Spatiotemporal Regulation of Early Lipolytic Signaling in Adipocytes

Abstract

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 β-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-HSLSer-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. 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 β-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-HSLSer-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. IntroductionThe regulation of lipid storage and lipolysis in adipocytes has important implications for the maintenance of whole body lipid homeostasis (1.Hajer G.R. van Haeften T.W. Visseren F.L. Eur. Heart J. 2008; 29: 2959-2971Crossref PubMed Scopus (1022) Google Scholar, 2.Langin D. Pharmacol. Res. 2006; 53: 482-491Crossref PubMed Scopus (272) Google Scholar). Dysregulation is associated with obesity and the onset of metabolic disease, insulin resistance, and type II diabetes. An important goal for the future is to design strategies that allow us to manipulate lipid storage and so control weight gain. Fundamental to a targeted approach to controlling the storage and mobilization of lipid in adipose tissue is a detailed understanding of the cellular mechanisms and machinery that regulate lipolysis. Although the key players in this process have been identified, the precise regulation of the lipolytic machinery is not yet fully understood. In this study we have begun to address this by analyzing the earliest events in the activation of lipolysis at the cellular level.Adipocytes are specialized lipid droplet (LD) 2The abbreviations used are: LDlipid dropletHSLhormone-sensitive lipaseP-HSLphospho-HSLTGtriglyceridePKAprotein kinase AIBMXisobutylmethylxanthineFskforskolinNEFAnonesterified fatty acid. -laden cells that store large amounts of neutral lipid, predominantly as triglycerides (TG) (3.Martin S. Parton R.G. Nat. Rev. Mol. Cell Biol. 2006; 7: 373-378Crossref PubMed Scopus (897) Google Scholar, 4.Murphy D.J. Prog. Lipid. Res. 2001; 40: 325-438Crossref PubMed Scopus (754) Google Scholar). In response to extracellular stimulation by catecholamines, adipocytes hydrolyze stored TGs to generate free fatty acids and glycerol. In rodent adipocytes, the hydrolysis of neutral lipids is tightly regulated by a series of signal transduction pathways from the G-protein-coupled β3-adrenergic receptor that culminate at the surface of the LD (2.Langin D. Pharmacol. Res. 2006; 53: 482-491Crossref PubMed Scopus (272) Google Scholar, 5.Collins S. Cao W. Robidoux J. Mol. Endocrinol. 2004; 18: 2123-2131Crossref PubMed Scopus (141) Google Scholar). A well characterized pathway from the β3-adrenergic receptor results in the elevation of cAMP levels, activating cAMP-dependent protein kinase/protein kinase A (PKA), which in turn phosphorylates downstream targets, including the lipid droplet scaffold/adaptor protein perilipin (6.Brasaemle D.L. J. Lipid Res. 2007; 48: 2547-2559Abstract Full Text Full Text PDF PubMed Scopus (750) Google Scholar) and the primary diacylglycerol lipase hormone-sensitive lipase (HSL) (7.Anthonsen M.W. Rönnstrand L. Wernstedt C. Degerman E. Holm C. J. Biol. Chem. 1998; 273: 215-221Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar). The array of phosphorylation sites present on HSL (7.Anthonsen M.W. Rönnstrand L. Wernstedt C. Degerman E. Holm C. J. Biol. Chem. 1998; 273: 215-221Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar, 8.Garton A.J. Campbell D.G. Carling D. Hardie D.G. Colbran R.J. Yeaman S.J. Eur. J. Biochem. 1989; 179: 249-254Crossref PubMed Scopus (228) Google Scholar, 9.Garton A.J. Campbell D.G. Cohen P. Yeaman S.J. FEBS Lett. 1988; 229: 68-72Crossref PubMed Scopus (79) Google Scholar, 10.Greenberg A.S. Shen W.J. Muliro K. Patel S. Souza S.C. Roth R.A. Kraemer F.B. J. Biol. Chem. 2001; 276: 45456-45461Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar, 11.Strålfors P. Björgell P. Belfrage P. Proc. Natl. Acad. Sci. U.S.A. 1984; 81: 3317-3321Crossref PubMed Scopus (197) Google Scholar) suggests a complex regulation with important implications for the control of lipolysis. In this study, we have investigated the spatial and temporal characteristics of HSL phosphorylation in 3T3-L1 adipocytes.In contrast to perilipin, which is constitutively associated with the LD surface, HSL is a cytosolic protein that translocates to LDs in response to catecholamine stimulation (12.Egan 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). Translocation of HSL is dependent upon phosphorylation (13.Su 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) and exquisitely regulates the functional activity of HSL in vivo. Phosphorylation of HSL has little effect on the intrinsic activity of the enzyme (14.Strålfors P. Honnor R.C. Eur. J. Biochem. 1989; 182: 379-385Crossref PubMed Scopus (114) Google Scholar). HSL is phosphorylated on up to three sites by PKA (Ser-563, 3Note that although this study analyzes murine HSL, by convention the amino acid numbering is derived from the rat HSL sequence, except where stated otherwise. Ser-659, and Ser-660 (7.Anthonsen M.W. Rönnstrand L. Wernstedt C. Degerman E. Holm C. J. Biol. Chem. 1998; 273: 215-221Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar)) and at a single site by extracellular signal-regulated kinase (ERK) (Ser-600 (10.Greenberg A.S. Shen W.J. Muliro K. Patel S. Souza S.C. Roth R.A. Kraemer F.B. J. Biol. Chem. 2001; 276: 45456-45461Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar)) in response to the activation of lipolysis. The phosphorylation of both Ser-659 and Ser-660 by PKA is necessary for activated lipolysis and the translocation of HSL to the LD surface (13.Su 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). However, although Ser-563 is phosphorylated in response to catecholamines, the functional significance of this is not known, as it triggers neither HSL translocation to LDs (13.Su 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) nor HSL activity in vitro (7.Anthonsen M.W. Rönnstrand L. Wernstedt C. Degerman E. Holm C. J. Biol. Chem. 1998; 273: 215-221Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar). HSL is also phosphorylated on Ser-565 by 5′-AMP-activated protein kinase in unstimulated adipocytes, although the precise role of HSLSer-565 phosphorylation by 5′-AMP-activated protein kinase remains elusive. Mutation of Ser-565 abolishes translocation of HSL to LDs in stimulated cells indicating a role in the activation of lipolysis (13.Su 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). Phosphorylation of peptides incorporating the Ser-565 site by 5′-AMP-activated protein kinase in vitro inhibits subsequent phosphorylation on the Ser-563 site by PKA (8.Garton A.J. Campbell D.G. Carling D. Hardie D.G. Colbran R.J. Yeaman S.J. Eur. J. Biochem. 1989; 179: 249-254Crossref PubMed Scopus (228) Google Scholar), indicating a functional relationship between these two sites.To examine the relationship between the specific phosphorylation events in HSL during activated lipolysis, we analyzed the distribution of phosphorylated HSL biochemically and by immunofluorescence microscopy using phospho-specific antibodies. We found both spatial and temporal differences in the progression of phosphorylation events upon acute activation of lipolysis in adipocytes. Furthermore, we found evidence that not only the phosphorylation but also the dephosphorylation of specific serines in HSL was tightly controlled. In light of these findings, we propose that distinct signaling complexes are involved in the activation of lipolysis, one at the LD surface and another distal to the LD, possibly at the cell surface. As stable association of HSL with LDs during lipolysis is dependent upon its phosphorylation on Ser-660 (13.Su 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), we propose that the distal phosphorylation complex is of primary importance in the initial activation of lipolysis, and we hypothesize that the LD-associated phosphorylation complexes are important in regulating the stability and/or duration of lipolysis.DISCUSSIONIn this study, we have described for the first time the earliest events regulating the acute activation of lipolysis by signal transduction from the β-adrenergic receptor in adipocytes. These studies provide new insights into fundamental aspects of lipolysis. By examining the phosphorylation of HSL within adipocytes, both spatially and temporally, we have identified several important aspects of HSL regulation. First, we describe the remarkably rapid, sequential phosphorylation of specific serine residues in HSL, at spatially distinct intracellular locales. Second, we provide evidence for the presence of two distinct PKA signaling complexes directly regulating lipolysis. Finally, we describe differences in the mutually exclusive phosphorylation of two serine residues in HSL, targeted by distinct protein kinases, upon activation of lipolysis by signal transduction from the cell surface compared with the direct elevation of intracellular cAMP levels. Together, these data show that the acute stimulation of lipolysis in adipocytes by β-adrenergic receptor agonists is controlled by a complex series of phosphorylation and dephosphorylation events, mediated by multiple signaling complexes. Although the later events occur at the surface of the LD itself, we propose that the primary signaling complex initiating lipolysis is located at the cell surface in close proximity to the β3-adrenergic receptor (Fig. 8).The phosphorylation of both HSL and perilipin A on multiple residues in basal adipocytes and during activated lipolysis has been well described (7.Anthonsen M.W. Rönnstrand L. Wernstedt C. Degerman E. Holm C. J. Biol. Chem. 1998; 273: 215-221Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar, 8.Garton A.J. Campbell D.G. Carling D. Hardie D.G. Colbran R.J. Yeaman S.J. Eur. J. Biochem. 1989; 179: 249-254Crossref PubMed Scopus (228) Google Scholar, 10.Greenberg A.S. Shen W.J. Muliro K. Patel S. Souza S.C. Roth R.A. Kraemer F.B. J. Biol. Chem. 2001; 276: 45456-45461Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar, 11.Strålfors P. Björgell P. Belfrage P. Proc. Natl. Acad. Sci. U.S.A. 1984; 81: 3317-3321Crossref PubMed Scopus (197) Google Scholar). However, the exact progression and spatial organization of distinct phosphorylation reactions is not well understood. By using the 3T3-L1 adipocyte model cell system, we have been able to examine the incredibly rapid kinetics and spatial organization of early phosphorylation of HSL, studies that would be difficult to accomplish in vivo or in primary adipocytes. Using explants of primary mouse adipose tissue, we show that identical phosphorylation responses are observed upon activation of lipolysis by β-adrenergic receptor signal transduction compared with the direct elevation of intracellular cAMP levels, as shown in 3T3-L1 adipocytes, confirming the relevancy of this system to primary adipose tissue. The results of these analyses suggest the presence of a highly organized signaling platform generating maximal phosphorylation and association with LDs within seconds rather than minutes. Our observation that P-HSLSer-660 initially appeared in the peripheral cytosol of the cell prior to its association with the LD, within the shortest time frame that we could reasonably measure (5 s), leads us to hypothesize that this phosphorylation event is mediated by a pool of PKA present at the cell surface, possibly in a complex with the β3-adrenergic receptor (Fig. 8). The appearance of both PKA-phosphorylated perilipin A and P-HSLSer-563 on the LD strongly suggests that either a second LD-associated pool of PKA is activated at the LD surface or that activated PKA is also capable of translocating to the LD surface.The initial phosphorylation of the LD scaffold/adaptor protein perilipin A was found to occur many seconds later than the phosphorylation of HSL on Ser-660, but it preceded the phosphorylation of HSL on Ser-563. As the maximal phosphorylation of HSLSer-563 closely follows the maximal phosphorylation of perilipin A, it is possible that the latter is required to stabilize HSLSer-563 at the LD surface. At present, it is unclear whether phosphorylation of Ser-563 occurs on nonphosphorylated HSL (Fig. 8A) or whether a phosphatase on the LD surface catalyzes the dephosphorylation of the Ser-565 site prior to phosphorylation on the Ser-563 (Fig. 8B). The low steady state level of Ser-563 phosphorylation of HSL in activated cells suggests that unlike Ser-660 phosphorylation, Ser-563 phosphorylation is not a requirement for translocation. At present, the functional significance of HSLSer-563 phosphorylation remains unknown.Cyclic AMP is a key signaling molecule in many different signal transduction pathways in numerous cell types. Specificity in cAMP signaling is achieved by anchoring PKA to the appropriate target compartment via an interaction with protein kinase A-anchoring proteins (23.Wong W. Scott J.D. Nat. Rev. Mol. Cell Biol. 2004; 5: 959-970Crossref PubMed Scopus (843) Google Scholar). Preventing PKA anchoring in adipocytes inhibits isoproterenol-stimulated lipolysis showing that correct localization is a requirement for signal transduction (24.Zhang J. Hupfeld C.J. Taylor S.S. Olefsky J.M. Tsien R.Y. Nature. 2005; 437: 569-573Crossref PubMed Scopus (250) Google Scholar). In adipocytes, D-AKAP1 has been proposed to anchor PKA to the LD surface (25.Bridges D. MacDonald J.A. Wadzinski B. Moorhead G.B. Biochem. Biophys. Res. Commun. 2006; 346: 351-357Crossref PubMed Scopus (21) Google Scholar) and could therefore mediate phosphorylation of perilipin A and P-HSLSer-563. The ability of protein kinase A-anchoring proteins to form complexes with multiple components of a signaling pathway, including kinases, phosphatases, and phosphodiesterases (reviewed in Refs. 26.Beene D.L. Scott J.D. Curr. Opin. Cell Biol. 2007; 19: 192-198Crossref PubMed Scopus (195) Google Scholar, 27.Diviani D. Curr. Opin. Pharmacol. 2008; 8: 166-173Crossref PubMed Scopus (24) Google Scholar) suggests that a signaling platform is likely to exist on the LD surface. Identifying the components and regulation of this complex will be an important future endeavor.Determining the molecular composition of the different lipolytic signaling complexes in adipocytes, and the mechanism by which agonist-mediated activation of lipolysis is controlled by protein kinases and phosphatases, is crucial to understanding lipolysis. Furthermore, the relationship between HSL translocation and the activity of a second major LD-associated lipase, adipocyte TG lipase, is also of primary importance. Adipocyte TG lipase catalyzes the first hydrolytic reaction of lipolysis resulting in the generation of diglyceride from TG (28.Zechner R. Kienesberger P.C. R. J. Lipid Res. Full Text Full Text PDF PubMed Scopus Google Scholar). As diglyceride is the major intracellular of HSL, and it has been shown that HSL an site to to the LD (13.Su 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), it is to hypothesize that the activity of adipocyte TG lipase is required to stabilize the interaction of phosphorylated HSL with the LD by providing the In we have shown that phosphorylation of key lipolytic proteins in 3T3-L1 adipocytes is to regulation both spatially and temporally, of in the regulation of lipolysis at the cellular IntroductionThe regulation of lipid storage and lipolysis in adipocytes has important implications for the maintenance of whole body lipid homeostasis (1.Hajer G.R. van Haeften T.W. Visseren F.L. Eur. Heart J. 2008; 29: 2959-2971Crossref PubMed Scopus (1022) Google Scholar, 2.Langin D. Pharmacol. Res. 2006; 53: 482-491Crossref PubMed Scopus (272) Google Scholar). Dysregulation is associated with obesity and the onset of metabolic disease, insulin resistance, and type II diabetes. An important goal for the future is to design strategies that allow us to manipulate lipid storage and so control weight gain. Fundamental to a targeted approach to controlling the storage and mobilization of lipid in adipose tissue is a detailed understanding of the cellular mechanisms and machinery that regulate lipolysis. Although the key players in this process have been identified, the precise regulation of the lipolytic machinery is not yet fully understood. In this study we have begun to address this by analyzing the earliest events in the activation of lipolysis at the cellular level.Adipocytes are specialized lipid droplet (LD) 2The abbreviations used are: LDlipid dropletHSLhormone-sensitive lipaseP-HSLphospho-HSLTGtriglyceridePKAprotein kinase AIBMXisobutylmethylxanthineFskforskolinNEFAnonesterified fatty acid. -laden cells that store large amounts of neutral lipid, predominantly as triglycerides (TG) (3.Martin S. Parton R.G. Nat. Rev. Mol. Cell Biol. 2006; 7: 373-378Crossref PubMed Scopus (897) Google Scholar, 4.Murphy D.J. Prog. Lipid. Res. 2001; 40: 325-438Crossref PubMed Scopus (754) Google Scholar). In response to extracellular stimulation by catecholamines, adipocytes hydrolyze stored TGs to generate free fatty acids and glycerol. In rodent adipocytes, the hydrolysis of neutral lipids is tightly regulated by a series of signal transduction pathways from the G-protein-coupled β3-adrenergic receptor that culminate at the surface of the LD (2.Langin D. Pharmacol. Res. 2006; 53: 482-491Crossref PubMed Scopus (272) Google Scholar, 5.Collins S. Cao W. Robidoux J. Mol. Endocrinol. 2004; 18: 2123-2131Crossref PubMed Scopus (141) Google Scholar). A well characterized pathway from the β3-adrenergic receptor results in the elevation of cAMP levels, activating cAMP-dependent protein kinase/protein kinase A (PKA), which in turn phosphorylates downstream targets, including the lipid droplet scaffold/adaptor protein perilipin (6.Brasaemle D.L. J. Lipid Res. 2007; 48: 2547-2559Abstract Full Text Full Text PDF PubMed Scopus (750) Google Scholar) and the primary diacylglycerol lipase hormone-sensitive lipase (HSL) (7.Anthonsen M.W. Rönnstrand L. Wernstedt C. Degerman E. Holm C. J. Biol. Chem. 1998; 273: 215-221Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar). The array of phosphorylation sites present on HSL (7.Anthonsen M.W. Rönnstrand L. Wernstedt C. Degerman E. Holm C. J. Biol. Chem. 1998; 273: 215-221Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar, 8.Garton A.J. Campbell D.G. Carling D. Hardie D.G. Colbran R.J. Yeaman S.J. Eur. J. Biochem. 1989; 179: 249-254Crossref PubMed Scopus (228) Google Scholar, 9.Garton A.J. Campbell D.G. Cohen P. Yeaman S.J. FEBS Lett. 1988; 229: 68-72Crossref PubMed Scopus (79) Google Scholar, 10.Greenberg A.S. Shen W.J. Muliro K. Patel S. Souza S.C. Roth R.A. Kraemer F.B. J. Biol. Chem. 2001; 276: 45456-45461Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar, 11.Strålfors P. Björgell P. Belfrage P. Proc. Natl. Acad. Sci. U.S.A. 1984; 81: 3317-3321Crossref PubMed Scopus (197) Google Scholar) suggests a complex regulation with important implications for the control of lipolysis. In this study, we have investigated the spatial and temporal characteristics of HSL phosphorylation in 3T3-L1 adipocytes.In contrast to perilipin, which is constitutively associated with the LD surface, HSL is a cytosolic protein that translocates to LDs in response to catecholamine stimulation (12.Egan 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). Translocation of HSL is dependent upon phosphorylation (13.Su 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) and exquisitely regulates the functional activity of HSL in vivo. Phosphorylation of HSL has little effect on the intrinsic activity of the enzyme (14.Strålfors P. Honnor R.C. Eur. J. Biochem. 1989; 182: 379-385Crossref PubMed Scopus (114) Google Scholar). HSL is phosphorylated on up to three sites by PKA (Ser-563, 3Note that although this study analyzes murine HSL, by convention the amino acid numbering is derived from the rat HSL sequence, except where stated otherwise. Ser-659, and Ser-660 (7.Anthonsen M.W. Rönnstrand L. Wernstedt C. Degerman E. Holm C. J. Biol. Chem. 1998; 273: 215-221Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar)) and at a single site by extracellular signal-regulated kinase (ERK) (Ser-600 (10.Greenberg A.S. Shen W.J. Muliro K. Patel S. Souza S.C. Roth R.A. Kraemer F.B. J. Biol. Chem. 2001; 276: 45456-45461Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar)) in response to the activation of lipolysis. The phosphorylation of both Ser-659 and Ser-660 by PKA is necessary for activated lipolysis and the translocation of HSL to the LD surface (13.Su 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). However, although Ser-563 is phosphorylated in response to catecholamines, the functional significance of this is not known, as it triggers neither HSL translocation to LDs (13.Su 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) nor HSL activity in vitro (7.Anthonsen M.W. Rönnstrand L. Wernstedt C. Degerman E. Holm C. J. Biol. Chem. 1998; 273: 215-221Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar). HSL is also phosphorylated on Ser-565 by 5′-AMP-activated protein kinase in unstimulated adipocytes, although the precise role of HSLSer-565 phosphorylation by 5′-AMP-activated protein kinase remains elusive. Mutation of Ser-565 abolishes translocation of HSL to LDs in stimulated cells indicating a role in the activation of lipolysis (13.Su 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). Phosphorylation of peptides incorporating the Ser-565 site by 5′-AMP-activated protein kinase in vitro inhibits subsequent phosphorylation on the Ser-563 site by PKA (8.Garton A.J. Campbell D.G. Carling D. Hardie D.G. Colbran R.J. Yeaman S.J. Eur. J. Biochem. 1989; 179: 249-254Crossref PubMed Scopus (228) Google Scholar), indicating a functional relationship between these two sites.To examine the relationship between the specific phosphorylation events in HSL during activated lipolysis, we analyzed the distribution of phosphorylated HSL biochemically and by immunofluorescence microscopy using phospho-specific antibodies. We found both spatial and temporal differences in the progression of phosphorylation events upon acute activation of lipolysis in adipocytes. Furthermore, we found evidence that not only the phosphorylation but also the dephosphorylation of specific serines in HSL was tightly controlled. In light of these findings, we propose that distinct signaling complexes are involved in the activation of lipolysis, one at the LD surface and another distal to the LD, possibly at the cell surface. As stable association of HSL with LDs during lipolysis is dependent upon its phosphorylation on Ser-660 (13.Su 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), we propose that the distal phosphorylation complex is of primary importance in the initial activation of lipolysis, and we hypothesize that the LD-associated phosphorylation complexes are important in regulating the stability and/or duration of lipolysis.