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AMP-Activated Kinase Regulates Lipid Droplet Localization and Stability of Adipose Triglyceride Lipase in C. elegans Dauer Larvae.

Xie M, Roy R - PLoS ONE (2015)

Bottom Line: Physical interaction of ATGL-1 with PAR-5 results in sequestration of ATGL-1 away from the lipid droplets and eventual proteasome-mediated degradation.In addition, we also show that the major AMPK phosphorylation site on ATGL-1, Ser 303, is required for both modification of its lipid droplet localization and its degradation.Our data provide mechanistic insight as to how AMPK functions to enhance survival through its ability to protect the accumulated triglyceride deposits from rapid hydrolysis to preserve the energy stores during periods of extended environmental duress.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, McGill University, 1205 avenue Docteur Penfield, Montreal, Canada.

ABSTRACT
Animals have developed diverse mechanisms to adapt to their changing environment. Like many organisms the free-living nematode C. elegans can alternate between a reproductive mode or a diapause-like "dauer" stage during larval development to circumvent harsh environmental conditions. The master metabolic regulator AMP-activated protein kinase (AMPK) is critical for survival during the dauer stage, where it phosphorylates adipose triglyceride lipase (ATGL-1) at multiple sites to block lipid hydrolysis and ultimately protect the cellular triglyceride-based energy depot from rapid depletion. However, how the AMPK-mediated phosphorylation affects the function of ATGL-1 has not been characterised at the molecular level. Here we show that AMPK phosphorylation leads to the generation of 14-3-3 binding sites on ATGL-1, which are recognized by the C. elegans 14-3-3 protein orthologue PAR-5. Physical interaction of ATGL-1 with PAR-5 results in sequestration of ATGL-1 away from the lipid droplets and eventual proteasome-mediated degradation. In addition, we also show that the major AMPK phosphorylation site on ATGL-1, Ser 303, is required for both modification of its lipid droplet localization and its degradation. Our data provide mechanistic insight as to how AMPK functions to enhance survival through its ability to protect the accumulated triglyceride deposits from rapid hydrolysis to preserve the energy stores during periods of extended environmental duress.

No MeSH data available.


AMPK-Mediated Phosphorylation of ATGL-1 Enhances Ubiquitin-Mediated Degradation via the Proteasome.(A)-(C) ATGL-1 protein levels are regulated by AMPK by enhancing ubiquitin-mediated proteasomal degradation. Individual proteasome components were compromised through RNAi, of which many, but not all, increase the levels of ATGL-1 in control daf-2 dauer larvae. (D) Ubiquitylated ATGL-1 intermediates accumulate in daf-2; aak(0) mutant dauer larvae. Immunoprecipitation of ATGL-1 from lysates obtained from both control daf-2 and daf-2; aak(0) mutant dauer larvae was analyzed by western blot analysis using anti-ubiquitin antibody. High molecular weight ubiquitin-conjugated entities are seen in immunoprecipitates obtained from daf-2; aak(0) but are more prominent in the daf-2 lysates. More lysate was loaded for control daf-2 animals to equalize the amount of ATGL-1 protein loaded with that of daf-2; aak(0) animals. “IP:ATGL-1” refers to the protein lysate that was subjected to immunoprecipitation with our anti-ATGL-1 polyclonal antibody. “Input” refers to the total protein lysate before performing the immunoprecipitation step.
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pone.0130480.g005: AMPK-Mediated Phosphorylation of ATGL-1 Enhances Ubiquitin-Mediated Degradation via the Proteasome.(A)-(C) ATGL-1 protein levels are regulated by AMPK by enhancing ubiquitin-mediated proteasomal degradation. Individual proteasome components were compromised through RNAi, of which many, but not all, increase the levels of ATGL-1 in control daf-2 dauer larvae. (D) Ubiquitylated ATGL-1 intermediates accumulate in daf-2; aak(0) mutant dauer larvae. Immunoprecipitation of ATGL-1 from lysates obtained from both control daf-2 and daf-2; aak(0) mutant dauer larvae was analyzed by western blot analysis using anti-ubiquitin antibody. High molecular weight ubiquitin-conjugated entities are seen in immunoprecipitates obtained from daf-2; aak(0) but are more prominent in the daf-2 lysates. More lysate was loaded for control daf-2 animals to equalize the amount of ATGL-1 protein loaded with that of daf-2; aak(0) animals. “IP:ATGL-1” refers to the protein lysate that was subjected to immunoprecipitation with our anti-ATGL-1 polyclonal antibody. “Input” refers to the total protein lysate before performing the immunoprecipitation step.

Mentions: In separate RNAi experiments we compromised individual members of the pas, pbs, rpt and rpn gene families and noted that the elimination of most, but not all of these individual proteasome components were associated with increases in the levels of ATGL-1 protein in control daf-2 dauers, whereby the levels became similar to those observed in daf-2; aak(0) dauers likely due to differential involvement of the various components [21], or potentially due to RNAi efficiency (Fig 5A–5C). This suggests that the AMPK-mediated reduction in ATGL-1 protein levels that we observed in control daf-2 dauers requires a functional proteasome.


AMP-Activated Kinase Regulates Lipid Droplet Localization and Stability of Adipose Triglyceride Lipase in C. elegans Dauer Larvae.

Xie M, Roy R - PLoS ONE (2015)

AMPK-Mediated Phosphorylation of ATGL-1 Enhances Ubiquitin-Mediated Degradation via the Proteasome.(A)-(C) ATGL-1 protein levels are regulated by AMPK by enhancing ubiquitin-mediated proteasomal degradation. Individual proteasome components were compromised through RNAi, of which many, but not all, increase the levels of ATGL-1 in control daf-2 dauer larvae. (D) Ubiquitylated ATGL-1 intermediates accumulate in daf-2; aak(0) mutant dauer larvae. Immunoprecipitation of ATGL-1 from lysates obtained from both control daf-2 and daf-2; aak(0) mutant dauer larvae was analyzed by western blot analysis using anti-ubiquitin antibody. High molecular weight ubiquitin-conjugated entities are seen in immunoprecipitates obtained from daf-2; aak(0) but are more prominent in the daf-2 lysates. More lysate was loaded for control daf-2 animals to equalize the amount of ATGL-1 protein loaded with that of daf-2; aak(0) animals. “IP:ATGL-1” refers to the protein lysate that was subjected to immunoprecipitation with our anti-ATGL-1 polyclonal antibody. “Input” refers to the total protein lysate before performing the immunoprecipitation step.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4476745&req=5

pone.0130480.g005: AMPK-Mediated Phosphorylation of ATGL-1 Enhances Ubiquitin-Mediated Degradation via the Proteasome.(A)-(C) ATGL-1 protein levels are regulated by AMPK by enhancing ubiquitin-mediated proteasomal degradation. Individual proteasome components were compromised through RNAi, of which many, but not all, increase the levels of ATGL-1 in control daf-2 dauer larvae. (D) Ubiquitylated ATGL-1 intermediates accumulate in daf-2; aak(0) mutant dauer larvae. Immunoprecipitation of ATGL-1 from lysates obtained from both control daf-2 and daf-2; aak(0) mutant dauer larvae was analyzed by western blot analysis using anti-ubiquitin antibody. High molecular weight ubiquitin-conjugated entities are seen in immunoprecipitates obtained from daf-2; aak(0) but are more prominent in the daf-2 lysates. More lysate was loaded for control daf-2 animals to equalize the amount of ATGL-1 protein loaded with that of daf-2; aak(0) animals. “IP:ATGL-1” refers to the protein lysate that was subjected to immunoprecipitation with our anti-ATGL-1 polyclonal antibody. “Input” refers to the total protein lysate before performing the immunoprecipitation step.
Mentions: In separate RNAi experiments we compromised individual members of the pas, pbs, rpt and rpn gene families and noted that the elimination of most, but not all of these individual proteasome components were associated with increases in the levels of ATGL-1 protein in control daf-2 dauers, whereby the levels became similar to those observed in daf-2; aak(0) dauers likely due to differential involvement of the various components [21], or potentially due to RNAi efficiency (Fig 5A–5C). This suggests that the AMPK-mediated reduction in ATGL-1 protein levels that we observed in control daf-2 dauers requires a functional proteasome.

Bottom Line: Physical interaction of ATGL-1 with PAR-5 results in sequestration of ATGL-1 away from the lipid droplets and eventual proteasome-mediated degradation.In addition, we also show that the major AMPK phosphorylation site on ATGL-1, Ser 303, is required for both modification of its lipid droplet localization and its degradation.Our data provide mechanistic insight as to how AMPK functions to enhance survival through its ability to protect the accumulated triglyceride deposits from rapid hydrolysis to preserve the energy stores during periods of extended environmental duress.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, McGill University, 1205 avenue Docteur Penfield, Montreal, Canada.

ABSTRACT
Animals have developed diverse mechanisms to adapt to their changing environment. Like many organisms the free-living nematode C. elegans can alternate between a reproductive mode or a diapause-like "dauer" stage during larval development to circumvent harsh environmental conditions. The master metabolic regulator AMP-activated protein kinase (AMPK) is critical for survival during the dauer stage, where it phosphorylates adipose triglyceride lipase (ATGL-1) at multiple sites to block lipid hydrolysis and ultimately protect the cellular triglyceride-based energy depot from rapid depletion. However, how the AMPK-mediated phosphorylation affects the function of ATGL-1 has not been characterised at the molecular level. Here we show that AMPK phosphorylation leads to the generation of 14-3-3 binding sites on ATGL-1, which are recognized by the C. elegans 14-3-3 protein orthologue PAR-5. Physical interaction of ATGL-1 with PAR-5 results in sequestration of ATGL-1 away from the lipid droplets and eventual proteasome-mediated degradation. In addition, we also show that the major AMPK phosphorylation site on ATGL-1, Ser 303, is required for both modification of its lipid droplet localization and its degradation. Our data provide mechanistic insight as to how AMPK functions to enhance survival through its ability to protect the accumulated triglyceride deposits from rapid hydrolysis to preserve the energy stores during periods of extended environmental duress.

No MeSH data available.