Spatiotemporal regulation of early lipolytic signaling in adipocytes.
Hormone-sensitive lipase (HSL) is a key enzyme regulating the acute activation of lipolysis. HSL functionality is controlled by multiple phosphorylation events, which regulate its association with the surface of lipid droplets (LDs). We determined the progression and stability of HSL phosphorylation on individual serine residues both spatially and temporally in adipocytes using phospho-specific antibodies. Within seconds of beta-adrenergic receptor activation, HSL was phosphorylated on Ser-660, the phosphorylated form appearing in the peripheral cytosol prior to rapid translocation to, and stable association with, LDs. In contrast, phosphorylation of HSL on Ser-563 was delayed, the phosphorylated protein was predominantly detected on LDs, and mutation of the Ser-659/Ser-660 site to Ala significantly reduced subsequent phosphorylation on Ser-563. Phosphorylation of HSL on Ser-565 was observed in control cells; the phosphorylated protein was translocated to LDs with similar kinetics to total HSL, and the degree of phosphorylation was inversely related to phospho-HSL(Ser-563). These results describe the remarkably rapid, sequential phosphorylation of specific serine residues in HSL at spatially distinct intracellular locales, providing new insight into the complex regulation of lipolysis.
Martin
Sally
S
Division of Molecular Cell Biology, Institute for Molecular Bioscience, University of Queensland, Queensland 4072, Australia. s.martin@uq.edu.au
Okano
Satomi
S
Kistler
Carol
C
Fernandez-Rojo
Manuel A
MA
Hill
Michelle M
MM
Parton
Robert G
RG
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D041721:3T3-L1 Cells; D017667:Adipocytes / Q000378:metabolism*; D058667:Adrenergic beta-3 Receptor Agonists; D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D004149:Dioxoles / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D007124:Immunoenzyme Techniques; D007545:Isoproterenol / Q000494:pharmacology; D008066:Lipolysis / Q000187:drug effects / Q000502:physiology*; D051379:Mice; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D015398:Signal Transduction; D002787:Sterol Esterase / Q000235:genetics / Q000378:metabolism*; D013347:Subcellular Fractions
D058667:Adrenergic beta-3 Receptor Agonists; D000318:Adrenergic beta-Agonists; D004149:Dioxoles; D010726:Phosphodiesterase Inhibitors; C076126:disodium (R,R)-5-(2-((2-(3-chlorophenyl)-2-hydroxyethyl)-amino)propyl)-1,3-benzodioxole-2,3-dicarboxylate; D002787:Sterol Esterase; D007545:Isoproterenol; D015056:1-Methyl-3-isobutylxanthine