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Hormonal Control of Reversible Translocation of Perilipin B to the Plasma Membrane in Primary Human Adipocytes*

In adipocytes, perilipin coats and protects the central lipid droplet, which stores triacylglycerol. Alternative mRNA splicing gives rise to perilipin A and B. Hormones such as catecholamines and insulin regulate triacylglycerol metabolism through reversible serine phosphorylation of perilipin A. It was recently shown that perilipin was also located in triacylglycerol-synthesizing caveolae of the plasma membrane. We now report that perilipin at the plasma membrane of primary human adipocytes was phosphorylated on a cluster of threonine residues (299, 301, and 306) within an acidic domain that forms part of the lipid targeting domain. Perilipin B comprised <10% of total perilipin but was the major isoform associated with the plasma membrane of human adipocytes. This association was controlled by insulin and catecholamine: perilipin B was specifically depleted from the plasma membrane in response to the catecholamine isoproterenol, while insulin increased the amount of threonine phosphorylated perilipin at the plasma membrane. The reversible translocation of perilipin B to and from the plasma membrane in response to insulin and isoproterenol, respectively, suggests a specific function for perilipin B to protect newly synthesized triacylglycerol in the plasma membrane.

In adipocytes, perilipin coats and protects the central lipid droplet, which stores triacylglycerol. Alternative mRNA splicing gives rise to perilipin A and B. Hormones such as catecholamines and insulin regulate triacylglycerol metabolism through reversible serine phosphorylation of perilipin A. It was recently shown that perilipin was also located in triacylglycerol-synthesizing caveolae of the plasma membrane. We now report that perilipin at the plasma membrane of primary human adipocytes was phosphorylated on a cluster of threonine residues (299, 301, and 306) within an acidic domain that forms part of the lipid targeting domain. Perilipin B comprised <10% of total perilipin but was the major isoform associated with the plasma membrane of human adipocytes. This association was controlled by insulin and catecholamine: perilipin B was specifically depleted from the plasma membrane in response to the catecholamine isoproterenol, while insulin increased the amount of threonine phosphorylated perilipin at the plasma membrane. The reversible translocation of perilipin B to and from the plasma membrane in response to insulin and isoproterenol, respectively, suggests a specific function for perilipin B to protect newly synthesized triacylglycerol in the plasma membrane.
Adipose tissue is the major site in the body for storage of fatty acids as triacylglycerols. When energy is needed hormones, such as the catecholamine noradrenaline, stimulate hydrolysis of triacylglycerol (lipolysis) to mobilize the stored fatty acids, which are the primary source of energy for different tissues. The storage of triacylglycerol in adipocytes, on the other hand, is favored by insulin stimulation of lipogenesis and inhibition of lipolysis. Catecholamine stimulation of lipolysis is mediated by activation of cAMP-dependent protein kinase (1) increasing the phosphorylation of hormone-sensitive lipase (2), which when phosphorylated is activated and translocated from the cytosol to the surface of the central lipid droplet in the adipocyte (3,4). The lipolytic activity of hormone-sensitive lipase is additionally regulated by perilipin, which also becomes phosphorylated by cAMP-dependent protein kinase (5,6). The anti-lipolytic effect of insulin is mediated via dephosphorylation of both hormone-sensitive lipase and perilipin (6,7).
Perilipin is largely located at the surface of the central lipid droplet in adipocytes. In the absence of lipolytic stimulation perilipin inhibits lipolysis by acting as a barrier against hydrolysis of the triacylglycerol by lipases, but when phosphorylated by cAMP-dependent protein kinase perilipin undergoes a conformational change that allows access of hormone-sensitive lipase to the surface of the lipid droplet and enhances the lipase activity (4,8). In adipocytes there are two forms of perilipin, perilipin A and perilipin B, expressed from differentially spliced mRNA. Perilipin A is present at a much higher concentration than perilipin B (9). While perilipin A affects both triacylglycerol lipases present in adipocytes, the effect of perilipin B is apparently restricted to hormone-sensitive lipase (10). Otherwise no specific function has been attributed to perilipin B. It has recently been shown that perilipin may also be located at the plasma membrane (11,12). Moreover, at the plasma membrane perilipin was found associated with caveolae membrane that contained triacylglycerol synthesized from exogenous fatty acids (12).
In the present study we identified phosphorylated proteins in the plasma membrane of primary human adipocytes by applying our vectorial proteomics approach and mass spectrometry (13). We found that perilipin, predominantly perilipin B, at the plasma membrane was phosphorylated on a cluster of three threonine residues (299, 301, and 306). Insulin and ␤-adrenergic treatment of adipocytes induced a reversible translocation of perilipin B to and from the plasma membrane, suggesting that the hormone-controlled association of perilipin B with the plasma membrane may act to protect the newly formed triacylglycerol in the plasma membrane from hydrolysis.

Isolation of Human Adipocytes and Preparation of Plasma Membrane
Fractions-Subcutaneous fat tissue was obtained during elective abdominal surgery on female patients. All participants gave their informed approval, and the Local Ethics Committee approved our study. Adipocytes were isolated by collagenase digestion (14) and the cells preincubated in Krebs-Ringer solution (0.12 M NaCl, 4.7 mM KCl, 2.5 mM CaCl 2 , 1.2 mM MgSO 4 , 1.2 mM KH 2 PO 4 ) containing 20 mM Hepes, pH 7.40, 3.5% (w/v) fatty acid-free bovine serum albumin, 100 nM phenylisopropyladenosine, 0.5 unit/ml adenosine deaminase with 2 mM glucose, at 37°C and then incubated for 10 min with or without 100 nM insulin or with 100 nM isoproterenol. Plasma membrane fractions were obtained by homogenization of the cells at room temperature (22°C) in 10 mM NaH 2 PO 4 , pH 7.4, 1 mM EDTA, 0.25 M sucrose, 25 mM NaF, 1 mM Na 4 P 2 O 7 , 2 mM Na 3 VO 4 , 0.5 mM EGTA, 4 mM iodoacetate. A plasma membrane-containing pellet obtained by centrifugation at 16,000 ϫ g for 20 min was resuspended in 10 mM Tris/HCl, 1 mM EDTA, and 2 mM Na 3 VO 4 . Plasma membranes were then purified by sucrose density-gradient centrifugation (15).
Whole cell lysates were obtained by centrifugation of the cells through dinonylphtalate to remove the incubation medium and the cells were then lysed and boiled in SDS sample buffer containing protease and phosphates inhibitors (14).
SDS-PAGE and Immunoblotting-Protein samples were subjected to SDS-PAGE (9% acrylamide) and transferred to polyvinylidene difluoride membranes (Millipore). The membranes were blocked with milk proteins or bovine serum albumin and then incubated with mouse anti-phosphotyrosine PY20 (Transduction Laboratories, Lexington, KY), rabbit anti-phosphoserine, rabbit anti-phosphothreonine (Zymed Laboratories Inc.), or guinea pig anti-perilipin polyclonal antibodies (Progen Biotechnik, Heidelberg, Germany), as indicated. Detection was done by incubation with secondary antibodies conjugated to horseradish peroxidase, followed by chemiluminescence detection using ECL plus according to the manufacturer's instructions (Amersham Biosciences, Amersham, UK), and evaluated by chemiluminescence imaging (Las 1000; Fuji, Tokyo, Japan).
Trypsin Treatment of Plasma Membranes and Phopshopeptide Enrichment-Isolated plasma membranes were washed three times with 25 mM NH 4 HCO 3 , pH 8, and the final protein concentration was adjusted to 3 mg/ml. Cysteines were reduced with 2 mM dithiothreitol and alkylated with 6 mM iodoacetamide, and the proteins were digested by trypsin (sequence-grade modified trypsin, Promega, at 1 mg per 50 mg of sample protein for 24 h at 37°C). The generated peptides were separated from the plasma membranes by centrifugation at 170,000 ϫ g for 1 h.
Phosphopeptides were enriched by IMAC 2 (immobilized metal affinity chromatography) (16) as modified in Ref. 17. The peptides were methyl-esterified by 2 M methanolic HCl and then subjected to IMAC using a microcolumn (GELoader tip; Eppendorf, Hamburg, Germany) containing 7 l of chelating Sepharose (Amersham Biosciences, Uppsala, Sweden) loaded with FeCl 3 . The methyl-esterified peptides were dried and dissolved in 10 l of methanol/water/acetonitrile (1:1:1, by volume) before loading on the column. Non-specifically bound peptides were removed with 2 ϫ 20 l of 0.1% acetic acid in water, 2 ϫ 20 l of 0.1% acetic acid in 20% (v/v) acetonitrile, and 2 ϫ 20 * This work was supported by the Ö stergö tland County Council, the Novo Nordisk Foundation, the Swedish Diabetes Association, the Graduate Research School in Genomics and Bioinformatics (Forskarskolan fö r Genomik och Bioinformatik), and the Swedish Research Council. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 To whom correspondence should be addressed. l of 20% acetonitrile in water. Phosphopeptides were eluted by four washings with 10 l of 20 mM Na 2 HPO 4 in 20% acetonitrile. The four eluted fractions were separately collected and desalted using a C 18 ZipTip (Millipore, Bedford, MA). Mass Spectrometry-The phosphopeptides were analyzed on a hybrid mass spectrometer API Q-STAR Pulsar i (Applied Biosystems, Foster City, CA) equipped with a nanoelectrospray ion source (MDS Protana, Odense, Denmark). The desalted peptides (2 l in 50% acetonitrile in water with 1% formic acid) were loaded into the nanoelectrospray capillaries. Mass spectra of the phosphopeptides fragmented by collision-induced dissociation were acquired with instrument settings recommended by Applied Biosystems with manual control of collision energy.

Identification of Phosphorylated Proteins in the Plasma Membrane of
Human Adipocytes-To identify phosphoproteins in the plasma membrane we subjected purified plasma membranes to proteolytic treatment with trypsin, which cleaves surface exposed domains of the membrane proteins (13,17,18). Generated peptides were separated from remaining membrane proteins by centrifugation and the phosphorylated peptides were then enriched by an IMAC procedure (16). The enriched phosphopeptides were sequenced by nanospray quadrupole time-of-flight mass spectrometry. We identified 26 phosphorylation sites in 10 proteins in the plasma membrane (Table 1). Serine phosphorylation at residues 5 and 37 of caveolin 1␣ and 1␤ isoforms in caveolae has previously been reported by us (17). Here we report that caveolin 2 was phosphorylated at serine 20. The cytoskeleton-binding proteins talin 2 and spectrin II␤ were phosphorylated on threonine 1843 and serine 2089, respectively. Novel phosphorylation sites were also identified in the signal transduction proteins Cbl-associated protein (CAP), protein kinase C␦-binding protein (SRBC), and protein kinase C␣-binding protein (SDPR). SDPR was phosphorylated on one threonine and three serine residues located in the same peptide (Table 1). In addition to four previously identified serine phosphorylation sites in polymerase I and transcript release factor (PTRF) (Ser-36, Ser-40, Ser-365, and Ser-366 (13)), we identified another five novel serine phosphorylation sites and one threonine phosphorylation in this protein that is associated with caveolae domains of the plasma membrane.
In the protein perilipin, known to be multiply serine phosphorylated (9), we found a tight cluster of threonine phosphorylation at residues 299, 301, and 306 as revealed by fragmentation and sequencing of the corresponding triply phosphorylated peptide (Fig. 1, Table 1). Perilipin A-containing bands after SDS-PAGE have been reported to contain phosphothreonine, but no phosphorylation sites have been mapped earlier (7).

Identification of Threonine-phosphorylated Perilipin in the Plasma
Membrane-To examine the effect of insulin on the phosphorylation of the identified phosphoproteins, we isolated plasma membranes from human adipocytes incubated with or without insulin and the protein phosphorylation was examined by immunoblotting. Antibodies against phosphotyrosine identified the insulin receptor ␤-subunit in cells treated with insulin, but insulin treatment had no detectable effect on serine phosphorylation as examined with anti-phosphoserine antibodies (data not shown). Antibodies against phosphothreonine, on the other hand, identified four proteins that were extensively phosphorylated in the plasma membrane of cells treated with insulin ( Fig. 2A).
We examined whether the phosphoproteins PTRF or perilipin, in which we identified threonine-phosphorylated residues (Table 1), corresponded to the major phosphothreonine protein bands. Immunoblotting analysis with antibodies against PTRF did not match any of the phosphothreonine protein bands in Fig. 2A (data not shown), whereas immunoblotting with anti-perilipin antibodies demonstrated that threonine-phosphorylated bands corresponded to perilipin proteins (Fig. 3). Perilipin B was the major isoform in the plasma membrane (Fig. 2B), while perilipin A was the dominant isoform (Ͼ90%) in whole cell lysates of the human adipocytes (Fig. 2B), in agreement with a previous report that more than 85% of total perilipin in rat adipocytes is perilipin A (9). This very marked enrichment of perilipin B at the plasma membrane indicates a specific function of this isoform in the plasma membrane.
Effect of Insulin or Isoproterenol-We examined the effect of insulin or the ␤-adrenergic agonist isoproterenol on perilipin in the plasma membrane. Adipocytes were incubated with insulin or isoproterenol and the plasma membranes were isolated. Immunoblotting with anti-perilipin antibodies revealed that insulin recruited threonine-phosphorylated perilipin B and perilipin A to the plasma membrane (Fig. 2B). The increase of perilipin B was 11.0 Ϯ 5.9-fold and of perilipin A 11.2 Ϯ 4.8-fold (mean Ϯ S.E., n ϭ 3) in the plasma membrane in response to insulin treatment of the intact adipocytes. Isoproterenol, on the other hand, induced a very marked and specific depletion of perilipin B from the plasma membrane, apparently without affecting perilipin A, which remained in the plasma membrane (Fig. 4A). The migration shift of perilipin A in the gel after separation by SDS-PAGE is likely the result of increased serine phosphorylation in response to isoproterenol stimulation, as has been demonstrated in whole cells (5).
When whole cell lysates were examined for effects of insulin or isoproterenol there was no detectable over-all effect of either hormone on the state of threonine-phosphorylation of perilipin B ( Figs. 2A and 4B), while insulin reduced the threonine-phosphorylation of perilipin A (Fig. 2A). Also there was no significant effect on the amount of perilipin in the cells by either hormone (Figs. 2B and 4A).

TABLE 1 Identification of phophoproteins in the plasma membrane of human adipocytes
The phosphopetides obtained by IMAC enrichment after cleavage from the plasma membranes by trypsin were subjected to collision induced fragmentation mass spectrometry. The peptide sequences obtained from the spectral data are listed below along with names of the proteins that the peptides originate from. The superscript numbers correspond to the amino acid position of peptides in the sequences of the corresponding protein. Lowercase boldface s and t indicate phosphorylation of serine and threonine.

DISCUSSION
In the present study we applied a proteomics approach (13) to study protein phosphorylation in the plasma membrane of human adipocytes. This allowed us to identify novel phosphorylation sites in a number of different proteins located at the plasma membrane, in particular threonine phosphorylation of perilipin. Key findings of this investigation were: first, that perilipin is triply phosphorylated on a tight cluster of threonine residues; second, that perilipin B is specifically associated with the plasma membrane of human adipocytes; and third, that this association is under insulin and ␤-adrenergic control.
Insulin increased the amount of perilipin A and B at the plasma membrane, while perilipin B was depleted at the plasma membrane in response to ␤-adrenergic stimulation. Our findings also suggest that the translocation of perilipin to the plasma membrane in response to insulin may be threonine phosphorylation-dependent. It has been shown that a central domain of perilipin consisting of amino acid residues 233-364 is important for targeting of perilipin to the central lipid droplet (19). The domain structure of perilipin is outlined in Fig. 5. Mass spectrometry analysis of protein phosphorylation in the plasma membrane revealed three phosphorylated threonine residues (299, 301, and 306) tightly clustered within this central domain of perilipin (Fig. 5). This cluster of phosphorylated threonine residues was also within an acidic domain (amino acids 292-319), which forms part of the lipid droplet targeting domain (19). The acidic character of this domain is further increased by introduction of the negative charges contributed by the threonine phosphorylation identified here. It remains to determine the function of such enhanced negative charge of the acidic domain in controlling perilipin targeting.
We have recently identified perilipin at the plasma membrane of primary rat adipocytes and demonstrated that perilipin is located to caveolae in insulintreated adipocytes and that added long-chain fatty acids are rapidly converted to triacylglycerol in the membrane of the perilipin-containing caveolae (12). Considering the established function of perilipin A to protect the central lipid FIGURE 1. Multiple threonine phosphorylation of perilipin at the plasma membrane. Phosphopeptide from perilipin was obtained by IMAC enrichment after cleavage from the plasma membranes by trypsin. The collision-induced fragmentation mass spectrum of the triply charged ion with m/z 1424.5 is shown. The peptide sequence obtained from the spectral data corresponds to perilipin with three phosphorylation sites on threonine residues 299, 301, and 306, as indicated in Table 1. The lowercase t in the sequence indicates phosphorylated threonine residues. The fragment y (C-terminal) and b (N-terminal) ions with charge state higher than 1ϩ are marked with corresponding superscript numbers. Asterisks indicate the fragments produced after neutral loss of H 3 PO 4 (98 Da). FIGURE 2. Effects of insulin on perilipin at the plasma membrane and in whole cell lysates of human adipocytes. Human adipocytes were incubated with or without 100 nM insulin, as indicated, and plasma membranes were isolated or whole cell lysates prepared, as indicated. Aliquots of 5 g of plasma membrane protein or equal amounts of cells were subjected to SDS-PAGE and immunoblotted with antibodies against phosphothreonine (P-Thr) (A) or perilipin (B). FIGURE 3. Identification of perilipin A and B in the plasma membrane as threoninephosphorylated proteins. 5 g of protein of the plasma membrane, isolated from primary human adipocytes incubated with insulin, was subjected to SDS-PAGE, and immunoblotting was performed with two antibodies after cutting the lane into two parts. One part was blotted with anti-perilipin and the other with anti-phosphothreonine antibody (P-Thr) as indicated. Perilipin A (Peri A) and perilipin B (Peri B) were identified by their relative electrophoretic mobilities (9,21). droplet triacylglycerol from hydrolysis in the absence of lipolytic stimulation, it is likely that perilipin B is recruited to the plasma membrane in response to insulin to protect the freshly synthesized triacylglycerol (12) from hydrolysis by the hormone-sensitive lipase. This lipase was found to localize to the triacylglycerol-synthesizing caveolae specifically. 3 The finding that lipolytic stimulation with isoproterenol specifically and completely depleted the plasma membrane of perilipin B also indicates such a function of perilipin B. In rat adipocytes almost 85% of the cellular perilipin has been reported to be the A isoform with the reminder constituting perilipin B (9). In the human adipocytes very little perilipin B (Ͻ10% of total perilipin) was detected in the whole cell lysates (Figs. 2B and 4A), demonstrating a very strong dominance of the A isoform in the human cells. The exact proportion of perilipin B to perilipin A at the plasma membrane was found to vary somewhat between preparations from different individuals, but perilipin B was invariably highly enriched at the plasma membrane compared with whole cells. With this in mind there is a remarkable preference for perilipin B in the plasma membrane supporting its specific function at the plasma membrane.
It is possible that the serine-phosphorylated perilipin A remaining in the plasma membrane after lipolytic activation by isoproterenol reflects a function of serine-phosphorylated perilipin A to support the hydrolysis of triacylglycerol in the plasma membrane, in agreement with a study showing that in perilipin null mice the hormone-sensitive lipase failed to translocate from the cytosol to the lipid droplet after stimulation of lipolysis but after introduction of perilipin A hormone-sensitive lipase was translocated to the lipid droplet (20). Furthermore, lipolytic stimulation of serine phosphorylation of perilipin A has been described to enhance the activity of hormone-sensitive lipase (10).
In conclusion, our findings suggest that perilipin B protects the triacylglycerol within the plasma membrane from being hydrolyzed during insulin-stimulated fatty acid uptake and triacylglycerol synthesis. The translocation of perilipin B to the plasma membrane in response to insulin may be threonine phosphorylation-dependent. Stimulation of lipolysis with isoproterenol caused translocation of perilipin B away from the plasma membrane, presumably to allow lipolytic access to lipases such as hormone-sensitive lipase. . Effects of isoproterenol on perilipin at the plasma membrane and in whole cell lysates of human adipocytes. Human adipocytes were incubated with or without 100 nM isoproterenol, as indicated, and plasma membranes were isolated or whole cell lysates prepared, as indicated. Aliquots of 5 g of plasma membrane protein or equal amounts of cells were subjected to SDS-PAGE and immunoblotted with antibodies against perilipin (A) or phosphothreonine (P-Thr) (B). Black boxes correspond to unique amino acid sequences. The positions of the predicted cAMP-dependent protein kinase serine phosphorylation sites (9) are indicated by S in the sequence of human perilipin A and B. The position of the identified threonine phosphorylation cluster is indicated by three vertical lines and T in perilipin B.