The Phosphorylation of Serine 492 of Perilipin A Directs Lipid Droplet Fragmentation and Dispersion

Perilipin A is a key regulator of triacylglycerol storage and hydrolysis in adipocytes; phosphorylation of perilipin A by protein kinase A facilitates maximal lipolysis. Chronic stimulation of lipolysis in 3T3-L1 adipocytes causes large perinuclear lipid droplets to fragment into myriad dispersed perilipin A-covered microlipid droplets. In cultured fibroblasts stably expressing ectopic perilipin A, clustered lipid droplets disperse throughout the cytoplasm upon incubation of the cells with forskolin and isobutylmethylxanthine (IBMX) to elevate levels of cAMP and activate protein kinase A, mirroring events observed in adipocytes. Furthermore, diethylum-belliferyl phosphate inhibits stimulated lipolysis but not the dispersion of lipid droplets, suggesting that products of lipolysis are not required for this remodeling process. We hypothesized that protein kinase A-mediated phosphorylation of perilipin A triggers the remodeling of lipid droplets. The mutation of serine 492 of perilipin A to alanine prevented the dispersion of clustered lipid droplets in fibroblasts stably expressing the mutated perilipin upon incubation with forskolin and IBMX. In contrast, the substitution of serines 81, 222, 276, or 433 with alanine, either singly or in combinations, did not affect the protein kinase A-mediated remodeling of lipid droplets. Interestingly, substitution of serines 433, 492, and 517 of perilipin A with glutamic acid residues blocked the dispersion of clustered lipid droplets in cells incubated with forskolin and IBMX, indicating that the addition of a negative charge does not mimic a phosphate group. We conclude that protein kinase A-mediated phosphorylation of serine 492 of perilipin A drives the fragmentation and dispersion of lipid droplets. Perilipin A is a key regulator of triacylglycerol storage and hydrolysis in adipocytes; phosphorylation of perilipin A by protein kinase A facilitates maximal lipolysis. Chronic stimulation of lipolysis in 3T3-L1 adipocytes causes large perinuclear lipid droplets to fragment into myriad dispersed perilipin A-covered microlipid droplets. In cultured fibroblasts stably expressing ectopic perilipin A, clustered lipid droplets disperse throughout the cytoplasm upon incubation of the cells with forskolin and isobutylmethylxanthine (IBMX) to elevate levels of cAMP and activate protein kinase A, mirroring events observed in adipocytes. Furthermore, diethylum-belliferyl phosphate inhibits stimulated lipolysis but not the dispersion of lipid droplets, suggesting that products of lipolysis are not required for this remodeling process. We hypothesized that protein kinase A-mediated phosphorylation of perilipin A triggers the remodeling of lipid droplets. The mutation of serine 492 of perilipin A to alanine prevented the dispersion of clustered lipid droplets in fibroblasts stably expressing the mutated perilipin upon incubation with forskolin and IBMX. In contrast, the substitution of serines 81, 222, 276, or 433 with alanine, either singly or in combinations, did not affect the protein kinase A-mediated remodeling of lipid droplets. Interestingly, substitution of serines 433, 492, and 517 of perilipin A with glutamic acid residues blocked the dispersion of clustered lipid droplets in cells incubated with forskolin and IBMX, indicating that the addition of a negative charge does not mimic a phosphate group. We conclude that protein kinase A-mediated phosphorylation of serine 492 of perilipin A drives the fragmentation and dispersion of lipid droplets. Adipose tissue contains the largest energy reserve in the body, stored as triacylglycerol in the intracellular lipid droplets of adipocytes. Triacylglycerol stores are hydrolyzed to mobilize energy during fasting and extended exercise. Catecholamines bind to β-adrenergic receptors on the plasma membranes of adipocytes to initiate a G-protein-mediated signaling cascade that activates adenylyl cyclase, thus increasing intracellular cAMP levels. Protein kinase A (or cAMP-dependent protein kinase) is then activated and phosphorylates several proteins required for the hydrolysis of triacylglycerol and consequent mobilization of fatty acids. Following phosphorylation by protein kinase A, hormone-sensitive lipase translocates from the cytosol onto lipid droplets (1Brasaemle D.L. Levin D.M. Adler-Wailes D.C. Londos C. Biochim. Biophys. Acta. 2000; 1483: 251-262Crossref PubMed Scopus (186) Google Scholar, 2Egan J.J. Greenberg A.S. Chang M.K. Wek S.A. Moos Jr., M.C. Londos C. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8537-8541Crossref PubMed Scopus (345) Google Scholar, 3Clifford G.M. Londos C. Kraemer F.B. Vernon R.G. Yeaman S.J. J. Biol. Chem. 2000; 275: 5011-5015Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 4Sztalryd C. Xu G. Dorward H. Tansey J.T. Contreras J.A. Kimmel A.R. Londos C. J. Cell Biol. 2003; 161: 1093-1103Crossref PubMed Scopus (415) Google Scholar, 5Su C.L. Sztalryd C. Contreras J.A. Holm C. Kimmel A.R. Londos C. J. Biol. Chem. 2003; 278: 43615-43619Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar), where it hydrolyzes triacylglycerol and diacylglycerol (6Holm C. Biochem. Soc. Trans. 2003; 31: 1120-1124Crossref PubMed Scopus (0) Google Scholar, 7Kraemer F.B. Shen W.J. J. Lipid Res. 2002; 43: 1585-1594Abstract Full Text Full Text PDF PubMed Scopus (371) Google Scholar, 8Yeaman S.J. Biochem. J. 2004; 379: 11-22Crossref PubMed Scopus (195) Google Scholar). Perilipin A is another major substrate for protein kinase A (9Greenberg A.S. Egan J.J. Wek S.A. Garty N.B. Blanchette-Mackie E.J. Londos C. J. Biol. Chem. 1991; 266: 11341-11346Abstract Full Text PDF PubMed Google Scholar, 10Greenberg A.S. Egan J.J. Wek S.A. Moos Jr., M.C. Londos C. Kimmel A.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 12035-12039Crossref PubMed Scopus (211) Google Scholar). Perilipin A localizes to lipid droplets in adipocytes and plays roles in facilitating both the storage and hydrolysis of triacylglycerol. In adipocytes, two isoforms of perilipin are derived from the alternate splicing of mRNA transcribed from a single gene (10Greenberg A.S. Egan J.J. Wek S.A. Moos Jr., M.C. Londos C. Kimmel A.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 12035-12039Crossref PubMed Scopus (211) Google Scholar, 11Lu X. Gruia-Gray J. Copeland N.G. Gilbert D.J. Jenkins N.A. Londos C. Kimmel A.R. Mamm. Genome. 2001; 12: 741-749Crossref PubMed Scopus (182) Google Scholar); perilipin A is the predominant protein isoform, whereas perilipin B is a minor isoform. Perilipins A and B share a common sequence through the first 405 amino acids of the amino terminus that contains three consensus sequences for protein kinase A; perilipin A has a unique carboxyl terminus with three additional consensus sequences for protein kinase A (10Greenberg A.S. Egan J.J. Wek S.A. Moos Jr., M.C. Londos C. Kimmel A.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 12035-12039Crossref PubMed Scopus (211) Google Scholar, 11Lu X. Gruia-Gray J. Copeland N.G. Gilbert D.J. Jenkins N.A. Londos C. Kimmel A.R. Mamm. Genome. 2001; 12: 741-749Crossref PubMed Scopus (182) Google Scholar). In adipocytes, perilipin A forms a barrier at the surfaces of lipid droplets that restricts the access of cytosolic lipases (12Brasaemle D.L. Rubin B. Harten I.A. Gruia-Gray J. Kimmel A.R. Londos C. J. Biol. Chem. 2000; 275: 38486-38493Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar, 13Tansey J.T. Sztalryd C. Gruia-Gray J. Roush D.L. Zee J.V. Gavrilova O. Reitman M.L. Deng C.X. Li C. Kimmel A.R. Londos C. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6494-6499Crossref PubMed Scopus (601) Google Scholar, 14Martinez-Botas J. Anderson J.B. Tessier D. Lapillonne A. Chang B.H. Quast M.J. Gorenstein D. Chen K.H. Chan L. Nat. Genet. 2000; 26: 474-479Crossref PubMed Scopus (488) Google Scholar, 15Souza S.C. de Vargas L.M. Yamamoto M.T. Lien P. Franciosa M.D. Moss L.G. Greenberg A.S. J. Biol. Chem. 1998; 273: 24665-24669Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar, 16Tansey J.T. Huml A.M. Vogt R. Davis K.E. Jones J.M. Fraser K.A. Brasaemle D.L. Kimmel A.R. Londos C. J. Biol. Chem. 2003; 278: 8401-8406Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 17Garcia A. Subramanian V. Sekowski A. Bhattacharyya S. Love M.W. Brasaemle D.L. J. Biol. Chem. 2004; 279: 8409-8416Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar), including hormone-sensitive lipase (18Souza S.C. Muliro K.V. Liscum L. Lien P. Yamamoto M.T. Schaffer J.E. Dallal G.E. Wang X. Kraemer F.B. Obin M. Greenberg A.S. J. Biol. Chem. 2002; 277: 8267-8272Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, 19Zhang H.H. Souza S.C. Muliro K.V. Kraemer F.B. Obin M.S. Greenberg A.S. J. Biol. Chem. 2003; 278: 51535-51542Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar), to the lipid droplet, thus promoting triacylglycerol storage under basal conditions. Consequently, perilipin null mice have a dramatically reduced mass of triacylglycerol in adipose tissue when compared with wild-type mice, and adipocytes isolated from perilipin null mice have a significantly elevated rate of basal lipolysis when compared with that of adipocytes isolated from wild-type mice (13Tansey J.T. Sztalryd C. Gruia-Gray J. Roush D.L. Zee J.V. Gavrilova O. Reitman M.L. Deng C.X. Li C. Kimmel A.R. Londos C. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6494-6499Crossref PubMed Scopus (601) Google Scholar, 14Martinez-Botas J. Anderson J.B. Tessier D. Lapillonne A. Chang B.H. Quast M.J. Gorenstein D. Chen K.H. Chan L. Nat. Genet. 2000; 26: 474-479Crossref PubMed Scopus (488) Google Scholar). Perilipin B does not protect stored triacylglycerol from hydrolysis by cytosolic lipases (16Tansey J.T. Huml A.M. Vogt R. Davis K.E. Jones J.M. Fraser K.A. Brasaemle D.L. Kimmel A.R. Londos C. J. Biol. Chem. 2003; 278: 8401-8406Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 19Zhang H.H. Souza S.C. Muliro K.V. Kraemer F.B. Obin M.S. Greenberg A.S. J. Biol. Chem. 2003; 278: 51535-51542Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar), suggesting that the unique 112-amino-acid carboxyl terminus of perilipin A is critical for this barrier function. Furthermore, studies with truncated forms of perilipin A have shown that sequences in both the amino and carboxyl termini are required for the protection of triacylglycerol stores (17Garcia A. Subramanian V. Sekowski A. Bhattacharyya S. Love M.W. Brasaemle D.L. J. Biol. Chem. 2004; 279: 8409-8416Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). When cAMP levels rise in adipocytes, perilipin A is phosphorylated by protein kinase A (9Greenberg A.S. Egan J.J. Wek S.A. Garty N.B. Blanchette-Mackie E.J. Londos C. J. Biol. Chem. 1991; 266: 11341-11346Abstract Full Text PDF PubMed Google Scholar) on as many as six serines (10Greenberg A.S. Egan J.J. Wek S.A. Moos Jr., M.C. Londos C. Kimmel A.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 12035-12039Crossref PubMed Scopus (211) Google Scholar, 11Lu X. Gruia-Gray J. Copeland N.G. Gilbert D.J. Jenkins N.A. Londos C. Kimmel A.R. Mamm. Genome. 2001; 12: 741-749Crossref PubMed Scopus (182) Google Scholar); the barrier function of perilipin A is attenuated, and lipolysis increases. Perilipin null mice show a blunted lipolytic response to β-adrenergic agonists (13Tansey J.T. Sztalryd C. Gruia-Gray J. Roush D.L. Zee J.V. Gavrilova O. Reitman M.L. Deng C.X. Li C. Kimmel A.R. Londos C. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6494-6499Crossref PubMed Scopus (601) Google Scholar, 14Martinez-Botas J. Anderson J.B. Tessier D. Lapillonne A. Chang B.H. Quast M.J. Gorenstein D. Chen K.H. Chan L. Nat. Genet. 2000; 26: 474-479Crossref PubMed Scopus (488) Google Scholar), indicating that phosphorylated perilipin plays a critical role in facilitating maximal lipolysis. Phosphorylation of one or more of three amino-terminal protein kinase A sites of perilipin A is required to assist docking of hormone-sensitive lipase onto lipid droplets (4Sztalryd C. Xu G. Dorward H. Tansey J.T. Contreras J.A. Kimmel A.R. Londos C. J. Cell Biol. 2003; 161: 1093-1103Crossref PubMed Scopus (415) Google Scholar) and to obtain maximal lipolysis (18Souza S.C. Muliro K.V. Liscum L. Lien P. Yamamoto M.T. Schaffer J.E. Dallal G.E. Wang X. Kraemer F.B. Obin M. Greenberg A.S. J. Biol. Chem. 2002; 277: 8267-8272Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, 19Zhang H.H. Souza S.C. Muliro K.V. Kraemer F.B. Obin M.S. Greenberg A.S. J. Biol. Chem. 2003; 278: 51535-51542Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Additionally, the protein kinase A-mediated phosphorylation of ectopic perilipin A on both amino and carboxyl-terminal consensus sites facilitates lipolysis in fibroblasts that do not express hormone-sensitive lipase (16Tansey J.T. Huml A.M. Vogt R. Davis K.E. Jones J.M. Fraser K.A. Brasaemle D.L. Kimmel A.R. Londos C. J. Biol. Chem. 2003; 278: 8401-8406Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 18Souza S.C. Muliro K.V. Liscum L. Lien P. Yamamoto M.T. Schaffer J.E. Dallal G.E. Wang X. Kraemer F.B. Obin M. Greenberg A.S. J. Biol. Chem. 2002; 277: 8267-8272Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, 19Zhang H.H. Souza S.C. Muliro K.V. Kraemer F.B. Obin M.S. Greenberg A.S. J. Biol. Chem. 2003; 278: 51535-51542Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar), suggesting that phosphorylated perilipin A promotes lipolysis by other cytosolic lipases that may include the recently identified adipose triglyceride lipase, also called desnutrin (20Jenkins C.M. Mancuso D.J. Yan W. Sims H.F. Gibson B. Gross R.W. J. Biol. Chem. 2004; 279: 48968-48975Abstract Full Text Full Text PDF PubMed Scopus (673) Google Scholar, 21Villena J.A. Roy S. Sarkadi-Nagy E. Kim K.H. Sul H.S. J. Biol. Chem. 2004; 279: 47066-47075Abstract Full Text Full Text PDF PubMed Scopus (502) Google Scholar, 22Zimmermann R. Strauss J.G. Haemmerle G. Schoiswohl G. Birner-Gruenberger R. Riederer M. Lass A. Neuberger G. Eisenhaber F. Hermetter A. Zechner R. Science. 2004; 306: 1383-1386Crossref PubMed Scopus (1486) Google Scholar). Clearly, there are uncharacterized mechanisms by which phosphorylated perilipin A facilitates lipolysis. Continuous stimulation of β-adrenergic receptors over several hours induces the large perinuclear lipid droplets of 3T3-L1 adipocytes to fragment into microlipid droplets and disperse throughout the cytoplasm (23Londos C. Brasaemle D.L. Schultz C.J. Adler-Wailes D.C. Levin D.M. Kimmel A.R. Rondinone C.M. Ann. N. Y. Acad. Sci. 1999; 892: 155-168Crossref PubMed Scopus (219) Google Scholar, 24Brasaemle D.L. Dolios G. Shapiro L. Wang R. J. Biol. Chem. 2004; 279: 46835-46842Abstract Full Text Full Text PDF PubMed Scopus (626) Google Scholar, 25Londos C. Brasaemle D.L. Schultz C.J. Segrest J.P. Kimmel A.R. Semin. Cell Dev. Biol. 1999; 10: 51-58Crossref PubMed Scopus (365) Google Scholar). A similar remodeling of lipid droplets has been observed in rat adipocytes following infusion of an agonist for β-3 adrenergic receptors into a subcutaneous fat pad or electroporation of cDNA encoding a constitutively active β-1 adrenergic receptor into the fat pads of live rats (26Granneman J.G. Li P. Lu Y. Tilak J. Am. J. Physiol. 2004; 287: E574-E582Crossref PubMed Scopus (34) Google Scholar). The mechanisms that promote the remodeling of lipid droplets in adipocytes are uncharacterized. In this study, we test the hypothesis that the fragmentation and dispersion of lipid droplets in lipolytically stimulated adipocytes is triggered by the phosphorylation of perilipin A by protein kinase A. Materials—Dulbecco's modified Eagle's medium and geneticin were obtained from Mediatech, Inc. (Herndon, VA). Forskolin, 3-isobutyl-1-methylxanthine (IBMX), 2The abbreviations used are: IBMX, 3-isobutyl-1-methylxanthine; DEUP, diethylumbelliferyl phosphate; BSA, bovine serum albumin. fetal bovine serum, and goat anti-rabbit immunoglobulin G peroxidase conjugate were purchased from Sigma. Fatty acid-free bovine serum albumin (BSA) was purchased from either Sigma or Biocell Laboratories, Inc. (Rancho Dominguez, CA). Diethylumbelliferyl phosphate (DEUP) was synthesized by Chem-Master International, Inc. (Stony Brook, NY). Triacsin C was obtained from BIOMOL Research Laboratories, Inc. (Plymouth Meeting, PA). Alexa Fluor 546-conjugated goat anti-rabbit IgG and Bodipy 493/503 were purchased from Molecular Probes, Inc. (Eugene, OR; catalog number D-3922). Rabbit anti-phosphoserine IgG was purchased from Research Diagnostics, Inc. (Flanders, NJ; catalog designation RDI-PHOSSERabr), and rabbit anti-phospho-(Ser/Thr) protein kinase A substrate IgG was purchased from Cell Signaling Technology, Inc. (Beverly, MA; catalog number 9621). A polyclonal antibody raised against calnexin was purchased from StressGen Biotechnologies Corp. (Victoria, BC, Canada). Polyclonal antibodies raised against adipose fatty acid-binding protein were kindly donated by Dr. Judith Storch (Rutgers University, New Brunswick, NJ). Antiserum raised against an amino-terminal peptide of mouse adipophilin (27Schultz C.J. Torres E. Londos C. Torday J.S. Am. J. Physiol. 2002; 283: L288-L296Crossref PubMed Scopus (102) Google Scholar) was kindly donated by Dr. Constantine Londos (National Institutes of Health, Bethesda, MD). Pfu DNA polymerase was purchased from Stratagene, Inc. (La Jolla, CA). Cell Culture—For most experiments, 3T3-L1 preadipocytes were used as a model of fibroblasts and were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mm glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin, as described previously (12Brasaemle D.L. Rubin B. Harten I.A. Gruia-Gray J. Kimmel A.R. Londos C. J. Biol. Chem. 2000; 275: 38486-38493Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar). 3T3-L1 fibroblasts stably expressing intact perilipin A delivered by a retroviral expression system, or the retroviral expression vector without a cDNA insert (control cells), were maintained in 0.6 mg/ml geneticin (12Brasaemle D.L. Rubin B. Harten I.A. Gruia-Gray J. Kimmel A.R. Londos C. J. Biol. Chem. 2000; 275: 38486-38493Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar). Confocal Microscopy of Adipocytes—Monolayers of 3T3-L1 preadipocytes were grown in culture medium with 8 μg/ml biotin, and upon reaching confluence, were induced to differentiate into adipocytes by the daily addition of 10 μg/ml insulin, 10 μm dexamethasone, and 0.5 mm IBMX in fresh culture medium for 3 days, followed by the removal of these agents and the daily addition of fresh culture medium without additives (28Brasaemle D.L. Barber T. Wolins N.E. Serrero G. Blanchette-Mackie E.J. Londos C. J. Lipid Res. 1997; 38: 2249-2263Abstract Full Text PDF PubMed Google Scholar). Six days following the initiation of differentiation, the cells were rinsed with phosphate-buffered saline, released with 0.25% trypsin and 1 mm EDTA, and seeded into culture dishes containing glass coverslips; cells from one 100-mm culture dish were seeded into three or four new 100-mm dishes. The cells were cultured for 24 h and then incubated in culture medium, either without additives or with 10 μm isoproterenol and 0.5 mm IBMX for 2, 4, or 8 h before fixation with 3% paraformaldehyde in phosphate-buffered saline, and prepared for microscopy (29Blanchette-Mackie E.J. Dwyer N.K. Barber T. Coxey R.A. Takeda T. Rondinone C.M. Theodorakis J.L. Greenberg A.S. Londos C. J. Lipid Res. 1995; 36: 1211-1226Abstract Full Text PDF PubMed Google Scholar). Fixed cells were probed with polyclonal antibodies raised against a recombinant amino-terminal peptide of perilipin A (28Brasaemle D.L. Barber T. Wolins N.E. Serrero G. Blanchette-Mackie E.J. Londos C. J. Lipid Res. 1997; 38: 2249-2263Abstract Full Text PDF PubMed Google Scholar) followed by Alexa Fluor 546-conjugated goat anti-rabbit IgG and Bodipy 493/503 to detect neutral lipids (30Gocze P.M. Freeman D.A. Cytometry. 1994; 17: 151-158Crossref PubMed Scopus (176) Google Scholar). Images of cells were captured in monochrome using a Zeiss LSM510 Meta confocal laser scanning microscope and processed using a Zeiss LSM image browser. Subcellular Fractionation of Adipocytes on Sucrose Gradients—Confluent 3T3-L1 preadipocytes in 100-mm culture dishes were induced to differentiate for 6 days, and adipocytes were then incubated in control medium or medium containing 10 μm isoproterenol and 0.5 mm IBMX for 6 h. Cells were harvested, lysed, and fractionated, as described previously (31Wolins N.E. Rubin B. Brasaemle D.L. J. Biol. Chem. 2001; 276: 5101-5108Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar). Briefly, post-mitochondrial supernatants were adjusted to 35% sucrose using a solution of 70% sucrose and then layered over 1-ml 50% sucrose cushions. Thereafter, 8 ml of a 0-30% linear sucrose gradient were layered over each sample, and gradients were centrifuged at 154,000 × g in a Beckman SW41Ti rotor for 4 h at 4°C. The floating lipid droplet fractions were collected by slicing off the top portions of the tubes with a Beckman tube slicer. The remainder of each gradient was collected in 1-ml fractions. Proteins from equivalent volumes of each fraction were separated by SDS-PAGE, transferred to nitrocellulose membranes, and probed with polyclonal antibodies raised against perilipin (28Brasaemle D.L. Barber T. Wolins N.E. Serrero G. Blanchette-Mackie E.J. Londos C. J. Lipid Res. 1997; 38: 2249-2263Abstract Full Text PDF PubMed Google Scholar), calnexin, and adipose fatty acid-binding protein, followed by horseradish peroxidase-conjugated secondary antibodies. Bound antibodies were detected using enhanced chemiluminescence reagents (Amersham Biosciences). Expression of Perilipin A cDNA Containing Alanine or Glutamic Acid Substitutions for Serines within Protein Kinase A Consensus Sequences— Perilipin A cDNA was amplified using polymerase chain reaction with Pfu polymerase and oligonucleotide primers complementary to the 5′ and 3′ ends of the coding sequence of perilipin A. Point mutations were introduced into the six protein kinase A consensus sequences using internal primers complementary to 20-36 nucleotides flanking and including the sequence encoding each serine; the codon for serine was altered to encode either alanine or glutamic acid. The mutated forms of the perilipin A cDNA were ligated into the unique HindIII site of the pSRαMSVtkneo retroviral expression vector (32Muller A.J. Young J.C. Pendergast A.M. Pondel M. Landau N.R. Littman D.R. Witte O.N. Mol. Cell. Biol. 1991; 11: 1785-1792Crossref PubMed Scopus (354) Google Scholar). All mutations were confirmed by sequencing the inserted cDNA. The retrovirus for each mutated form of perilipin A was packaged in 293T cells and used to transduce 3T3-L1 fibroblasts (12Brasaemle D.L. Rubin B. Harten I.A. Gruia-Gray J. Kimmel A.R. Londos C. J. Biol. Chem. 2000; 275: 38486-38493Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar). 3T3-L1 fibroblasts stably expressing the mutated forms of perilipin A were selected by growth in 0.6 mg/ml geneticin (12Brasaemle D.L. Rubin B. Harten I.A. Gruia-Gray J. Kimmel A.R. Londos C. J. Biol. Chem. 2000; 275: 38486-38493Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar, 33Garcia A. Sekowski A. Subramanian V. Brasaemle D.L. J. Biol. Chem. 2003; 278: 625-635Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Incubation of 3T3-L1 Fibroblasts to Study Lipid Droplet Morphology and Lipolysis—Prior to experiments, 3T3-L1 fibroblasts stably expressing either perilipin A, a mutated form of perilipin A, or the retroviral expression vector without an added cDNA sequence (control cells) were incubated with culture medium containing oleic acid complexed to fatty acid-free BSA at a 6:1 molar ratio (12Brasaemle D.L. Rubin B. Harten I.A. Gruia-Gray J. Kimmel A.R. Londos C. J. Biol. Chem. 2000; 275: 38486-38493Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar) for 16-18 h (lipid-loading). The cells were incubated with 200 μm oleate for fluorescence microscopy experiments; for all other experiments, the cells were incubated with 400-600 μm oleate. The latter lipid-loading conditions were selected to fully stabilize ectopic perilipin A (34Brasaemle D.L. Barber T. Kimmel A.R. Londos C. J. Biol. Chem. 1997; 272: 9378-9387Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar) but also increased protein levels of endogenous adipophilin on lipid droplets. To increase intracellular levels of cAMP, 10 μm forskolin in 0.1% dimethyl sulfoxide (Me2SO) and 0.5 mm IBMX were added to the cells in fresh culture medium. In some experiments, the cells were incubated with 800 μm DEUP added in 0.5% Me2SO without further additions or for 1 h prior to and during incubation with 10 μm forskolin and 0.5 mm IBMX. For each experiment, all cells were incubated with the same concentration of Me2SO. Fluorescence Microscopy of 3T3-L1 Fibroblasts—Control 3T3-L1 fibroblasts and cells expressing perilipin A or a mutated form of perilipin A were cultured on glass coverslips, lipid-loaded overnight, and then incubated in the experimental medium described above prior to fixation with 3% paraformaldehyde in phosphate-buffered saline and preparation for microscopy (29Blanchette-Mackie E.J. Dwyer N.K. Barber T. Coxey R.A. Takeda T. Rondinone C.M. Theodorakis J.L. Greenberg A.S. Londos C. J. Lipid Res. 1995; 36: 1211-1226Abstract Full Text PDF PubMed Google Scholar). Fixed cells were probed with antibodies raised against perilipin, followed by Alexa Fluor 546-conjugated goat anti-rabbit IgG and Bodipy 493/503 to detect neutral lipids (30Gocze P.M. Freeman D.A. Cytometry. 1994; 17: 151-158Crossref PubMed Scopus (176) Google Scholar). Cells were viewed with a Nikon Eclipse E800 fluorescence microscope equipped with a Hamamatsu Orca digital camera interfaced with a Power Macintosh G4. Images were captured in monochrome and processed using Improvision Openlab software. Cells co-stained with Alexa Fluor 546 secondary antibodies and Bodipy 493/503 are depicted in colors opposite to those observed for esthetic reasons. Measurement of the Rate of Triacylglycerol Hydrolysis in 3T3-L1 Fibroblasts—Confluent monolayers of cells expressing perilipin A and control cells were lipid-loaded for 16-18 h. Exogenous fatty acids were removed, and the cells were incubated in medium containing 6 μm triacsin C (12Brasaemle D.L. Rubin B. Harten I.A. Gruia-Gray J. Kimmel A.R. Londos C. J. Biol. Chem. 2000; 275: 38486-38493Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar, 35Igal R.A. Wang P. Coleman R.A. Biochem. J. 1997; 324: 529-534Crossref PubMed Scopus (125) Google Scholar), 1% fatty acid-free BSA to bind released fatty acids, and either forskolin and IBMX, DEUP, or forskolin and IBMX with DEUP. Cells were harvested, total lipids were extracted (36Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42361) Google Scholar), and triacylglycerol was quantified by thin layer chromatography, as described previously (34Brasaemle D.L. Barber T. Kimmel A.R. Londos C. J. Biol. Chem. 1997; 272: 9378-9387Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). Detection of Phosphorylation of Unmodified and Mutated Forms of Perilipin A—Confluent monolayers of control 3T3-L1 fibroblasts and cells stably expressing perilipin A or mutated forms of perilipin A were lipid-loaded and then incubated in control medium or medium containing forskolin and IBMX in the presence or absence of DEUP. Cells from six 100-mm culture dishes were collected and pooled for each condition and disrupted by homogenization in a hypotonic solution containing 10 mm Tris, pH 7.4, 1 mm EDTA, 10 mm sodium fluoride, 10 μg/ml leupeptin, 1 mm benzamidine, and 100 μm (4-(2-aminoethyl)benzenesulfonylfluoride) hydrochloride (lysis solution), as described previously (33Garcia A. Sekowski A. Subramanian V. Brasaemle D.L. J. Biol. Chem. 2003; 278: 625-635Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). The lysate was adjusted to 20% sucrose by dilution with 60% sucrose in lysis solution, transferred to an ultracentrifuge tube, overlaid with 9 ml of 5% sucrose in lysis solution, and then centrifuged for 30 min at 26,000 × g at 4 °C in a Sorvall TH-641 rotor. The floating lipid droplet layer was collected after slicing off the top portions of the tubes with a Beckman tube slicer. Lipid droplet fractions were delipidated (33Garcia A. Sekowski A. Subramanian V. Brasaemle D.L. J. Biol. Chem. 2003; 278: 625-635Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar) and the proteins solubilized in 2-fold-concentrated Laemmli's sample buffer (37Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206602) Google Scholar). Proteins were separated by SDS-PAGE and transferred electrophoretically to nitrocellulose membranes. Immunoblots were probed with antibodies raised against phosphorylated serine and horseradish peroxidase-conjugated goat anti-rabbit IgG, followed by enhanced chemiluminescence detection. Blots were stripped and reprobed with polyclonal antibodies raised against phosphoserine within a protein kinase A consensus sequence (RRXS), perilipin (28Brasaemle D.L. Barber T. Wolins N.E. Serrero G. Blanchette-Mackie E.J. Londos C. J. Lipid Res. 1997; 38: 2249-2263Abstract Full Text PDF PubMed Google Scholar), and adipophilin (27Schultz C.J. Torres E. Londos C. Torday J.S. Am. J. Physiol. 2002; 283: L288-L296Crossref PubMed Scopus (102) Google Scholar). Northern Blot Analysis—Total RNA was extracted from 3T3-L1 fibroblasts using RNeasy minicolumns (Qiagen) following the manufacturer's protocol,