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Cellular energy stress induces AMPK-mediated regulation of YAP and the Hippo pathway.

Mo JS, Meng Z, Kim YC, Park HW, Hansen CG, Kim S, Lim DS, Guan KL - Nat. Cell Biol. (2015)

Bottom Line: YAP (Yes-associated protein) is a transcription co-activator in the Hippo tumour suppressor pathway and controls cell growth, tissue homeostasis and organ size.AMPK-induced YAP inhibition can suppress oncogenic transformation of Lats- cells with high YAP activity.Our study establishes a molecular mechanism and functional significance of AMPK in linking cellular energy status to the Hippo-YAP pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Moores Cancer Center, University of California, San Diego, La Jolla, California 92093, USA.

ABSTRACT
YAP (Yes-associated protein) is a transcription co-activator in the Hippo tumour suppressor pathway and controls cell growth, tissue homeostasis and organ size. YAP is inhibited by the kinase Lats, which phosphorylates YAP to induce its cytoplasmic localization and proteasomal degradation. YAP induces gene expression by binding to the TEAD family transcription factors. Dysregulation of the Hippo-YAP pathway is frequently observed in human cancers. Here we show that cellular energy stress induces YAP phosphorylation, in part due to AMPK-dependent Lats activation, thereby inhibiting YAP activity. Moreover, AMPK directly phosphorylates YAP Ser 94, a residue essential for the interaction with TEAD, thus disrupting the YAP-TEAD interaction. AMPK-induced YAP inhibition can suppress oncogenic transformation of Lats- cells with high YAP activity. Our study establishes a molecular mechanism and functional significance of AMPK in linking cellular energy status to the Hippo-YAP pathway.

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Energy stress induces YAP phosphorylation via both Lats dependent and independent mechanisms(a) Deletion of Lats1/2 diminishes, but not completely abolishes YAP phosphorylation by 2-DG. Lats1−/−;Lats2fl/fl (denoted as Lats1 KO) MEFs and Cre- transduced Lats1−/−;Lats2fl/fl (denoted as Lst1/2 DKO) MEFs were treated with 10 and 25 mM 2-DG. YAP phosphorylation was detected by phospho-YAP (S127) antibody and phos-tag gel. Lats2 deletion was confirmed by Western blot. All lysates were run on the same gel and the vertical line in YAP phos-tag blot indicated different exposures of the same Western Blot (the YAP phos-tag blot of Lats1/2 DKO samples was exposed shorter due to high YAP protein levels in these samples). (b) Overexpressed of kinase-inactive Lats2 (Lats2-KR) abolishes 2-DG-induced S127 phosphorylation, but not total phosphorylation, in YAP. Flag-YAP was co-transfected with Lats2-WT or Lats2-KR mutant. After transfection, cells were treated with 25 mM 2-DG for 1 hr. (c) AMPK induces mobility shift of YAP-5SA. Flag-YAP-5SA was co-transfected with AMPKα WT or AMPKα DN in HEK293A as indicated. (d, e) 2-DG stimulates the interaction of YAP and AMPKα. HEK293A cells transfected with the indicated plasmids were immunoprecipitated with anti-FLAG (for Flag-YAP) or anti-HA (for HA-AMPK) and blotted with indicated antibodies for co-immunoprecipitation. (f) AICAR or Metformin promotes the association between endogenous AMPK and YAP. H2.35 cells were treated with AICAR or metformin. Cell lysates were immunoprecipitated with YAP or control IgG and Western blotted with AMPKα. All experiments are representatives of three independent experiments except panel e and f. The experiments in e, f were performed two times with similar results.
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Figure 3: Energy stress induces YAP phosphorylation via both Lats dependent and independent mechanisms(a) Deletion of Lats1/2 diminishes, but not completely abolishes YAP phosphorylation by 2-DG. Lats1−/−;Lats2fl/fl (denoted as Lats1 KO) MEFs and Cre- transduced Lats1−/−;Lats2fl/fl (denoted as Lst1/2 DKO) MEFs were treated with 10 and 25 mM 2-DG. YAP phosphorylation was detected by phospho-YAP (S127) antibody and phos-tag gel. Lats2 deletion was confirmed by Western blot. All lysates were run on the same gel and the vertical line in YAP phos-tag blot indicated different exposures of the same Western Blot (the YAP phos-tag blot of Lats1/2 DKO samples was exposed shorter due to high YAP protein levels in these samples). (b) Overexpressed of kinase-inactive Lats2 (Lats2-KR) abolishes 2-DG-induced S127 phosphorylation, but not total phosphorylation, in YAP. Flag-YAP was co-transfected with Lats2-WT or Lats2-KR mutant. After transfection, cells were treated with 25 mM 2-DG for 1 hr. (c) AMPK induces mobility shift of YAP-5SA. Flag-YAP-5SA was co-transfected with AMPKα WT or AMPKα DN in HEK293A as indicated. (d, e) 2-DG stimulates the interaction of YAP and AMPKα. HEK293A cells transfected with the indicated plasmids were immunoprecipitated with anti-FLAG (for Flag-YAP) or anti-HA (for HA-AMPK) and blotted with indicated antibodies for co-immunoprecipitation. (f) AICAR or Metformin promotes the association between endogenous AMPK and YAP. H2.35 cells were treated with AICAR or metformin. Cell lysates were immunoprecipitated with YAP or control IgG and Western blotted with AMPKα. All experiments are representatives of three independent experiments except panel e and f. The experiments in e, f were performed two times with similar results.

Mentions: Lats is the major kinase responsible for YAP/TAZ inhibition, although other kinases have also been implicated in their regulation20, 40, 41. To confirm the role of Lats in energy stress-induced YAP phosphorylation, we compared the Lats1−/−;Lats2fl/fl (Lats1 KO) MEFs with the Lats1/2−/− (Lats1/2 DKO) MEFs42 generated by infection of the Cre virus in the Lats1−/−;Lats2fl/fl (Lats1 KO) MEFs. As expected, deletion of Lats1/2 completely abolished YAP S127 phosphorylation and increased YAP protein levels (Fig. 3a). Moreover, the 2-DG-induced YAP mobility shift was significantly diminished in Lats1/2 DKO MEFs, supporting a role of Lats in energy-stress-induced YAP phosphorylation. However, 2-DG still induced a visible YAP mobility shift in the Lats1/2 DKO cells (Fig. 3a), suggesting that 2-DG could induce YAP phosphorylation independent of Lats. Interestingly, AMPK activation was noticeably enhanced in the Lats1/2 DKO MEFs compared to Lats1 KO MEFs (Fig. 3a). The enhanced AMPK activation in the Lats1/2 DKO MEFs indicates that these cells are under more severe energy stress. This phenomenon can be explained by a model in which the high YAP activity in the Lats1/2 DKO cells promotes cell growth and increases energy expenditure, thereby reading to energy stress in these cells.


Cellular energy stress induces AMPK-mediated regulation of YAP and the Hippo pathway.

Mo JS, Meng Z, Kim YC, Park HW, Hansen CG, Kim S, Lim DS, Guan KL - Nat. Cell Biol. (2015)

Energy stress induces YAP phosphorylation via both Lats dependent and independent mechanisms(a) Deletion of Lats1/2 diminishes, but not completely abolishes YAP phosphorylation by 2-DG. Lats1−/−;Lats2fl/fl (denoted as Lats1 KO) MEFs and Cre- transduced Lats1−/−;Lats2fl/fl (denoted as Lst1/2 DKO) MEFs were treated with 10 and 25 mM 2-DG. YAP phosphorylation was detected by phospho-YAP (S127) antibody and phos-tag gel. Lats2 deletion was confirmed by Western blot. All lysates were run on the same gel and the vertical line in YAP phos-tag blot indicated different exposures of the same Western Blot (the YAP phos-tag blot of Lats1/2 DKO samples was exposed shorter due to high YAP protein levels in these samples). (b) Overexpressed of kinase-inactive Lats2 (Lats2-KR) abolishes 2-DG-induced S127 phosphorylation, but not total phosphorylation, in YAP. Flag-YAP was co-transfected with Lats2-WT or Lats2-KR mutant. After transfection, cells were treated with 25 mM 2-DG for 1 hr. (c) AMPK induces mobility shift of YAP-5SA. Flag-YAP-5SA was co-transfected with AMPKα WT or AMPKα DN in HEK293A as indicated. (d, e) 2-DG stimulates the interaction of YAP and AMPKα. HEK293A cells transfected with the indicated plasmids were immunoprecipitated with anti-FLAG (for Flag-YAP) or anti-HA (for HA-AMPK) and blotted with indicated antibodies for co-immunoprecipitation. (f) AICAR or Metformin promotes the association between endogenous AMPK and YAP. H2.35 cells were treated with AICAR or metformin. Cell lysates were immunoprecipitated with YAP or control IgG and Western blotted with AMPKα. All experiments are representatives of three independent experiments except panel e and f. The experiments in e, f were performed two times with similar results.
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Figure 3: Energy stress induces YAP phosphorylation via both Lats dependent and independent mechanisms(a) Deletion of Lats1/2 diminishes, but not completely abolishes YAP phosphorylation by 2-DG. Lats1−/−;Lats2fl/fl (denoted as Lats1 KO) MEFs and Cre- transduced Lats1−/−;Lats2fl/fl (denoted as Lst1/2 DKO) MEFs were treated with 10 and 25 mM 2-DG. YAP phosphorylation was detected by phospho-YAP (S127) antibody and phos-tag gel. Lats2 deletion was confirmed by Western blot. All lysates were run on the same gel and the vertical line in YAP phos-tag blot indicated different exposures of the same Western Blot (the YAP phos-tag blot of Lats1/2 DKO samples was exposed shorter due to high YAP protein levels in these samples). (b) Overexpressed of kinase-inactive Lats2 (Lats2-KR) abolishes 2-DG-induced S127 phosphorylation, but not total phosphorylation, in YAP. Flag-YAP was co-transfected with Lats2-WT or Lats2-KR mutant. After transfection, cells were treated with 25 mM 2-DG for 1 hr. (c) AMPK induces mobility shift of YAP-5SA. Flag-YAP-5SA was co-transfected with AMPKα WT or AMPKα DN in HEK293A as indicated. (d, e) 2-DG stimulates the interaction of YAP and AMPKα. HEK293A cells transfected with the indicated plasmids were immunoprecipitated with anti-FLAG (for Flag-YAP) or anti-HA (for HA-AMPK) and blotted with indicated antibodies for co-immunoprecipitation. (f) AICAR or Metformin promotes the association between endogenous AMPK and YAP. H2.35 cells were treated with AICAR or metformin. Cell lysates were immunoprecipitated with YAP or control IgG and Western blotted with AMPKα. All experiments are representatives of three independent experiments except panel e and f. The experiments in e, f were performed two times with similar results.
Mentions: Lats is the major kinase responsible for YAP/TAZ inhibition, although other kinases have also been implicated in their regulation20, 40, 41. To confirm the role of Lats in energy stress-induced YAP phosphorylation, we compared the Lats1−/−;Lats2fl/fl (Lats1 KO) MEFs with the Lats1/2−/− (Lats1/2 DKO) MEFs42 generated by infection of the Cre virus in the Lats1−/−;Lats2fl/fl (Lats1 KO) MEFs. As expected, deletion of Lats1/2 completely abolished YAP S127 phosphorylation and increased YAP protein levels (Fig. 3a). Moreover, the 2-DG-induced YAP mobility shift was significantly diminished in Lats1/2 DKO MEFs, supporting a role of Lats in energy-stress-induced YAP phosphorylation. However, 2-DG still induced a visible YAP mobility shift in the Lats1/2 DKO cells (Fig. 3a), suggesting that 2-DG could induce YAP phosphorylation independent of Lats. Interestingly, AMPK activation was noticeably enhanced in the Lats1/2 DKO MEFs compared to Lats1 KO MEFs (Fig. 3a). The enhanced AMPK activation in the Lats1/2 DKO MEFs indicates that these cells are under more severe energy stress. This phenomenon can be explained by a model in which the high YAP activity in the Lats1/2 DKO cells promotes cell growth and increases energy expenditure, thereby reading to energy stress in these cells.

Bottom Line: YAP (Yes-associated protein) is a transcription co-activator in the Hippo tumour suppressor pathway and controls cell growth, tissue homeostasis and organ size.AMPK-induced YAP inhibition can suppress oncogenic transformation of Lats- cells with high YAP activity.Our study establishes a molecular mechanism and functional significance of AMPK in linking cellular energy status to the Hippo-YAP pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Moores Cancer Center, University of California, San Diego, La Jolla, California 92093, USA.

ABSTRACT
YAP (Yes-associated protein) is a transcription co-activator in the Hippo tumour suppressor pathway and controls cell growth, tissue homeostasis and organ size. YAP is inhibited by the kinase Lats, which phosphorylates YAP to induce its cytoplasmic localization and proteasomal degradation. YAP induces gene expression by binding to the TEAD family transcription factors. Dysregulation of the Hippo-YAP pathway is frequently observed in human cancers. Here we show that cellular energy stress induces YAP phosphorylation, in part due to AMPK-dependent Lats activation, thereby inhibiting YAP activity. Moreover, AMPK directly phosphorylates YAP Ser 94, a residue essential for the interaction with TEAD, thus disrupting the YAP-TEAD interaction. AMPK-induced YAP inhibition can suppress oncogenic transformation of Lats- cells with high YAP activity. Our study establishes a molecular mechanism and functional significance of AMPK in linking cellular energy status to the Hippo-YAP pathway.

Show MeSH
Related in: MedlinePlus