Activation of hormone-sensitive lipase requires two steps, protein phosphorylation and binding to the PAT-1 domain of lipid droplet coat proteins

Hong Wang, Liping Hu, Knut Dalen, Heidi Dorward, Amy Marcinkiewicz, Deanna Russell, Dawei Gong, Constantine Londos, Tomohiro Yamaguchi, Cecilia Holm, Mark A. Rizzo, Dawn Brasaemle, Carole Sztalryd

Research output: Contribution to journalArticle

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Abstract

Lipolysis is an important metabolic pathway controlling energy homeostasis through degradation of triglycerides stored in lipid droplets and release of fatty acids. Lipid droplets of mammalian cells are coated with one or more members of the PAT protein family, which serve important functions in regulating lipolysis. In this study, we investigate the mechanisms by which PAT family members, perilipin A, adipose differentiation-related protein (ADFP), and LSDP5, control lipolysis catalyzed by hormone-sensitive lipase (HSL), a major lipase in adipocytes and several non-adipose cells. We applied fluorescence microscopic tools to analyze proteins in situ in cultured Chinese hamster ovary cells using fluorescence recovery after photobleaching and anisotropy Forster resonance energy transfer. Fluorescence recovery after photobleaching data show that ADFP and LSDP5 exchange between lipid droplet and cytoplasmic pools, whereas perilipin A does not. Differences in protein mobility do not correlate with PAT protein-mediated control of lipolysis catalyzed by HSL or endogenous lipases. Forster resonance energy transfer and co-immunoprecipitation experiments reveal that each of the three PAT proteins bind HSL through interaction of the lipase with amino acids within the highly conserved amino-terminal PAT-1 domain. ADFP and LSDP5 bind HSL under basal conditions, whereas phosphorylation of serine residues within three amino-terminal protein kinase A consensus sequences of perilipin A is required for HSL binding and maximal lipolysis. Finally, protein kinase A-mediated phosphorylation of HSL increases lipolysis in cells expressing ADFP or LSDP5; in contrast, phosphorylation of perilipin A exerts the major control over HSL-mediated lipolysis when perilipin is the main lipid droplet protein.

Original languageEnglish (US)
Pages (from-to)32116-32125
Number of pages10
JournalJournal of Biological Chemistry
Volume284
Issue number46
DOIs
StatePublished - Nov 30 2009

Fingerprint

Sterol Esterase
Phosphorylation
Lipolysis
Capsid Proteins
Protein Binding
Chemical activation
Lipids
Lipase
Proteins
Fluorescence Recovery After Photobleaching
Fluorescence Resonance Energy Transfer
Cyclic AMP-Dependent Protein Kinases
Photobleaching
Fluorescence
Cells
Anisotropy
Consensus Sequence
Cricetulus
Metabolic Networks and Pathways
Lipid Droplet Associated Proteins

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Wang, Hong ; Hu, Liping ; Dalen, Knut ; Dorward, Heidi ; Marcinkiewicz, Amy ; Russell, Deanna ; Gong, Dawei ; Londos, Constantine ; Yamaguchi, Tomohiro ; Holm, Cecilia ; Rizzo, Mark A. ; Brasaemle, Dawn ; Sztalryd, Carole. / Activation of hormone-sensitive lipase requires two steps, protein phosphorylation and binding to the PAT-1 domain of lipid droplet coat proteins. In: Journal of Biological Chemistry. 2009 ; Vol. 284, No. 46. pp. 32116-32125.
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abstract = "Lipolysis is an important metabolic pathway controlling energy homeostasis through degradation of triglycerides stored in lipid droplets and release of fatty acids. Lipid droplets of mammalian cells are coated with one or more members of the PAT protein family, which serve important functions in regulating lipolysis. In this study, we investigate the mechanisms by which PAT family members, perilipin A, adipose differentiation-related protein (ADFP), and LSDP5, control lipolysis catalyzed by hormone-sensitive lipase (HSL), a major lipase in adipocytes and several non-adipose cells. We applied fluorescence microscopic tools to analyze proteins in situ in cultured Chinese hamster ovary cells using fluorescence recovery after photobleaching and anisotropy Forster resonance energy transfer. Fluorescence recovery after photobleaching data show that ADFP and LSDP5 exchange between lipid droplet and cytoplasmic pools, whereas perilipin A does not. Differences in protein mobility do not correlate with PAT protein-mediated control of lipolysis catalyzed by HSL or endogenous lipases. Forster resonance energy transfer and co-immunoprecipitation experiments reveal that each of the three PAT proteins bind HSL through interaction of the lipase with amino acids within the highly conserved amino-terminal PAT-1 domain. ADFP and LSDP5 bind HSL under basal conditions, whereas phosphorylation of serine residues within three amino-terminal protein kinase A consensus sequences of perilipin A is required for HSL binding and maximal lipolysis. Finally, protein kinase A-mediated phosphorylation of HSL increases lipolysis in cells expressing ADFP or LSDP5; in contrast, phosphorylation of perilipin A exerts the major control over HSL-mediated lipolysis when perilipin is the main lipid droplet protein.",
author = "Hong Wang and Liping Hu and Knut Dalen and Heidi Dorward and Amy Marcinkiewicz and Deanna Russell and Dawei Gong and Constantine Londos and Tomohiro Yamaguchi and Cecilia Holm and Rizzo, {Mark A.} and Dawn Brasaemle and Carole Sztalryd",
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Wang, H, Hu, L, Dalen, K, Dorward, H, Marcinkiewicz, A, Russell, D, Gong, D, Londos, C, Yamaguchi, T, Holm, C, Rizzo, MA, Brasaemle, D & Sztalryd, C 2009, 'Activation of hormone-sensitive lipase requires two steps, protein phosphorylation and binding to the PAT-1 domain of lipid droplet coat proteins', Journal of Biological Chemistry, vol. 284, no. 46, pp. 32116-32125. https://doi.org/10.1074/jbc.M109.006726

Activation of hormone-sensitive lipase requires two steps, protein phosphorylation and binding to the PAT-1 domain of lipid droplet coat proteins. / Wang, Hong; Hu, Liping; Dalen, Knut; Dorward, Heidi; Marcinkiewicz, Amy; Russell, Deanna; Gong, Dawei; Londos, Constantine; Yamaguchi, Tomohiro; Holm, Cecilia; Rizzo, Mark A.; Brasaemle, Dawn; Sztalryd, Carole.

In: Journal of Biological Chemistry, Vol. 284, No. 46, 30.11.2009, p. 32116-32125.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Activation of hormone-sensitive lipase requires two steps, protein phosphorylation and binding to the PAT-1 domain of lipid droplet coat proteins

AU - Wang, Hong

AU - Hu, Liping

AU - Dalen, Knut

AU - Dorward, Heidi

AU - Marcinkiewicz, Amy

AU - Russell, Deanna

AU - Gong, Dawei

AU - Londos, Constantine

AU - Yamaguchi, Tomohiro

AU - Holm, Cecilia

AU - Rizzo, Mark A.

AU - Brasaemle, Dawn

AU - Sztalryd, Carole

PY - 2009/11/30

Y1 - 2009/11/30

N2 - Lipolysis is an important metabolic pathway controlling energy homeostasis through degradation of triglycerides stored in lipid droplets and release of fatty acids. Lipid droplets of mammalian cells are coated with one or more members of the PAT protein family, which serve important functions in regulating lipolysis. In this study, we investigate the mechanisms by which PAT family members, perilipin A, adipose differentiation-related protein (ADFP), and LSDP5, control lipolysis catalyzed by hormone-sensitive lipase (HSL), a major lipase in adipocytes and several non-adipose cells. We applied fluorescence microscopic tools to analyze proteins in situ in cultured Chinese hamster ovary cells using fluorescence recovery after photobleaching and anisotropy Forster resonance energy transfer. Fluorescence recovery after photobleaching data show that ADFP and LSDP5 exchange between lipid droplet and cytoplasmic pools, whereas perilipin A does not. Differences in protein mobility do not correlate with PAT protein-mediated control of lipolysis catalyzed by HSL or endogenous lipases. Forster resonance energy transfer and co-immunoprecipitation experiments reveal that each of the three PAT proteins bind HSL through interaction of the lipase with amino acids within the highly conserved amino-terminal PAT-1 domain. ADFP and LSDP5 bind HSL under basal conditions, whereas phosphorylation of serine residues within three amino-terminal protein kinase A consensus sequences of perilipin A is required for HSL binding and maximal lipolysis. Finally, protein kinase A-mediated phosphorylation of HSL increases lipolysis in cells expressing ADFP or LSDP5; in contrast, phosphorylation of perilipin A exerts the major control over HSL-mediated lipolysis when perilipin is the main lipid droplet protein.

AB - Lipolysis is an important metabolic pathway controlling energy homeostasis through degradation of triglycerides stored in lipid droplets and release of fatty acids. Lipid droplets of mammalian cells are coated with one or more members of the PAT protein family, which serve important functions in regulating lipolysis. In this study, we investigate the mechanisms by which PAT family members, perilipin A, adipose differentiation-related protein (ADFP), and LSDP5, control lipolysis catalyzed by hormone-sensitive lipase (HSL), a major lipase in adipocytes and several non-adipose cells. We applied fluorescence microscopic tools to analyze proteins in situ in cultured Chinese hamster ovary cells using fluorescence recovery after photobleaching and anisotropy Forster resonance energy transfer. Fluorescence recovery after photobleaching data show that ADFP and LSDP5 exchange between lipid droplet and cytoplasmic pools, whereas perilipin A does not. Differences in protein mobility do not correlate with PAT protein-mediated control of lipolysis catalyzed by HSL or endogenous lipases. Forster resonance energy transfer and co-immunoprecipitation experiments reveal that each of the three PAT proteins bind HSL through interaction of the lipase with amino acids within the highly conserved amino-terminal PAT-1 domain. ADFP and LSDP5 bind HSL under basal conditions, whereas phosphorylation of serine residues within three amino-terminal protein kinase A consensus sequences of perilipin A is required for HSL binding and maximal lipolysis. Finally, protein kinase A-mediated phosphorylation of HSL increases lipolysis in cells expressing ADFP or LSDP5; in contrast, phosphorylation of perilipin A exerts the major control over HSL-mediated lipolysis when perilipin is the main lipid droplet protein.

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