysates were extracted for immunoprecipitation with Atg14L-specific antibody, Vps34-specific antibody or control IgG, followed by probing with Beclin1-specific antibody. (e) Cardiomyocytes were transduced with or without Ad-Mst1. Forty-eight hours after transduction, lysates were extracted for immunoprecipitation with Beclin1 antibody or control IgG, followed by probing with antibodies to Bcl-2, Bcl-xL, Atg14, UVRAG, or Rubicon. Representative images are shown. Results represent means from 3 independent experiments. (f) Heart homogenates obtained from NTg, Tg-Mst1, Tg-DN-Mst1, or Mst1−/− mice were immunoprecipitated with Atg14L antibody or control IgG, followed by probing with Beclin1 antibody. Representative images are shown. Results represent means from 3 independent experiments. (g) Cardiomyocytes were transduced with Ad-GFP-LC3 with or without Ad-Mst1 either in the absence or presence of Ad-sh-Bcl-2 or Ad-sh-Bcl-xL. Left: Representative images of GFP-LC3 puncta are shown. Right: Quantitative analysis of the number of GFP-LC3 puncta is shown (n = 6 in each group). Data are reported as mean ± SEM. * P < 0.05 vs untreated control; † P < 0.05 vs Ad-Mst1; § P < 0.05 vs Ad-shBcl-2; ¶ P < 0.05 vs Ad-shBcl-xL. (h) Cardiomyocytes transduced with or without Ad-Mst1 were treated with ABT-737 (0, 0.1, 1, 10 µM) for 12 hours. Representative immunoblots with antibodies to p62/SQSTM1, LC3 and GAPDH are shown. Results represent means from 3 independent experiments. (i) An assay for Beclin1 dimerization. Cardiomyocytes were transduced with Ad-3xFlag-Beclin1-WT and Ad-3xHA-Beclin1-WT with or without Ad-Mst1. Forty-eight hours after transduction, lysates were extracted for immunoprecipitation with HA-specific or Flag-specific antibody, followed by probing with Flag-specific or HA-specific antibody, respectively. Representative images are shown. Results represent means from 3 independent experiments.
Figure 4
Figure 4. Mst1 phosphorylates Beclin1 at Thr108, located in its BH3 domain
(a) In vitro kinase assays were carried out by incubating recombinant GST-Beclin1-WT protein with recombinant Mst1 in the presence of 32P-labeled ATP. Reactions were analyzed by SDS-PAGE followed by autoradiography. Representative images are shown. Results represent means from 3 independent experiments. (b) The indicated recombinant partial proteins of Beclin1 were incubated with recombinant Mst1 in the presence of 32P-labeled ATP for in vitro kinase assays. Representative images are shown. Results represent means from 3 independent experiments. (c) The MS/MS spectrum of the Beclin1-NT2 peptide [102–121]. The peptide treated with Mst1 contained a phosphorylated Thr108. (d) Upper: Cardiomyocytes were transduced with wild-type 3x Flag-tagged Beclin1 or T108A mutant. Ectopically expressed Beclin1 proteins were immunoprecipitated with a Flag-M2 antibody and detected with an antibody against either Beclin1 phosphorylated at Thr108 or total Beclin1. Middle: Cardiomyocytes were transduced with Ad-Mst1, Ad-DN-Mst1, or Ad-LacZ. Expression and phosphorylation of Beclin1 was examined by immunoblots with specific antibodies. Phospho-Beclin1 (Thr108) antibody detects Beclin1 phosphorylated by Mst1. Lower: Immunoblot analysis of heart homogenates with phospho-Beclin1 (Thr108)-specific antibody. Representative images are shown. Results represent means from 3 independent experiments. (e) Representative immunofluorescent images of staining with phospho-Beclin1 (Thr108)-specific antibody (green), DAPI (blue), and Troponin T (red), a cardiac-specific marker, in NTg and Tg-Mst1 hearts are shown. Arrows indicate positive staining of phospho-Beclin1 (Thr108) in the perinuclear region. Magnification of a perinuclear region is shown in the inset. Results represent means from 4 independent experiments. (f) Representative immunofluorescent images of staining with phospho-Beclin1 (Thr108)-specific antibody labeled with Alexa Fluor® 488 (green), DAPI (blue), Alexa Fluor® 555 phalloidin (violet), and either KDEL-specific antibody labeled with Alexa Fluor® 647 (red, a marker of endoplasmic reticulum), TGN46-specific antibody labeled with Alexa Fluor® 647 (red, a marker of golgi apparatus), or MitoTracker® Deep Red FM (red, a marker of mitochondria) in Ad-shScramble-transduced cardiomyocytes are shown. Results represent means from 4 independent experiments.
Figure 5
Figure 5. Beclin1 phosphorylated by Mst1 at Thr108 can interact with Bcl-2 family proteins, thereby inhibiting autophagy
(a-f) Pull-down assays after kinase reaction. (a) GST-Beclin1-WT was incubated with either Mst1 or death-associated protein kinase (DAPK) in the presence of ATP, and then incubated with a recombinant Bcl-2 protein to conduct pull-down assays. (b) GST-Beclin1-WT, GST-Beclin1-T108D, or GST-Beclin1-T108A was incubated with or without Mst1 in the presence of ATP, and then incubated with either Bcl-2 or Bcl-xL protein. (c) GST-Beclin1-WT was incubated with or without Mst1 in the presence of ATP, and then incubated with Bcl-2-WT or Bcl-2-H117A proteins. (d) The indicated recombinant GST-tagged partial proteins of Beclin1 were incubated with or without Mst1 in the presence of ATP, and then incubated with Bcl-2 protein. (e) GST-Beclin1-N2 or GST-Beclin1-MID was incubated with or without Mst1 in the presence of ATP, and then incubated with either His-Bcl-2-WT or His-Bcl-2-ΔBH4 proteins. (f) Non-tagged Beclin1-WT after kinase reaction with or without Mst1, cross-linked with or without Bcl-2, were separated by Native-PAGE. The gels were then visualized by both immunoblotting (Upper) and Coomassie Brilliant Blue staining (Lower). In (a) to (f), Representative images are shown. Results represent means from 3 independent experiments. (g) Lipid kinase assay of the Beclin1-Vps34 complex I. Cardiomyocytes were co-transduced with Ad-Beclin1-WT, Ad-Beclin1-T108A, or Ad-Beclin1-T108D either in the absence or presence of Ad-Mst1. Upper: Endogenous Vps34 complex was immunoprecipitated with Atg14L-specific antibody for the in vitro kinase assay. The resulting radioactive PtdIns(3,4,5)P3 was separated by TLC. Lower: Radioactive PtdIns(3,4,5)P3 was excised from thin layer plates and the radioactivity was measured in an LSC (n = 4, respectively). Data are reported as mean ± SEM. (h) Membrane-associated PI3K assay in situ. Cardiomyocytes were co-transduced with Ad-Beclin1-WT, Ad-Beclin1-T108A, or Ad-Beclin1-T108D in the absence or presence of Ad-Mst1 24 hours after Ad-GFP-2xFYVE transduction. Quantitative analysis of the number of GFP-2xFYVE dots is shown (n = 6 in each group). Data are reported as mean ± SEM.
Figure 6
Figure 6. Mst1-induced phosphorylation of Beclin1 inhibits autophagy and induces apoptosis by sequestering Bcl-2 from Bax, thereby causing deterioration of cardiac function in failing hearts
(a) Cardiomyocytes were transduced with Ad-sh-Beclin1 and either Ad-Beclin1-WT, Ad-Beclin1-T108A, or Ad-Beclin1-T108D in the absence or presence of Ad-Mst1 24 hours after Ad-GFP-LC3 transduction, under either nutrient rich or starvation conditions. Upper: Representative images of GFP-LC3 puncta are shown. Lower: Quantitative analysis of the number of GFP-LC3 puncta is shown (n = 6 in each group). Data are reported as mean ± SEM. * P < 0.05 vs Ad-3xFlag-Beclin1-WT, Starvation (−), # P < 0.05 vs Ad-3xFlag-Beclin1-WT, Starvation (+), † P < 0.05 vs Ad-3xFlag-Beclin1-WT+Ad-Mst1, Starvation (+). (b) Cardiomyocytes were transduced with Ad-myc-Bcl-2-WT either in the absence or presence of Ad-Mst1, and Ad-Beclin1-WT, Ad-Beclin1-T108D or Ad-Beclin1-T108A. Forty-eight hours after transduction, lysates were extracted for immunoprecipitation with antibodies to myc or Bax (clone 6A7), which recognizes only the activated form of Bax, followed by probing with Beclin1 or Bax antibodies. (c) Cardiomyocytes were transduced with or without Ad-Mst1 either in the absence or presence of Ad-Beclin1-WT, Ad-Beclin1-T108D or Ad-Beclin1-T108A. Quantitative analysis of the number of TUNEL-positive myocytes and immunoblot analysis of cardiomyocytes with cleaved caspase-3 antibody are shown (n = 3 in each group). Data are reported as mean ± SEM. * P < 0.05 vs Ad-Beclin1-WT, Ad-Mst1 (−). (d) Representative immunofluorescent images of staining with phospho-Beclin1 (Thr108)-specific antibody (green), DAPI (blue), and Troponin T antibody (red) in NTg, Tg-DN-Mst1 and Mst1−/− mice 6 weeks after coronary artery ligation are shown. Arrows and inset indicate positive staining of phospho-Beclin1 (Thr108) in the perinuclear region. Results represent means from 4 independent experiments. (e) Immunoblot analysis of heart homogenates in Tg-DN-Mst1, Mst1−/−, and NTg mice after 6 weeks of MI with phospho-Beclin1 (Thr108), total Beclin1, and GAPDH antibodies. Representative images are shown. Results represent means from 3 independent experiments. (f) Immunoblot analyses of myocardial tissue homogenates from explanted hearts with antibodies to phospho-Mst1 (Thr183), total Mst1, phospho-Beclin1 (Thr108), total Beclin1, p62/SQSTM1, LC3, and tubulin are shown. Six samples (Case #1b - #6b) were from heart transplant recipients, and 6 samples (Case #1a - #6a) were from heart donors. Representative images are shown. Results represent means from 3 independent experiments. (g) Representative immunofluorescent images of staining with p62/SQSTM1 (green), DAPI (blue), and ProteoStat® aggresome detection reagent (red) in myocardial tissue samples from a heart transplant recipient (Case #1b) and donor (Case #1a) are shown. Yellow arrows indicate co-localization of aggresomes with p62 in the perinuclear region of CMs. Results represent means from 3 independent experiments.
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References

    1. Levine B, Klionsky DJ. Development by self-digestion: molecular mechanisms and biological functions of autophagy. Dev Cell. 2004;6:463–477. - PubMed
    1. Nakai A, et al. The role of autophagy in cardiomyocytes in the basal state and in response to hemodynamic stress. Nat Med. 2007;13:619–624. - PubMed
    1. Yan L, et al. Autophagy in chronically ischemic myocardium. Proc Natl Acad Sci U S A. 2005;102:13807–13812. - PMC - PubMed
    1. Matsui Y, et al. Distinct roles of autophagy in the heart during ischemia and reperfusion: roles of AMP-activated protein kinase and Beclin 1 in mediating autophagy. Circ Res. 2007;100:914–922. - PubMed
    1. Zhu H, et al. Cardiac autophagy is a maladaptive response to hemodynamic stress. J Clin Invest. 2007;117:1782–1793. - PMC - PubMed

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