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. 2009 Nov 13;284(46):32116-25.
doi: 10.1074/jbc.M109.006726. Epub 2009 Aug 29.

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

Hong Wang  1 , Liping Hu, Knut Dalen, Heidi Dorward, Amy Marcinkiewicz, Deanna Russell, Dawei Gong, Constantine Londos, Tomohiro Yamaguchi, Cecilia Holm, Megan A Rizzo, Dawn Brasaemle, Carole Sztalryd
Affiliations

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

Hong Wang et al. J Biol Chem. .

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.

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Figures

FIGURE 1.
FIGURE 1.
PAT protein association with lipid droplets and effects on lipolysis. A, FRAP analysis of ADFP-YFP, perilipin-YFP (under basal and stimulated conditions), and LDSP5-YFP in CHO-K1 cells following overnight incubation with 400 μm oleic acid. The cells were incubated with 5 μm triacsin C in the absence of supplemental fatty acids, and perilipin-YFP cells were stimulated for 15 min with 10 μm forskolin, 1 mm IBMX, and 5 μm triacsin C. Live cells were examined with a confocal laser microscope using a ×40 oil immersion objective. Red boxed regions were bleached at time 0, and fluorescence within these regions was monitored at 15-s intervals. Bar, 10 μm. B, wild-type or CHO-K1 cells stably expressing ADFP-YFP, perilipin-YFP, or LSDP5-YFP were incubated overnight with 400 μm [3H]oleic acid. Supplemental fatty acids were withdrawn, and cells were incubated with 5 μm triacsin C with (stimulated) or without (basal) 1 mm IBMX and 10 μm forskolin; the amount of [3H]oleic acid released into the medium was measured after 3 h. Data represent means ± S.E. (n = 8) (*, p < 0.05, for PKA-stimulated value compared with basal value).
FIGURE 2.
FIGURE 2.
Recruitment of HSL to lipid droplets is dependent on the PAT protein composition of the droplet. Recruitment of HSL to perilipin-covered lipid droplets requires activation of PKA. CHO-K1 cells were co-transfected with HSL-CFP and a PAT fusion protein as follows: perilipin-YFP (A), ADFP-YFP (B), or LDSP-5-YFP (C). Cells were incubated overnight with 400 μm oleic acid. The following day, supplemental fatty acids were removed, and the cells were incubated with 5 μm triacsin C and no further additions (basal) or stimulated for 30 min with 10 μm forskolin, 1 mm IBMX, and 5 μm triacsin C (stimulated). Live cells were examined with a confocal microscope as in Fig. 1. Bar, 10 μm. Top, middle, and bottom rows show representative cells from 6 to 12 separate experiments.
FIGURE 3.
FIGURE 3.
HSL binds to PAT family proteins, as determined by in situ AFRET and co-immunoprecipitation (IP). A, images were collected by confocal microscopy of live cells expressing pairs of fluorescent fusion proteins shown along the x axis and calculations for AFRET were performed (29). The gray line indicates the threshold of significance. Data are means ± S.E. from 6 to 12 experiments. *, p < 0.05; **, p < 0.01. B, CHO-K1 cells expressing HSL-CFP were transfected with a PAT fusion protein as follows: perilipin-YFP, ADFP-YFP, or LDSP-5-YFP. The cells were then incubated with 400 μm oleic acid overnight. The following day, cells were incubated with 5 μm triacsin C (B, basal conditions) or with 10 μm forskolin, 1 mm IBMX, and 5 μm triacsin C for 30 min (S, stimulated conditions). Cell lysates were incubated with rabbit anti-HSL IgG (basal or stimulated) or rabbit pre-immune control IgG. Immunoprecipitates were analyzed by Western blot for PAT family proteins and HSL using a commercial GFP antibody that cross-reacts with YFP. One of three similar experiments is shown. IP, immunoprecipitation; WB, Western blot; Ab, antibody. C, Western blot ECL signals were quantified by densitometry using ImageJ software. Each data point represents the average of the calculated ratio of the amount of each PAT protein recovered in stimulated conditions relative to the amount recovered in basal conditions. Data represent means ± S.E. of three experiments (*, p < 0.05, basal versus stimulated conditions).
FIGURE 4.
FIGURE 4.
Activation of PKA is required for increased lipolysis. CHO-Flip-In cells expressing ADFP-YFP, LSDP5-YFP, or perilipin-YFP were transduced with an adenoviral HSL-CFP construct for 48 h prior to lipolysis measurements. Control cells (without HSL-CFP) were infected with an adenoviral construct for lacZ. A, cells were loaded overnight with 400 μm [3H]oleic acid and then incubated in the absence of supplemental fatty acids with 5 μm triacsin C for 2 h. Basal activities are shown in the left graph, and activities from cells stimulated with IBMX and forskolin are expressed as a percentage over basal and shown in the right graph. Data are means ± S.E. from three separate experiments (a, p < 0.01 comparing lipolysis of cells lacking HSL but containing peri-YFP or LSDP5-YFP-coated droplets to lipolysis of cells containing ADFP-YFP-coated lipid droplets; b, p < 0.01 comparing lipolysis of HSL-expressing cells containing peri-YFP or LSDP5-coated droplets to lipolysis of HSL-expressing cells containing ADFP-YFP-coated droplets; c, p < 0.05 comparing % change over basal from cells lacking HSL to % change over basal in HSL-expressing cells; d, p < 0.05 comparing % change over basal in HSL-expressing cells containing peri-YFP or LSDP5-YFP-coated droplets to HSL-expressing cells containing ADFP-YFP-coated droplets; e, p < 0.01 comparing % change over basal in cells lacking HSL but containing peri-YFP-coated lipid droplets to % change over basal in cells lacking HSL but containing ADFP-YFP- or LSDP5-YFP-coated lipid droplets. B, immunoblot of cellular proteins extracted from CHO-Flip-In cells expressing ADFP-YFP, LSDP5-YFP, or perilipin-YFP and transduced with an adenoviral HSL-CFP (or lacZ) construct. Immunoblot shown is representative of three separate experiments.
FIGURE 5.
FIGURE 5.
AT-1 domain is the site of HSL binding. A, Western blot ECL signals were quantified by densitometry using ImageJ software. Each data point represents the average of the calculated ratio of the amount of each truncated form of perilipin-YFP recovered in IP performed with HSL IgGs relative to the amount recovered in IP performed with control rabbit IgGs. Plasmids for fusion constructs of perilipin-(1–121), perilipin-(1–199), perilipin-(1–302), and perilipin-(233–517) with YFP were transfected into CHO-K1 cells stably expressing HSL-GFP. Cells were incubated under stimulated conditions, extracts prepared for co-IP with rabbit anti-HSL antibodies, and blots probed with a GFP antibody. Data represent means ± S.E. of three experiments. B, in situ AFRET measurements; data were collected in live cells incubated in basal conditions and expressing pairs of fluorescent fusion proteins, as described in Fig. 3. Measurements were acquired solely in the cytoplasm. The gray line indicates the threshold of significance. Data are means ± S.E. from three experiments for each condition. *, p < 0.05, for the comparison of HSL-CFP/Peri-(1–121)-YFP to LSDP-5-CFP/Peri-YFP.
FIGURE 6.
FIGURE 6.
HSL recruitment to lipid droplets requires the phosphorylation of serines 81, 222, and 276 of perilipin A. A, CHO-K1 cells were co-transfected with HSL-CFP and one of the following mutated phosphorylation site perilipin fusion proteins: perilipin-YFP (panels a and a′); perilipin S81A-YFP (panels b and b′); perilipin S222A-YFP (panels c and c′); perilipin S276A-YFP (panels d and d′); perilipin STriA-YFP (Ser-81, Ser-222, and Ser-276 mutated to Ala) (panels e and e′); perilipin SQuadA-YFP (Ser-81, Ser-222, Ser-276, and Ser-433 mutated to Ala) (panels f and f′). Cells were incubated overnight with 400 μm oleic acid. The following day, the cells were incubated with 5 μm triacsin C with no further additions (basal) or were stimulated for 10 min with 10 μm forskolin, 1 mm IBMX, and 5 μm triacsin C; live cells were examined with a confocal microscope as in Fig. 1. Bar, 10 μm. B, quantitative analysis for HSL binding to lipid droplets covered with perilipin A or mutated variants of perilipin. Data are means ± S.E. from 2 to 14 experiments. HSL binding to lipid droplets coated with each of the mutated variants of perilipin was significantly different from the binding of HSL to unmodified perilipin A (**, p < 0.001).
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