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AMPK-dependent activation of the Cyclin Y/CDK16 complex controls autophagy

Dohmen, Marc, Krieg, Sarah, Agalaridis, Georgios, Zhu, Xiaoqing, Shehata, Saifeldin N., Pfeiffenberger, Elisabeth, Amelang, Jan, Bütepage, Mareike, Buerova, Elena, Pfaff, Carolina M., Chanda, Dipanjan, Geley, Stephan, Preisinger, Christian, Sakamoto, Kei, Lüscher, Bernhard, Neumann, Dietbert, Vervoorts, Jörg·2020·Nature Communications
DOI 10.1038/s41467-020-14812-0PMC PMC7042329
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Abstract

The AMP-activated protein kinase (AMPK) is a master sensor of the cellular energy status that is crucial for the adaptive response to limited energy availability. AMPK is implicated in the regulation of many cellular processes, including autophagy. However, the precise mechanisms by which AMPK controls these processes and the identities of relevant substrates are not fully understood. Using protein microarrays, we identify Cyclin Y as an AMPK substrate that is phosphorylated at Serine 326 (S326) both in vitro and in cells. Phosphorylation of Cyclin Y at S326 promotes its interaction with the Cyclin-dependent kinase 16 (CDK16), thereby stimulating its catalytic activity. When expressed in cells, Cyclin Y/CDK16 is sufficient to promote autophagy. Moreover, Cyclin Y/CDK16 is necessary for efficient AMPK-dependent activation of autophagy. This functional interaction is mediated by AMPK phosphorylating S326 of Cyclin Y. Collectively, we define Cyclin Y/CDK16 as downstream effector of AMPK for inducing autophagy.

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Fig. 1 Protein microarray screen for the identification of AMPK substrates. a Schematic representation of the ProtoArray based screen with approximately 9000 human proteins using AMPK (see also Supplementary Data 1). b Details of two sub-arrays incubated with or without AMPK with marked substrates are shown. c GST-CDK16, Cyclin Y-His6 and GST were incubated in the presence of [γ-32P]-ATP with AMPK. Phosphorylation was determined by autoradiography (32P, top). Proteins were visualized by Coomassie blue staining (CB, bottom; n = 2). d HeLa cells were transfected with vectors expressing GFP-CDK16 and Cyclin Y-Flag and treated for 1 h with 0.5 mM AICAR/50 µM A769662 (A769) as indicated. Cyclin Y-Flag was immunoprecipitated with Flag antibodies (IP) and immunoblotted against CDK16 and Cyclin Y or used for in vitro kinase assays with myeloid basic protein (MBP) as substrate. Autoradiographs (32P) and Coomassie blue staining (CB) of MBP are displayed. Whole cell lysates (WCL) were immunoblotted with the indicated antibodies (n = 3). e Quantification of CDK16 co-immunoprecipitated with Cyclin Y. Statistical significance was measured via unpaired and two-tailed Student’s t-tests and is presented as follows: **p < 0.01, and ***p < 0.001. All error bars indicate SD (n = 3; Cyclin Y + AICAR/A769 vs. Cyclin Y/CDK16: t = 8.719, df = 4; Cyclin Y/CDK16 vs. Cyclin Y/CDK16 + AICAR/A769: t = 5.595, df = 4). n biological independent replicate. SD standard deviation. Source data are provided as a Source Data file.
Fig. 1 Protein microarray screen for the identification of AMPK substrates. a Schematic representation of the ProtoArray based screen with approximately 9000 human proteins using AMPK (see also Supplementary Data 1). b Details of two sub-arrays incubated with or without AMPK with marked substrates are shown. c GST-CDK16, Cyclin Y-His6 and GST were incubated in the presence of [γ-32P]-ATP with AMPK. Phosphorylation was determined by autoradiography (32P, top). Proteins were visualized by Coomassie blue staining (CB, bottom; n = 2). d HeLa cells were transfected with vectors expressing GFP-CDK16 and Cyclin Y-Flag and treated for 1 h with 0.5 mM AICAR/50 µM A769662 (A769) as indicated. Cyclin Y-Flag was immunoprecipitated with Flag antibodies (IP) and immunoblotted against CDK16 and Cyclin Y or used for in vitro kinase assays with myeloid basic protein (MBP) as substrate. Autoradiographs (32P) and Coomassie blue staining (CB) of MBP are displayed. Whole cell lysates (WCL) were immunoblotted with the indicated antibodies (n = 3). e Quantification of CDK16 co-immunoprecipitated with Cyclin Y. Statistical significance was measured via unpaired and two-tailed Student’s t-tests and is presented as follows: **p < 0.01, and ***p < 0.001. All error bars indicate SD (n = 3; Cyclin Y + AICAR/A769 vs. Cyclin Y/CDK16: t = 8.719, df = 4; Cyclin Y/CDK16 vs. Cyclin Y/CDK16 + AICAR/A769: t = 5.595, df = 4). n biological independent replicate. SD standard deviation. Source data are provided as a Source Data file.CC BY · PubMed Central
Fig. 2 Verification of the Cyclin Y S326 phosphorylation site in vitro and in cells. a GST-Cyclin Y wt and S326A were incubated ± AMPK. S326-P was detected using a phospho-specific antibody (n = 2). b HeLa cells were transfected with vectors expressing Cyclin Y-Flag, the S326A mutant or an empty control vector and treated with 0.5 mM AICAR/50 µM A769662 (A769) for 1 h as indicated. Cyclin Y was immunoprecipitated with a Flag antibody (IP). The blots were immunoblotted with the indicated antibodies (WCL). (n = 2). c HeLa cells were transfected with a vector expressing Cyclin Y-Flag as indicated. After 72 h cells were treated with 0.5 mM AICAR/50 µM A769662 for 1 h. Cyclin Y was immunoprecipitated with a Flag antibody. The blots were immunoblotted with the indicated antibodies (n = 2). d HeLa cells were transfected with a vector expressing Cyclin Y-Flag and siRNA against AMPK-α1/2 or control siRNA as indicated. After 72 h cells were treated with 0.5 mM AICAR/50 µM A769662 for 1 h. The blots were immunoblotted with the indicated antibodies (n = 2). e NIH3T3 cells were transfected with the indicated siRNAs. After 72 h cells were treated with 0.5 mM AICAR/50 µM A769662 for 1 h prior to protein analysis by immunoblotting (n = 2). f NIH3T3 cells were treated with 0.5 mM AICAR/50 µM A769662, EBSS or Ionomycin for 1 h or were grown in glucose-free medium for 16 h and lysates were immunoblotted as indicated (n = 2). n biological independent replicate. Source data are provided as a Source Data file.
Fig. 2 Verification of the Cyclin Y S326 phosphorylation site in vitro and in cells. a GST-Cyclin Y wt and S326A were incubated ± AMPK. S326-P was detected using a phospho-specific antibody (n = 2). b HeLa cells were transfected with vectors expressing Cyclin Y-Flag, the S326A mutant or an empty control vector and treated with 0.5 mM AICAR/50 µM A769662 (A769) for 1 h as indicated. Cyclin Y was immunoprecipitated with a Flag antibody (IP). The blots were immunoblotted with the indicated antibodies (WCL). (n = 2). c HeLa cells were transfected with a vector expressing Cyclin Y-Flag as indicated. After 72 h cells were treated with 0.5 mM AICAR/50 µM A769662 for 1 h. Cyclin Y was immunoprecipitated with a Flag antibody. The blots were immunoblotted with the indicated antibodies (n = 2). d HeLa cells were transfected with a vector expressing Cyclin Y-Flag and siRNA against AMPK-α1/2 or control siRNA as indicated. After 72 h cells were treated with 0.5 mM AICAR/50 µM A769662 for 1 h. The blots were immunoblotted with the indicated antibodies (n = 2). e NIH3T3 cells were transfected with the indicated siRNAs. After 72 h cells were treated with 0.5 mM AICAR/50 µM A769662 for 1 h prior to protein analysis by immunoblotting (n = 2). f NIH3T3 cells were treated with 0.5 mM AICAR/50 µM A769662, EBSS or Ionomycin for 1 h or were grown in glucose-free medium for 16 h and lysates were immunoblotted as indicated (n = 2). n biological independent replicate. Source data are provided as a Source Data file.CC BY · PubMed Central
Fig. 3 Cyclin Y and CDK16 are required for efficient induction of autophagy. a NIH3T3 cells were transfected with siRNA against Cdk16, Cyclin Y or a control for 72 h and grown in EBSS for 2 h. Proteins were detected as indicated (n = 3). b Representative confocal images of the NIH3T3 treated as in panel a. Endogenous LC3 was stained with the 4E12 antibody to monitor autophagy. Scale bar: 50 µm. c Quantification of the LC3 dots shown in panel b. Statistical significance was measured via unpaired and two-tailed Student’s t-tests and is presented as follows: ***p < 0.001. All error bars indicate SD (n = 3; 100 cells counted for each replicate; siControl vs. siCdk16: t = 10.09, df = 4; siControl vs. siCyclin Y: t = 9.866, df = 4). d NIH3T3 cells were transfected with siControl or siCyclin Y, treated with 200 nM Bafilomycin A1 (Baf. A1) or 3-MA for 2 h, and grown in EBSS for additional 2 h as indicated. Lysates were immunoblotted with the indicated antibodies (n = 2). e Immortalized Cdk16+/+ and Cdk16−/− MEFs were treated with 200 nM Baf. A1 for 4 h prior to growth in EBSS for additional 2 h. Proteins were measured by immunoblotting (n = 3). f Representative confocal images of the Cdk16+/+ and Cdk16−/− MEFs treated as in panel e. Endogenous LC3 was stained (antibody 4E12) to monitor autophagy. Scale bar: 50 µm. g Quantification of the LC3 dots shown in panel f. Statistical significance was measured via unpaired and two-tailed Student’s t-tests and is presented as follows: *p < 0.05 and ***p < 0.001. All error bars indicate SD (n = 3; 100 cells counted for each replicate; EBSS: t = 10.850, df = 4; Baf. A1: t = 4.58, df = 4; EBSS/Baf. A1: t = 3.978, df = 4). h Human CDK16 was stably expressed in immortalized Cdk16−/− MEFs ( + CDK16). Control cells were infected with an empty virus. Cells were treated with 200 nM Baf. A1 and grown in EBSS. Proteins were detected as indicated (n = 2). n biological independent replicate. SD standard deviation. Source data are provided as a Source Data file.
Fig. 3 Cyclin Y and CDK16 are required for efficient induction of autophagy. a NIH3T3 cells were transfected with siRNA against Cdk16, Cyclin Y or a control for 72 h and grown in EBSS for 2 h. Proteins were detected as indicated (n = 3). b Representative confocal images of the NIH3T3 treated as in panel a. Endogenous LC3 was stained with the 4E12 antibody to monitor autophagy. Scale bar: 50 µm. c Quantification of the LC3 dots shown in panel b. Statistical significance was measured via unpaired and two-tailed Student’s t-tests and is presented as follows: ***p < 0.001. All error bars indicate SD (n = 3; 100 cells counted for each replicate; siControl vs. siCdk16: t = 10.09, df = 4; siControl vs. siCyclin Y: t = 9.866, df = 4). d NIH3T3 cells were transfected with siControl or siCyclin Y, treated with 200 nM Bafilomycin A1 (Baf. A1) or 3-MA for 2 h, and grown in EBSS for additional 2 h as indicated. Lysates were immunoblotted with the indicated antibodies (n = 2). e Immortalized Cdk16+/+ and Cdk16−/− MEFs were treated with 200 nM Baf. A1 for 4 h prior to growth in EBSS for additional 2 h. Proteins were measured by immunoblotting (n = 3). f Representative confocal images of the Cdk16+/+ and Cdk16−/− MEFs treated as in panel e. Endogenous LC3 was stained (antibody 4E12) to monitor autophagy. Scale bar: 50 µm. g Quantification of the LC3 dots shown in panel f. Statistical significance was measured via unpaired and two-tailed Student’s t-tests and is presented as follows: *p < 0.05 and ***p < 0.001. All error bars indicate SD (n = 3; 100 cells counted for each replicate; EBSS: t = 10.850, df = 4; Baf. A1: t = 4.58, df = 4; EBSS/Baf. A1: t = 3.978, df = 4). h Human CDK16 was stably expressed in immortalized Cdk16−/− MEFs ( + CDK16). Control cells were infected with an empty virus. Cells were treated with 200 nM Baf. A1 and grown in EBSS. Proteins were detected as indicated (n = 2). n biological independent replicate. SD standard deviation. Source data are provided as a Source Data file.CC BY · PubMed Central
Fig. 4 Active Cyclin Y/CDK16 complexes induce autophagy. a NIH3T3 cells stably expressing mCherry-GFP-LC3 were transfected with HA-CDK16 and Cyclin Y-Flag as indicated or treated for 2 h with EBSS or for 4 h with 200 nM Bafilomycin A1 (Baf. A1) and lysates were immunoblotted as indicated. KR kinase-deficient CDK16 mutant, AA CDK16 binding deficient Cyclin Y mutant (n = 3). b Representative confocal images of the NIH3T3-mCherry-GFP-LC3 cells treated as in panel a. Staining of the HA-CDK16 in purple identified transfected cells. Autophagosomes (yellow dots) and autolysosomes (red dots) were detected by an overlay of the GFP and mCherry fluorescent signals. Scale bar: 20 µm. c Quantification of autophagosomes (yellow dots) and autolysosomes (red dots) of cells shown in panel b. Statistical significance was measured via unpaired and two-tailed Student’s t-tests and is presented as follows: **p < 0.01. All error bars indicate SD (n = 3; 50 cells counted for each replicate; wt vs. KR: t = 5.707, df = 4; wt vs. AA: t = 5.557, df = 4). d GFP-tagged CDK14, CDK15 or CDK16 were expressed with or without Cyclin Y-Flag in HeLa cells. Cyclin Y was immunoprecipitated with a Flag antibody (IP). Lysates were immunoblotted with the indicated antibodies (WCL). (n = 4) e Representative confocal images of HeLa cells treated as in panel d. Fluorescent GFP signals identified CDK expressing cells. Endogenous LC3 (red) was used to measure autophagy with the 4E12 antibody. Scale bar: 50 µm. f Quantification of the LC3 dots shown in panel e. Statistical significance was measured via unpaired and two-tailed Student’s t-tests and is presented as follows: ****p < 0.0001. All error bars indicate SD. (n = 1; 100 cells were counted for each treatment; control vs. Cyclin Y/CDK16: t = 6.771, df = 14; Cyclin Y/CDK14 vs. Cyclin Y/CDK16: t = 6.855, df = 14; Cyclin Y/CDK15 vs. Cyclin Y/CDK16: t = 7.139, df = 15). n biological independent replicate. SD standard deviation. Source data are provided as a Source Data file.
Fig. 4 Active Cyclin Y/CDK16 complexes induce autophagy. a NIH3T3 cells stably expressing mCherry-GFP-LC3 were transfected with HA-CDK16 and Cyclin Y-Flag as indicated or treated for 2 h with EBSS or for 4 h with 200 nM Bafilomycin A1 (Baf. A1) and lysates were immunoblotted as indicated. KR kinase-deficient CDK16 mutant, AA CDK16 binding deficient Cyclin Y mutant (n = 3). b Representative confocal images of the NIH3T3-mCherry-GFP-LC3 cells treated as in panel a. Staining of the HA-CDK16 in purple identified transfected cells. Autophagosomes (yellow dots) and autolysosomes (red dots) were detected by an overlay of the GFP and mCherry fluorescent signals. Scale bar: 20 µm. c Quantification of autophagosomes (yellow dots) and autolysosomes (red dots) of cells shown in panel b. Statistical significance was measured via unpaired and two-tailed Student’s t-tests and is presented as follows: **p < 0.01. All error bars indicate SD (n = 3; 50 cells counted for each replicate; wt vs. KR: t = 5.707, df = 4; wt vs. AA: t = 5.557, df = 4). d GFP-tagged CDK14, CDK15 or CDK16 were expressed with or without Cyclin Y-Flag in HeLa cells. Cyclin Y was immunoprecipitated with a Flag antibody (IP). Lysates were immunoblotted with the indicated antibodies (WCL). (n = 4) e Representative confocal images of HeLa cells treated as in panel d. Fluorescent GFP signals identified CDK expressing cells. Endogenous LC3 (red) was used to measure autophagy with the 4E12 antibody. Scale bar: 50 µm. f Quantification of the LC3 dots shown in panel e. Statistical significance was measured via unpaired and two-tailed Student’s t-tests and is presented as follows: ****p < 0.0001. All error bars indicate SD. (n = 1; 100 cells were counted for each treatment; control vs. Cyclin Y/CDK16: t = 6.771, df = 14; Cyclin Y/CDK14 vs. Cyclin Y/CDK16: t = 6.855, df = 14; Cyclin Y/CDK15 vs. Cyclin Y/CDK16: t = 7.139, df = 15). n biological independent replicate. SD standard deviation. Source data are provided as a Source Data file.CC BY · PubMed Central
Fig. 5 ULK1 and Beclin1 are required for Cyclin Y/CDK16-induced autophagy. a HeLa cells were co-transfected with siRNA against ULK1, Beclin1 or a control and vectors expressing GFP-CDK16 and Cyclin Y-Flag for 72 h. Lysates were immunoblotted with the indicated antibodies. (n = 3). b Representative confocal images of HeLa cells treated as in panel a. GFP-CDK16 identified transfected cells. Endogenous LC3 staining (red) with the 4E12 antibody monitored autophagy. Scale bar: 50 µm. c Quantification of the LC3 dots shown in panel b. Statistical significance was measured via unpaired and two-tailed Student’s t-tests and is presented as follows: ****p < 0.0001. All error bars indicate SD. (n = 1; 250 cells were analyzed for each treatment; siControl vs. siControl Cyclin Y/CDK16: t = 13.49, df = 8; siControl Cyclin Y/CDK16 vs. siULK1 Cyclin Y/CDK16: t = 13.53, df = 8; siControl Cyclin Y/CDK16 vs. siBeclin1 Cyclin Y/CDK16: t = 13.15, df = 8). n biological independent replicate. SD standard deviation. Source data are provided as a Source Data file.
Fig. 5 ULK1 and Beclin1 are required for Cyclin Y/CDK16-induced autophagy. a HeLa cells were co-transfected with siRNA against ULK1, Beclin1 or a control and vectors expressing GFP-CDK16 and Cyclin Y-Flag for 72 h. Lysates were immunoblotted with the indicated antibodies. (n = 3). b Representative confocal images of HeLa cells treated as in panel a. GFP-CDK16 identified transfected cells. Endogenous LC3 staining (red) with the 4E12 antibody monitored autophagy. Scale bar: 50 µm. c Quantification of the LC3 dots shown in panel b. Statistical significance was measured via unpaired and two-tailed Student’s t-tests and is presented as follows: ****p < 0.0001. All error bars indicate SD. (n = 1; 250 cells were analyzed for each treatment; siControl vs. siControl Cyclin Y/CDK16: t = 13.49, df = 8; siControl Cyclin Y/CDK16 vs. siULK1 Cyclin Y/CDK16: t = 13.53, df = 8; siControl Cyclin Y/CDK16 vs. siBeclin1 Cyclin Y/CDK16: t = 13.15, df = 8). n biological independent replicate. SD standard deviation. Source data are provided as a Source Data file.CC BY · PubMed Central
Fig. 6 AMPK-induced autophagy requires Cyclin Y/CDK16. a Immortalized Cdk16+/+ and Cdk16−/− MEFs were treated with 1 mM AICAR for 1 or 16 h. Lysates were immunoblotted with the indicated antibodies (n = 3). b Representative confocal images of the Cdk16+/+ and Cdk16−/− MEFs treated as in panel a. Endogenous LC3 Staining with the 4E12 antibody monitored autophagy. Scale bar: 50 µm. c LC3 dots of experiments as shown in panel b were quantified. Statistical significance was measured via unpaired and two-tailed Student’s t-tests and is presented as follows: **p < 0.01. All error bars indicate SD. (n = 3; 100 cells counted for each replicate; Cdk16+/+ + AICAR vs. Cdk16−/− + AICAR: t = 4.964; df = 4). d Immortalized Cdk16+/+ and Cdk16−/− MEFs were treated with 1 mM AICAR for 1 h and/or with 200 nM Bafilomycin A1 (Baf. A1) for 6 h as indicated. For the combination, AICAR was added during the last hour of Baf. A1 treatment. Lysates were immunoblotted with the indicated antibodies (n = 1). e NIH3T3 cells were transfected with siRNA against Cyclin Y or control siRNA and stimulated with 0.5 mM AICAR/50 µM A769662 (A769) or with 200 nM Baf. A1 or a combination. Proteins were analyzed as indicated. (n = 3). f Representative confocal images of the NIH3T3 cells treated as in panel e. Endogenous LC3 was stained with the 4E12 antibody to depict autophagy. Scale bar: 50 µm. g LC3 dots as shown in panel f were quantified. Statistical significance was measured via unpaired and two-tailed Student’s t-tests and is presented as follows: **p < 0.01, ***p < 0.001. All error bars indicate SD. (n = 3; 100 cells counted for each replicate; AICAR/A769: t = 9.494, df = 6; Baf. A1: t = 6.549, df = 6; AICAR/A769 + Baf. A1: t = 9.484, df = 4). n biological independent replicate. SD standard deviation. Source data are provided as a Source Data file.
Fig. 6 AMPK-induced autophagy requires Cyclin Y/CDK16. a Immortalized Cdk16+/+ and Cdk16−/− MEFs were treated with 1 mM AICAR for 1 or 16 h. Lysates were immunoblotted with the indicated antibodies (n = 3). b Representative confocal images of the Cdk16+/+ and Cdk16−/− MEFs treated as in panel a. Endogenous LC3 Staining with the 4E12 antibody monitored autophagy. Scale bar: 50 µm. c LC3 dots of experiments as shown in panel b were quantified. Statistical significance was measured via unpaired and two-tailed Student’s t-tests and is presented as follows: **p < 0.01. All error bars indicate SD. (n = 3; 100 cells counted for each replicate; Cdk16+/+ + AICAR vs. Cdk16−/− + AICAR: t = 4.964; df = 4). d Immortalized Cdk16+/+ and Cdk16−/− MEFs were treated with 1 mM AICAR for 1 h and/or with 200 nM Bafilomycin A1 (Baf. A1) for 6 h as indicated. For the combination, AICAR was added during the last hour of Baf. A1 treatment. Lysates were immunoblotted with the indicated antibodies (n = 1). e NIH3T3 cells were transfected with siRNA against Cyclin Y or control siRNA and stimulated with 0.5 mM AICAR/50 µM A769662 (A769) or with 200 nM Baf. A1 or a combination. Proteins were analyzed as indicated. (n = 3). f Representative confocal images of the NIH3T3 cells treated as in panel e. Endogenous LC3 was stained with the 4E12 antibody to depict autophagy. Scale bar: 50 µm. g LC3 dots as shown in panel f were quantified. Statistical significance was measured via unpaired and two-tailed Student’s t-tests and is presented as follows: **p < 0.01, ***p < 0.001. All error bars indicate SD. (n = 3; 100 cells counted for each replicate; AICAR/A769: t = 9.494, df = 6; Baf. A1: t = 6.549, df = 6; AICAR/A769 + Baf. A1: t = 9.484, df = 4). n biological independent replicate. SD standard deviation. Source data are provided as a Source Data file.CC BY · PubMed Central
Fig. 7 AMPK-dependent phosphorylation of Cyclin Y at S326 is required for autophagy. a HeLa cells were transfected with GFP-CDK16, Cyclin Y-Flag or phosphosite mutants as indicated. The interaction of CDK16 and Cyclin Y was analyzed in GFP-specific immunoprecipitations (IP). Proteins were analyzed by immunoblotting as specified (WCL). (n = 2). b Representative confocal images of HeLa cells from panel a. GFP-CDK16 identified transfected cells and staining for endogenous LC3 (red, antibody 4E12) monitored autophagy. Scale bar: 50 µm. c Quantification of LC3 dots per cell treated as displayed in panel b. Statistical significance was measured via unpaired and two-tailed Student’s t-tests and is presented as follows: **p < 0.01; ***p < 0.001; ****p < 0.0001. All error bars indicate SD. (n = 1; 100 cells were analyzed for each treatment; control vs. Cyclin Y/CDK16: t = 7.274, df = 11; control vs. Cyclin Y S324A/CDK16: t = 4.356, df = 8; Cyclin Y/CDK16 vs. Cyclin Y S100A/CDK16: t = 8.644, df = 13; Cyclin Y/CDK16 vs. Cyclin Y S326A/CDK16: t = 7.765, df = 13; Cyclin Y S100A/CDK16 vs. Cyclin Y S324A/CDK16: t = 5.214, df = 10; Cyclin Y S324A/CDK16 vs. Cyclin Y S326A/CDK16: t = 4.734, df = 10). d HeLa cells were transfected with GFP-CDK16 and Cyclin Y-Flag-wt or the S326A mutant and siRNA against AMPK-α1/2 or control. The interaction of CDK16 and Cyclin Y was analyzed in GFP-specific immunoprecipitations. Proteins were analyzed by immunoblotting as specified. (n = 2). e Representative confocal images of the HeLa cells from panel d. GFP-CDK16 identified the transfected cells and staining for endogenous LC3 (red, antibody 4E12) measured autophagy. Scale bar: 50 µm. f Quantification of the LC3 dots shown in panel e. Statistical significance was measured via unpaired and two-tailed Student’s t-tests and is presented as follows: ****p < 0.0001. All error bars indicate SD. (n = 1; 250 cells were analyzed for each treatment; siControl vs. siControl Cyclin Y/CDK16: t = 8.113, df = 8; siControl Cyclin Y/CDK16 vs. siAMPK-α1/2 Cyclin Y/CDK16: t = 8.290, df = 8). n biological independent replicate. SD standard deviation. Source data are provided as a Source Data file.
Fig. 7 AMPK-dependent phosphorylation of Cyclin Y at S326 is required for autophagy. a HeLa cells were transfected with GFP-CDK16, Cyclin Y-Flag or phosphosite mutants as indicated. The interaction of CDK16 and Cyclin Y was analyzed in GFP-specific immunoprecipitations (IP). Proteins were analyzed by immunoblotting as specified (WCL). (n = 2). b Representative confocal images of HeLa cells from panel a. GFP-CDK16 identified transfected cells and staining for endogenous LC3 (red, antibody 4E12) monitored autophagy. Scale bar: 50 µm. c Quantification of LC3 dots per cell treated as displayed in panel b. Statistical significance was measured via unpaired and two-tailed Student’s t-tests and is presented as follows: **p < 0.01; ***p < 0.001; ****p < 0.0001. All error bars indicate SD. (n = 1; 100 cells were analyzed for each treatment; control vs. Cyclin Y/CDK16: t = 7.274, df = 11; control vs. Cyclin Y S324A/CDK16: t = 4.356, df = 8; Cyclin Y/CDK16 vs. Cyclin Y S100A/CDK16: t = 8.644, df = 13; Cyclin Y/CDK16 vs. Cyclin Y S326A/CDK16: t = 7.765, df = 13; Cyclin Y S100A/CDK16 vs. Cyclin Y S324A/CDK16: t = 5.214, df = 10; Cyclin Y S324A/CDK16 vs. Cyclin Y S326A/CDK16: t = 4.734, df = 10). d HeLa cells were transfected with GFP-CDK16 and Cyclin Y-Flag-wt or the S326A mutant and siRNA against AMPK-α1/2 or control. The interaction of CDK16 and Cyclin Y was analyzed in GFP-specific immunoprecipitations. Proteins were analyzed by immunoblotting as specified. (n = 2). e Representative confocal images of the HeLa cells from panel d. GFP-CDK16 identified the transfected cells and staining for endogenous LC3 (red, antibody 4E12) measured autophagy. Scale bar: 50 µm. f Quantification of the LC3 dots shown in panel e. Statistical significance was measured via unpaired and two-tailed Student’s t-tests and is presented as follows: ****p < 0.0001. All error bars indicate SD. (n = 1; 250 cells were analyzed for each treatment; siControl vs. siControl Cyclin Y/CDK16: t = 8.113, df = 8; siControl Cyclin Y/CDK16 vs. siAMPK-α1/2 Cyclin Y/CDK16: t = 8.290, df = 8). n biological independent replicate. SD standard deviation. Source data are provided as a Source Data file.CC BY · PubMed Central
Fig. 8 Model of the AMPK-dependent activation of Cyclin Y/CDK16 for the induction of autophagy. AMPK is activated under energy stress situations (- amino acids; - glucose) or by allosteric activators (AICAR/A769) and phosphorylates Cyclin Y at S326. This phosphorylation allows the interaction of Cyclin Y with CDK16 and stimulates the kinase activity of CDK16, as illustrated by the phosphorylation of Cyclin Y at S336. The CDK16 activity downstream of AMPK is required for the efficient induction of autophagy.
Fig. 8 Model of the AMPK-dependent activation of Cyclin Y/CDK16 for the induction of autophagy. AMPK is activated under energy stress situations (- amino acids; - glucose) or by allosteric activators (AICAR/A769) and phosphorylates Cyclin Y at S326. This phosphorylation allows the interaction of Cyclin Y with CDK16 and stimulates the kinase activity of CDK16, as illustrated by the phosphorylation of Cyclin Y at S336. The CDK16 activity downstream of AMPK is required for the efficient induction of autophagy.CC BY · PubMed Central

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Metadata

Authors
Dohmen, M., Krieg, S., Agalaridis, G., Zhu, X., Shehata, S. N., Pfeiffenberger, E., Amelang, J., Bütepage, M., Buerova, E., Pfaff, C. M., Chanda, D., Geley, S., Preisinger, C., Sakamoto, K., Lüscher, B., Neumann, D., & Vervoorts, J.
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2020-02-25
Volume
11
Issue
1
ISSN
2041-1723
DOI
10.1038/s41467-020-14812-0
PMC
PMC7042329
Cited by
40
References
5
Language
en
Licence
Funders
  • Chinese Scholarship Council
  • Marie Curie fellowship
  • Deutsche Forschungsgemeinschaft (LU 466/16-2)
  • Nederlandse Organisatie voor Wetenschappelijk Onderzoek (864.10.007)

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