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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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AMPK phosphorylates S94 in YAP(a) AMPK directly phosphorylates YAP in vitro. The indicated various YAP mutant proteins were purified from E.coli and used as substrates for in vitro phosphorylation by AMPK. Phosphorylation was determined by 32P-autoradiography and the amount of each GST-YAP protein was detected by coomassie staining. (b) Confirmation of AMPK phosphorylation sites in YAP by mutagenesis. Schematic representation of YAP domains and deletion constructs used to map phosphorylation sites are shown on the top. The human YAP protein contains a TEAD Binding Domain (TBD; residues 48–102), two WW domains (WW1, 171–204; WW2, 230–263) domains and an Activation Domain (AD; 292–488). Three different YAP deletion constructs and multiple amino acid mutations in these YAP truncations were expressed, purified, and used as substrates for in vitro AMPK phosphorylation in the presence of 32P-ATP. Phosphorylation was detected by autoradiography. GST-YAP fragment protein was detected by Coomassie staining.
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Figure 4: AMPK phosphorylates S94 in YAP(a) AMPK directly phosphorylates YAP in vitro. The indicated various YAP mutant proteins were purified from E.coli and used as substrates for in vitro phosphorylation by AMPK. Phosphorylation was determined by 32P-autoradiography and the amount of each GST-YAP protein was detected by coomassie staining. (b) Confirmation of AMPK phosphorylation sites in YAP by mutagenesis. Schematic representation of YAP domains and deletion constructs used to map phosphorylation sites are shown on the top. The human YAP protein contains a TEAD Binding Domain (TBD; residues 48–102), two WW domains (WW1, 171–204; WW2, 230–263) domains and an Activation Domain (AD; 292–488). Three different YAP deletion constructs and multiple amino acid mutations in these YAP truncations were expressed, purified, and used as substrates for in vitro AMPK phosphorylation in the presence of 32P-ATP. Phosphorylation was detected by autoradiography. GST-YAP fragment protein was detected by Coomassie staining.

Mentions: To gain mechanistic insight into AMPK-mediated regulation of YAP, we tested whether YAP is a direct substrate of AMPK. AMPK phosphorylated recombinant YAP-WT in vitro (Fig. 4a). The YAP-S127A and YAP-5SA mutants were also phosphorylated by AMPK, though more weakly, indicating that AMPK can phosphorylate YAP on residues distinct from the Lats phosphorylation sites. In vitro phosphorylation by AMPK altered the migration of YAP-5SA (Supplementary Fig. 4a). Mass spectrometry experiments of several batches of in vitro phosphorylated YAP-5SA identified several phosphorylation sites on YAP, including high- and low-confidence sites (Supplementary Fig. 4b). Further in vitro phosphorylation experiments confirmed that serine residues 94, 366 and 463 were direct AMPK targets (Fig. 4b and Supplementary 4c). We examined the robustness of YAP as an AMPK substrate by comparing with known physiological AMPK substrates TSC2 and ULK143, 44. AMPK phosphorylated YAP as efficiently as ULK1 (Supplementary Fig. 4d–f); the efficiency of YAP (51–270) phosphorylation by AMPK was comparable with the TSC2 fragment (1300–1367). Because energy starvation or AMPK co-transfection inhibits YAP transcriptional activity (Fig.1d, 2d, 2e), we examined the effect of the putative AMPK phosphorylation sites on YAP activity. Mutation of YAP S94 to glutamate or alanine diminished the ability of YAP to activate the TEAD1 reporter, whereas mutation of S366 or S463 did not significantly affect YAP activity (Fig. 5a, b). Therefore, we focused our further studies on S94. Notably, substitution of S94 by any residue, including alanine or glutamate, abolished YAP-TEAD interaction (Fig. 5c) and decreased TEAD reporter activity. We propose that phosphorylation of YAP-S94 by AMPK disrupts its interaction with TEAD.


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)

AMPK phosphorylates S94 in YAP(a) AMPK directly phosphorylates YAP in vitro. The indicated various YAP mutant proteins were purified from E.coli and used as substrates for in vitro phosphorylation by AMPK. Phosphorylation was determined by 32P-autoradiography and the amount of each GST-YAP protein was detected by coomassie staining. (b) Confirmation of AMPK phosphorylation sites in YAP by mutagenesis. Schematic representation of YAP domains and deletion constructs used to map phosphorylation sites are shown on the top. The human YAP protein contains a TEAD Binding Domain (TBD; residues 48–102), two WW domains (WW1, 171–204; WW2, 230–263) domains and an Activation Domain (AD; 292–488). Three different YAP deletion constructs and multiple amino acid mutations in these YAP truncations were expressed, purified, and used as substrates for in vitro AMPK phosphorylation in the presence of 32P-ATP. Phosphorylation was detected by autoradiography. GST-YAP fragment protein was detected by Coomassie staining.
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Related In: Results  -  Collection

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Figure 4: AMPK phosphorylates S94 in YAP(a) AMPK directly phosphorylates YAP in vitro. The indicated various YAP mutant proteins were purified from E.coli and used as substrates for in vitro phosphorylation by AMPK. Phosphorylation was determined by 32P-autoradiography and the amount of each GST-YAP protein was detected by coomassie staining. (b) Confirmation of AMPK phosphorylation sites in YAP by mutagenesis. Schematic representation of YAP domains and deletion constructs used to map phosphorylation sites are shown on the top. The human YAP protein contains a TEAD Binding Domain (TBD; residues 48–102), two WW domains (WW1, 171–204; WW2, 230–263) domains and an Activation Domain (AD; 292–488). Three different YAP deletion constructs and multiple amino acid mutations in these YAP truncations were expressed, purified, and used as substrates for in vitro AMPK phosphorylation in the presence of 32P-ATP. Phosphorylation was detected by autoradiography. GST-YAP fragment protein was detected by Coomassie staining.
Mentions: To gain mechanistic insight into AMPK-mediated regulation of YAP, we tested whether YAP is a direct substrate of AMPK. AMPK phosphorylated recombinant YAP-WT in vitro (Fig. 4a). The YAP-S127A and YAP-5SA mutants were also phosphorylated by AMPK, though more weakly, indicating that AMPK can phosphorylate YAP on residues distinct from the Lats phosphorylation sites. In vitro phosphorylation by AMPK altered the migration of YAP-5SA (Supplementary Fig. 4a). Mass spectrometry experiments of several batches of in vitro phosphorylated YAP-5SA identified several phosphorylation sites on YAP, including high- and low-confidence sites (Supplementary Fig. 4b). Further in vitro phosphorylation experiments confirmed that serine residues 94, 366 and 463 were direct AMPK targets (Fig. 4b and Supplementary 4c). We examined the robustness of YAP as an AMPK substrate by comparing with known physiological AMPK substrates TSC2 and ULK143, 44. AMPK phosphorylated YAP as efficiently as ULK1 (Supplementary Fig. 4d–f); the efficiency of YAP (51–270) phosphorylation by AMPK was comparable with the TSC2 fragment (1300–1367). Because energy starvation or AMPK co-transfection inhibits YAP transcriptional activity (Fig.1d, 2d, 2e), we examined the effect of the putative AMPK phosphorylation sites on YAP activity. Mutation of YAP S94 to glutamate or alanine diminished the ability of YAP to activate the TEAD1 reporter, whereas mutation of S366 or S463 did not significantly affect YAP activity (Fig. 5a, b). Therefore, we focused our further studies on S94. Notably, substitution of S94 by any residue, including alanine or glutamate, abolished YAP-TEAD interaction (Fig. 5c) and decreased TEAD reporter activity. We propose that phosphorylation of YAP-S94 by AMPK disrupts its interaction with TEAD.

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