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a The phosphorylation of ULK1 at Ser758 by mTORC1 stabilizes the AMPK-ULK1 interaction. HEK293T cells lacking ULK1 were transiently transfected with either myc-tagged WT or mutant ULK1, with or without Rheb. Anti-myc immunoprecipitates were analyzed for the presence of endogenous AMPKα. b AMPK activation stabilizes the AMPK-ULK1 interaction. HEK293T cells lacking ULK1 were transiently transfected with myc-tagged ULK1. The cells were incubated in either full or amino acid-deprived medium, or treatment with Torin1 in the presence or absence of A769662 for 2 h. Anti-myc immunoprecipitates were analyzed for endogenous AMPKα. c Quantitative analysis of the AMPK-ULK1 interaction and ULK1 activity from b . d AMPK kinase activity is required to stabilize the AMPK-ULK1 interaction. HEK293T cells lacking AMPKα were transiently transfected with either WT or KD <t>AMPKα1</t> and myc-tagged ULK1. The cells were treated as described in b . e , f AMPK activation stabilizes the AMPK-ULK1 interaction. ULK1 KO HEK293T cells that were transduced with myc-ULK1 were treated with the indicated conditions for 1 h. g Schematics summarizing how mTORC1 and AMPK regulate the AMPK-ULK1 interaction and ULK1 activity. h AMPK-dependent events, beyond the phosphorylation of ULK1 Ser758 by mTORC1, stabilize the AMPK-ULK1 interaction. ULK1 KO HEK293T cells were transiently transfected with myc-tagged WT or the S758A ULK1 mutant. The cells were treated as described in b . i Summary of AMPK- and mTORC1-mediated phosphorylation sites on ULK1 and the impact of their alanine substitution on Ser758 phosphorylation and the stability of the AMPK-ULK1 interaction. The residue numbers are based on the human ULK1 sequence. j , k The phosphorylation of ULK1 at either Ser556 or Thr660 is necessary for AMPK to stabilize the AMPK-ULK1 interaction. l Quantitative analysis of the AMPK-ULK1 interaction from k . The statistical analysis in this figure was performed as described in Fig. . The concentrations of the chemicals used for the experiments in this figure are the same as described in Figs. 1, 2 and Supplementary Fig. 2. Source data are provided as a Source Data file.
Kd Ampkα1, supplied by Addgene inc, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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a The phosphorylation of ULK1 at Ser758 by mTORC1 stabilizes the AMPK-ULK1 interaction. HEK293T cells lacking ULK1 were transiently transfected with either myc-tagged WT or mutant ULK1, with or without Rheb. Anti-myc immunoprecipitates were analyzed for the presence of endogenous AMPKα. b AMPK activation stabilizes the AMPK-ULK1 interaction. HEK293T cells lacking ULK1 were transiently transfected with myc-tagged ULK1. The cells were incubated in either full or amino acid-deprived medium, or treatment with Torin1 in the presence or absence of A769662 for 2 h. Anti-myc immunoprecipitates were analyzed for endogenous AMPKα. c Quantitative analysis of the AMPK-ULK1 interaction and ULK1 activity from b . d AMPK kinase activity is required to stabilize the AMPK-ULK1 interaction. HEK293T cells lacking AMPKα were transiently transfected with either WT or KD AMPKα1 and myc-tagged ULK1. The cells were treated as described in b . e , f AMPK activation stabilizes the AMPK-ULK1 interaction. ULK1 KO HEK293T cells that were transduced with myc-ULK1 were treated with the indicated conditions for 1 h. g Schematics summarizing how mTORC1 and AMPK regulate the AMPK-ULK1 interaction and ULK1 activity. h AMPK-dependent events, beyond the phosphorylation of ULK1 Ser758 by mTORC1, stabilize the AMPK-ULK1 interaction. ULK1 KO HEK293T cells were transiently transfected with myc-tagged WT or the S758A ULK1 mutant. The cells were treated as described in b . i Summary of AMPK- and mTORC1-mediated phosphorylation sites on ULK1 and the impact of their alanine substitution on Ser758 phosphorylation and the stability of the AMPK-ULK1 interaction. The residue numbers are based on the human ULK1 sequence. j , k The phosphorylation of ULK1 at either Ser556 or Thr660 is necessary for AMPK to stabilize the AMPK-ULK1 interaction. l Quantitative analysis of the AMPK-ULK1 interaction from k . The statistical analysis in this figure was performed as described in Fig. . The concentrations of the chemicals used for the experiments in this figure are the same as described in Figs. 1, 2 and Supplementary Fig. 2. Source data are provided as a Source Data file.

Journal: Nature Communications

Article Title: Redefining the role of AMPK in autophagy and the energy stress response

doi: 10.1038/s41467-023-38401-z

Figure Lengend Snippet: a The phosphorylation of ULK1 at Ser758 by mTORC1 stabilizes the AMPK-ULK1 interaction. HEK293T cells lacking ULK1 were transiently transfected with either myc-tagged WT or mutant ULK1, with or without Rheb. Anti-myc immunoprecipitates were analyzed for the presence of endogenous AMPKα. b AMPK activation stabilizes the AMPK-ULK1 interaction. HEK293T cells lacking ULK1 were transiently transfected with myc-tagged ULK1. The cells were incubated in either full or amino acid-deprived medium, or treatment with Torin1 in the presence or absence of A769662 for 2 h. Anti-myc immunoprecipitates were analyzed for endogenous AMPKα. c Quantitative analysis of the AMPK-ULK1 interaction and ULK1 activity from b . d AMPK kinase activity is required to stabilize the AMPK-ULK1 interaction. HEK293T cells lacking AMPKα were transiently transfected with either WT or KD AMPKα1 and myc-tagged ULK1. The cells were treated as described in b . e , f AMPK activation stabilizes the AMPK-ULK1 interaction. ULK1 KO HEK293T cells that were transduced with myc-ULK1 were treated with the indicated conditions for 1 h. g Schematics summarizing how mTORC1 and AMPK regulate the AMPK-ULK1 interaction and ULK1 activity. h AMPK-dependent events, beyond the phosphorylation of ULK1 Ser758 by mTORC1, stabilize the AMPK-ULK1 interaction. ULK1 KO HEK293T cells were transiently transfected with myc-tagged WT or the S758A ULK1 mutant. The cells were treated as described in b . i Summary of AMPK- and mTORC1-mediated phosphorylation sites on ULK1 and the impact of their alanine substitution on Ser758 phosphorylation and the stability of the AMPK-ULK1 interaction. The residue numbers are based on the human ULK1 sequence. j , k The phosphorylation of ULK1 at either Ser556 or Thr660 is necessary for AMPK to stabilize the AMPK-ULK1 interaction. l Quantitative analysis of the AMPK-ULK1 interaction from k . The statistical analysis in this figure was performed as described in Fig. . The concentrations of the chemicals used for the experiments in this figure are the same as described in Figs. 1, 2 and Supplementary Fig. 2. Source data are provided as a Source Data file.

Article Snippet: Human WT AMPKα1 (Addgene #79010) and KD AMPKα1 (Addgene #79011) DNAs and rat WT AMPKα2 (Addgene #15991) and K45R AMPKα2 (kinase inactive mutant) (Addgene #15992) DNAs were subcloned into pLV-EF1a-IRES-Puro or pLV-EF1a-IRES-blast.

Techniques: Phospho-proteomics, Transfection, Mutagenesis, Activation Assay, Incubation, Activity Assay, Transduction, Residue, Sequencing