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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 Generates More Phospho-(Ser) 14-3-3 Binding motifs and Enhances ATGL-1 Interaction with the C. elegans 14-3-3 Protein, PAR-5.(A) An increase in both the abundance and the number of bands that were detected by the P-14-3-3 antibody was observed in lysates obtained from N2 animals treated with the potent phosphatase inhibitor calyculin A. (B) More phospho-(Ser) 14-3-3 binding motif was generated in the ATGL-1 immunoprecipitates from control daf-2 dauers compared to daf-2; aak(0) mutant dauers. Immunoprecipitation of ATGL-1 from total lysates obtained from control daf-2 and daf-2; aak(0) mutant dauer larvae were immunoblotted with antisera that recognizes phospho-(Ser) 14-3-3 binding motif. 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. (C) The anti-PAR-5 antibody recognized a single band at approximately 30kD, corresponding to its predicted molecular weight, which disappeared following par-5(RNAi). (D) PAR-5 protein levels are the same in control daf-2 and daf-2; aak(0) dauers. (E) and (F) Immunoprecipitation of PAR-5 (D) or ATGL-1 (E) from protein lysates obtained from control daf-2 and daf-2; aak(0) mutant dauer larvae were subjected to immunoblot analysis using ATGL-1 or PAR-5 antibody as indicated on the panels. 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.g007: AMPK-Mediated Phosphorylation of ATGL-1 Generates More Phospho-(Ser) 14-3-3 Binding motifs and Enhances ATGL-1 Interaction with the C. elegans 14-3-3 Protein, PAR-5.(A) An increase in both the abundance and the number of bands that were detected by the P-14-3-3 antibody was observed in lysates obtained from N2 animals treated with the potent phosphatase inhibitor calyculin A. (B) More phospho-(Ser) 14-3-3 binding motif was generated in the ATGL-1 immunoprecipitates from control daf-2 dauers compared to daf-2; aak(0) mutant dauers. Immunoprecipitation of ATGL-1 from total lysates obtained from control daf-2 and daf-2; aak(0) mutant dauer larvae were immunoblotted with antisera that recognizes phospho-(Ser) 14-3-3 binding motif. 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. (C) The anti-PAR-5 antibody recognized a single band at approximately 30kD, corresponding to its predicted molecular weight, which disappeared following par-5(RNAi). (D) PAR-5 protein levels are the same in control daf-2 and daf-2; aak(0) dauers. (E) and (F) Immunoprecipitation of PAR-5 (D) or ATGL-1 (E) from protein lysates obtained from control daf-2 and daf-2; aak(0) mutant dauer larvae were subjected to immunoblot analysis using ATGL-1 or PAR-5 antibody as indicated on the panels. 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: Many of the downstream effects of AMPK phosphorylation that have been characterized are mediated through the generation of 14-3-3 protein binding sites followed by changes in subcellular localization [22, 23]. Therefore, since we observed a change in the localization of ATGL-1 in response to AMPK we questioned whether a similar mechanism might underlie the dissociation of ATGL-1 from the lipid droplets. We first performed bioinformatic analysis of the ATGL-1 protein sequence using the online Motif Scan tool (http://scansite.mit.edu/motifscan_seq.phtml), which revealed the presence of several regions that corresponded to potential 14-3-3 protein binding sites (data not shown). To address whether AMPK might generate 14-3-3 sites on ATGL-1 to dissociate it away from the lipid droplets, we analyzed our ATGL-1 immunoprecipitates from control daf-2 and daf-2; aak(0) dauers with a 14-3-3 motif antibody that was generated against peptides bearing R-X-Y/F-X-pS sequence (Fig 7B). Western analysis suggested that 14-3-3 binding sites were less prominent in daf-2; aak(0) dauer larvae compared to control daf-2 dauer larvae, consistent with a model wherein phosphorylation of ATGL-1 by AMPK generates a 14-3-3 site(s) that potentially leads to 14-3-3 Protein binding to modify ATGL-1 localization by dissociating it from the droplets (Fig 7B).


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 Generates More Phospho-(Ser) 14-3-3 Binding motifs and Enhances ATGL-1 Interaction with the C. elegans 14-3-3 Protein, PAR-5.(A) An increase in both the abundance and the number of bands that were detected by the P-14-3-3 antibody was observed in lysates obtained from N2 animals treated with the potent phosphatase inhibitor calyculin A. (B) More phospho-(Ser) 14-3-3 binding motif was generated in the ATGL-1 immunoprecipitates from control daf-2 dauers compared to daf-2; aak(0) mutant dauers. Immunoprecipitation of ATGL-1 from total lysates obtained from control daf-2 and daf-2; aak(0) mutant dauer larvae were immunoblotted with antisera that recognizes phospho-(Ser) 14-3-3 binding motif. 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. (C) The anti-PAR-5 antibody recognized a single band at approximately 30kD, corresponding to its predicted molecular weight, which disappeared following par-5(RNAi). (D) PAR-5 protein levels are the same in control daf-2 and daf-2; aak(0) dauers. (E) and (F) Immunoprecipitation of PAR-5 (D) or ATGL-1 (E) from protein lysates obtained from control daf-2 and daf-2; aak(0) mutant dauer larvae were subjected to immunoblot analysis using ATGL-1 or PAR-5 antibody as indicated on the panels. 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.g007: AMPK-Mediated Phosphorylation of ATGL-1 Generates More Phospho-(Ser) 14-3-3 Binding motifs and Enhances ATGL-1 Interaction with the C. elegans 14-3-3 Protein, PAR-5.(A) An increase in both the abundance and the number of bands that were detected by the P-14-3-3 antibody was observed in lysates obtained from N2 animals treated with the potent phosphatase inhibitor calyculin A. (B) More phospho-(Ser) 14-3-3 binding motif was generated in the ATGL-1 immunoprecipitates from control daf-2 dauers compared to daf-2; aak(0) mutant dauers. Immunoprecipitation of ATGL-1 from total lysates obtained from control daf-2 and daf-2; aak(0) mutant dauer larvae were immunoblotted with antisera that recognizes phospho-(Ser) 14-3-3 binding motif. 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. (C) The anti-PAR-5 antibody recognized a single band at approximately 30kD, corresponding to its predicted molecular weight, which disappeared following par-5(RNAi). (D) PAR-5 protein levels are the same in control daf-2 and daf-2; aak(0) dauers. (E) and (F) Immunoprecipitation of PAR-5 (D) or ATGL-1 (E) from protein lysates obtained from control daf-2 and daf-2; aak(0) mutant dauer larvae were subjected to immunoblot analysis using ATGL-1 or PAR-5 antibody as indicated on the panels. 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: Many of the downstream effects of AMPK phosphorylation that have been characterized are mediated through the generation of 14-3-3 protein binding sites followed by changes in subcellular localization [22, 23]. Therefore, since we observed a change in the localization of ATGL-1 in response to AMPK we questioned whether a similar mechanism might underlie the dissociation of ATGL-1 from the lipid droplets. We first performed bioinformatic analysis of the ATGL-1 protein sequence using the online Motif Scan tool (http://scansite.mit.edu/motifscan_seq.phtml), which revealed the presence of several regions that corresponded to potential 14-3-3 protein binding sites (data not shown). To address whether AMPK might generate 14-3-3 sites on ATGL-1 to dissociate it away from the lipid droplets, we analyzed our ATGL-1 immunoprecipitates from control daf-2 and daf-2; aak(0) dauers with a 14-3-3 motif antibody that was generated against peptides bearing R-X-Y/F-X-pS sequence (Fig 7B). Western analysis suggested that 14-3-3 binding sites were less prominent in daf-2; aak(0) dauer larvae compared to control daf-2 dauer larvae, consistent with a model wherein phosphorylation of ATGL-1 by AMPK generates a 14-3-3 site(s) that potentially leads to 14-3-3 Protein binding to modify ATGL-1 localization by dissociating it from the droplets (Fig 7B).

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.