Lipase-selective Functional Domains of Perilipin A Differentially Regulate Constitutive and Protein Kinase A-stimulated Lipolysis*

Perilipin (Peri) A is a lipid droplet-associated phosphoprotein that acts dually as a suppressor of basal (constitutive) lipolysis and as an enhancer of cyclic AMP-dependent protein kinase (PKA)-stimulated lipolysis by both hormone-sensitive lipase (HSL) and non-HSL(s). To identify domains of Peri A that mediate these multiple actions, we introduced adenoviruses expressing truncated or mutated Peri A and HSL into NIH 3T3 fibroblasts lacking endogenous perilipins and HSL but overexpressing acyl-CoA synthetase 1 and fatty acid transporter 1. We identified two lipase-selective functional domains: 1) Peri A (amino acids 1–300), which inhibits basal lipolysis and promotes PKA-stimulated lipolysis by HSL, and 2) Peri A (amino acids 301–517), which inhibits basal lipolysis by non-HSL and promotes PKA-stimulated lipolysis by both HSL and non-HSL. PKA site mutagenesis revealed that PKA-stimulated lipolysis by HSL requires phosphorylation of one or more sites within Peri 1–300 (Ser81, Ser222, and Ser276). PKA-stimulated lipolysis by non-HSL additionally requires phosphorylation of one or more PKA sites within Peri 301–517 (Ser433, Ser492, and Ser517). Peri 301–517 promoted PKA-stimulated lipolysis by HSL yet did not block HSL-mediated basal lipolysis, indicating that an additional region(s) within Peri 301–517 promotes hormone-stimulated lipolysis by HSL. These results suggest a model of Peri A function in which 1) lipase-specific “barrier” domains block basal lipolysis by HSL and non-HSL, 2) differential PKA site phosphorylation allows PKA-stimulated lipolysis by HSL and non-HSL, respectively, and 3) additional domains within Peri A further facilitate PKA-stimulated lipolysis, again with lipase selectivity.

Perilipin (Peri) A is a lipid droplet-associated phosphoprotein that acts dually as a suppressor of basal (constitutive) lipolysis and as an enhancer of cyclic AMPdependent protein kinase (PKA)-stimulated lipolysis by both hormone-sensitive lipase (HSL) and non-HSL(s). To identify domains of Peri A that mediate these multiple actions, we introduced adenoviruses expressing truncated or mutated Peri A and HSL into NIH 3T3 fibroblasts lacking endogenous perilipins and HSL but overexpressing acyl-CoA synthetase 1 and fatty acid transporter 1. We identified two lipase-selective functional domains: 1) Peri A (amino acids 1-300), which inhibits basal lipolysis and promotes PKA-stimulated lipolysis by HSL, and 2) Peri A (amino acids 301-517), which inhibits basal lipolysis by non-HSL and promotes PKA-stimulated lipolysis by both HSL and non-HSL. PKA site mutagenesis revealed that PKA-stimulated lipolysis by HSL requires phosphorylation of one or more sites within Peri 1-300 (Ser 81 , Ser 222 , and Ser 276 ). PKAstimulated lipolysis by non-HSL additionally requires phosphorylation of one or more PKA sites within Peri 301-517 (Ser 433 , Ser 492 , and Ser 517 ). Peri 301-517 promoted PKA-stimulated lipolysis by HSL yet did not block HSL-mediated basal lipolysis, indicating that an additional region(s) within Peri 301-517 promotes hormone-stimulated lipolysis by HSL. These results suggest a model of Peri A function in which 1) lipase-specific "barrier" domains block basal lipolysis by HSL and non-HSL, 2) differential PKA site phosphorylation allows PKA-stimulated lipolysis by HSL and non-HSL, respectively, and 3) additional domains within Peri A further facilitate PKA-stimulated lipolysis, again with lipase selectivity.
Hydrolysis of triacylglycerol (TAG) 1 (lipolysis) in adipocytes is a key event that supplies the primary source of energy, free fatty acids, for other tissues. In times of energy need such as fasting (1)(2)(3) and exercise (4,5), adipocyte lipolysis is regulated by hormones such as catecholamines, which activate cAMP-dependent protein kinase (PKA) (6,7). Activation of PKA results in a marked increase in lipolysis as compared with spontaneous lipolysis in the absence of hormones (basal or constitutive lipolysis). Thus, basal and PKA-stimulated lipolysis reflect the lipolytic response of adipocytes to the changing energy requirements of the body. In obesity, basal lipolysis is increased, and PKA-stimulated lipolysis is blunted (8). This dysregulation of adipocyte lipolysis is associated with the development of insulin resistance and type 2 diabetes (9).
TAG breakdown (lipolysis) is mediated by lipases, which hydrolyze TAG sequestered in intracellular lipid droplets. Approximately 50% of the neutral triglyceride lipase activity in white adipose tissue is attributable to hormone-sensitive lipase (HSL) (10 -12). HSL-mediated lipolysis is under the tight control of PKA. In the absence of PKA activation, constitutive HSL activity has been thought to mediate basal lipolysis (13). When PKA is activated, it phosphorylates HSL (13,14), resulting in enhanced hydrolytic activity (15), translocation of HSL from cytosol to the lipid droplet surface (16 -18), and enhanced TAG breakdown. HSL was previously considered the rate-limiting enzyme in adipocyte lipolysis. This view, however, has recently been challenged by the following observations: 1) HSL-deficient mice are not obese, suggesting significant activity of lipase(s) other than HSL (10,12,19); 2) adipocytes derived from epididymal fat pads or embryonic fibroblasts of HSL null mice retain 50% of basal TAG lipase activity and are responsive to PKA activation (10,11,12,19); and 3) cell lines lacking endogenous HSL, i.e. 3T3-L1 preadipocytes (21), NIH 3T3 fibroblasts (22), and Chinese hamster ovary cells (23), exhibit significant TAG hydrolysis under basal and PKA-stimulated conditions. Thus, both HSL and non-HSL(s) play important roles in basal and PKA-stimulated lipolysis.
The hydrolytic actions of HSL and non-HSLs are regulated at the lipid droplet surface by perilipins, a family of lipid droplet-associated phosphoproteins (24,25). Although not required for lipid droplet formation and TAG storage, perilipin association with the lipid droplet regulates the magnitude of lipolysis and thus levels of stored TAG (21,22). Peri A and Peri B are the predominant isoforms of murine adipocytes, with Peri A constituting ϳ85% of total perilipin. Derived from alternative splicing of a single gene, Peri A and Peri B share a common N-terminal region of 1-405 amino acids (aa) but possess unique C termini (26,27) (Fig. 1). Under nonstimulated (basal) conditions, Peri A acts as a barrier to HSL (22) and non-HSL(s) (21)(22)(23), thereby decreasing basal lipolysis (22,23) and increasing intracellular (TAG) storage (21,22). Upon lipolytic stimulation, Peri A is phosphorylated at up to six sites by PKA (25, 28) (Fig. 1). This phosphorylation facilitates lipase(s) access to the lipid droplet, thereby promoting both HSL-mediated and non-HSL-mediated lipolysis (22,29,30). Peri A therefore functions as both a suppressor of basal lipolysis and as an enhancer of PKA-stimulated lipolysis. The functions of Peri B are less defined.
How does the dual function of perilipin in regulating lipolyis arise from perilipin protein structure? Recent data have suggested a model of Peri A function in which the phosphorylation of N-terminal PKA sites (PKA1, PKA2, and PKA3; Fig. 1) promotes PKA-stimulated lipolysis (22,23), and the C terminus of Peri A (aa 406 -517) acts as a barrier to all lipases (inhibits basal lipolysis) (23). In contrast, novel data in the present study identify multiple, lipase-selective domains within Peri A that function coordinately to regulate basal and stimulated lipolysis. Our data support a model of perilipin function in which 1) discrete lipase-selective protein domains of perilipin act as barriers to HSL and non-HSL(s), respectively, 2) phosphorylation of different PKA sites allows PKA-stimulated lipolysis mediated by HSL and non-HSL(s), respectively, and 3) additional "facilitative" domains within Peri A enhance PKA-stimulated lipolysis, again with lipase selectivity.

Materials
A polyclonal antibody (MSM) specific for the N terminus of Peri A was generated using an N-terminal peptide (aa 1-21, MSMNKGPTLL-DGDLPEQENVL). A polyclonal antibody (PREK) specific for the C terminus of Peri A was generated using a C-terminal peptide (aa 483-517, PREKPARRVSDSFFRPSVC). A polyclonal anti-HSL antibody was generated using a peptide based upon the rat HSL sequence KDLS-FKGNSEPSDSPEM. The antibodies were subsequently affinity-purified and used for Western blotting (1:1000). Tissue culture reagents and media were purchased from Invitrogen. LipofectAMINE Plus TM reagent was purchased from Invitrogen. Horseradish-linked anti-rabbit IgG was from Amersham Biosciences; SuperSignal® Chemiluminescent Substrate was obtained from Pierce. All other chemicals were purchased from Sigma.

Methods
Generation of Truncation and Mutational Constructs of Peri A-Truncations and mutations of Peri A constructed for the study are shown in Fig. 1. A C-terminal deletion expressing 1-300 aa of Peri A (Peri 1-300) was generated by PCR amplification of mouse Peri A cDNA using a 5Ј-primer containing a BglII restriction site (5Ј-GCG TCT GCC AGA TCT AGC TGC TTT CTC-3Ј) and a 3Ј-primer that contains a KpnI restriction site and a FLAG epitope tag sequence: 5Ј-CTC TTC GGT ACC TCA CTT ATC GTC GTC ATC CTT GTA ATC TGT GTC TGT CTG GTC GTC ATG-3Ј. N-terminal deletions of Peri A were generated by PCR amplification of a cDNA that encodes Peri A-FLAG. A common 3Ј-primer containing a SalI restriction site (5Ј-CGA CCT GTC GAC TCA CTT ATC GTC GTC-3Ј) and the following 5Ј-primers containing a BglII restriction site and the Kozak translation initiation sequence were used: Peri 142-517, Generation of Recombinant Adenoviruses-Adenoviruses expressing ␤-galactosidase (Ad ␤-gal), the Aequoria Victoria green fluorescent protein (Ad GFP), Peri A (Ad Peri A), Peri B (Ad Peri B), and HSL (Ad HSL) were generated as described previously (22,31). Adenoviruses expressing 1-300 aa of Peri A (Ad 1-300) and N-terminal PKA site-mutated Peri A (Ad Peri A⌬123) were constructed using the two-cosmid system (32) in which perilipin cDNAs were subcloned into BglII-KpnI-opened pLEP-CMV vector. Adenoviruses expressing the C-terminal regions of Peri A (Ad 142-517, Ad 201-517, Ad 233-517, Ad 251-517, and Ad 301-517) and PKA site-mutated Peri A (Ad Peri A⌬456 and Ad Peri A⌬1-6) were generated using the AdEasy TM adenoviral vector system (Stratagene, La Jolla, CA). In brief, perilipin cDNAs were subcloned into pShuttle-CMV vector cleaved with BglII and SalI. The expression vector was linearized with PmeI and co-transformed into BJ5183 bacterial cells with supercoiled viral DNA plasmid pAdEasy. Recombinant plasmid DNA was then used to transfect HEK293 cells in which deleted viral assembly genes were complemented. Adenoviral amplification was performed as described (22).
Adenovirus-mediated Expression of Perilipins and HSL-Adenoviruses expressing truncated/mutated Peri A and HSL were expressed in ACS1/FATP1 cells (kindly provided by Dr. Jean E. Schaffer from Washington University, St. Louis, MO). ACS1/FATP1 are NIH 3T3 fibroblasts that are engineered to stably overexpress acyl-CoA synthetase 1 (ACS1) and long chain fatty acid (FA) transport protein 1 (FATP1) (33). These cells were selected for use because 1) they lack endogenous perilipins and HSL, and 2) overexpression of ACS1/FATP1 stimulates FA import and substantial TAG accumulation in lipid droplets (22). These features make ACS1/FATP1 cells powerful tools for elucidating the relative roles of adenovirally expressed perilipins in HSL and non-HSL(s)-mediated lipolysis.
ACS1/FATP1 cells in confluent cultures were transduced with either Ad Control (Ad GFP or Ad ␤-gal) to control for nonspecific effects of adenoviral infection or Ad HSL in combination with adenoviruses expressing various perilipins. Transduction was performed as described (22). To minimize differences in the expression levels of HSL and perilipins among samples and assays, each adenovirus was pretitered, and an optimal titer was maintained thereafter to transduce ACS1/ FATP1 cells. The optimal titer for Ad HSL was defined as the titer that maximally increased basal lipolysis, which could be inhibited by Peri A (22). The optimal titers for adenoviruses expressing various perilipins were chosen so that all these perilipins were expressed at similar levels.
FA Loading-Transduced ACS1/FATP1 cells were incubated for 48 h of incubation in Dulbecco's modified Eagle's medium containing 5 mM FIG. 1. Schematic diagram of recombinant adenoviruses expressing Peri A, Peri B, and mutated/truncated Peri A. Consensus PKA phosphorylation sites are indicated by arrows. Peri A⌬123 represents mutation of Ser 81 , Ser 222 , and Ser 276 to Ala. Peri A⌬456 represents mutation of Ser 433 , Ser 492 , and Ser 517 to Ala. Peri A⌬1-6 represents mutation of all six serine sites to Ala. Peri B shares 1-405 aa with Peri A but has a unique C terminus (black box). Serial truncations were made from the N and C termini of Peri A. glucose, 10% calf serum, and 1% fatty acid-free bovine serum albumin bound to palmitic and oleic acid (240 M each). Incubation was terminated by removing lipid loading medium and washing cells with phosphate-buffered saline.
Lipolysis-After transduction and 48 h of incubation with FA, the ACS1/FATP1 cells were washed with phosphate-buffered saline and treated for 4 h with or without a PKA activator, forskolin (20 m) (35), in Dulbecco's modified Eagle's medium containing 5 mM glucose, 2% FA-free bovine serum albumin. The medium was collected, and glycerol was measured as an index of lipolysis as described (22).
Western Blotting-The cellular proteins were extracted and quantified as described (36). The total lysates (10 g/sample) were subjected to SDS-PAGE and Western blotting (37).
Statistical Analyses-The results are expressed as the mean Ϯ S.E. The data were analyzed by one-way analysis of variance using Graph-Pad InStat Software (version 2.04a, Neoptolemos, University of Birmingham, Birmingham, UK). Multiple comparisons were performed with Tukey's honestly significant differences procedure. p values less than 0.05 were considered statistically significant.

Localization of Truncated and Mutated Perilipins on the
Lipid Droplet Surface-ACS1/FATP1 cells were transduced with adenoviruses expressing either Peri A, Peri A truncations, Peri B (a naturally occurring Peri A truncation), or Peri A PKA site mutants. Because association with the lipid droplet membrane is a prerequisite for the ability of perilipin to regulate lipolysis, we initially assessed the ability of these perilipin constructs to target to and associate with lipid droplets. Transduced cells were fixed and prepared for confocal microscopy after incubation with FA for 48 h. Simultaneous fluorescent detection of neutral lipids and perilipins allowed us to correlate immunoreactivity with intracellular structure (Fig. 2). Neutral lipids were stained with Bodipy (red fluorescence). Perilipins were detected with specific antibodies directed against the N or C terminus of Peri A (green fluorescence). Immunostaining for Peri 1-300, Peri 142-517, Peri 233-517, Peri 251-517, and Peri 301-517 revealed a ring-like pattern that was similarly observed with Peri A and Peri B (Fig. 2). The distinct perilipin rings appear localizing around the perimeters of lipid droplets as shown in the overlay of perlilipin and lipid staining. Coincident staining of perilipin and neutral lipid (yellow color) was also evident in the overlay. These staining patterns are clearly distinct from the diffuse cytoplasmic staining observed with Peri 1-200, which serves as a negative control for perilipin association with lipid droplets. Thus, Peri 1-300 and N-terminal truncated Peri A retain the ability to target and associate with lipid droplets. Mutation of PKA phosphorylation sites had no effect on the ability of Peri A to target and associate with lipid droplets, because Peri A⌬123, Peri A⌬456, and Peri A⌬1-6 all localized to the surface of lipid droplets (data not shown). No perilipin immunoreactivity was evident in control cells lacking perilipin expression constructs (data not shown).
Effects of Truncated/Mutated Peri A on Non-HSL-mediated Lipolysis-We next investigated the relative roles of truncated and mutated Peri A in lipolysis mediated by non-HSL(s). ACS1/ FATP1 cells (which do not express endogenous perilipins or HSL) were transduced with either Ad Control (Ad ␤-gal or Ad GFP) or with adenoviruses expressing Peri A, Peri A truncations, or Peri B. Expression of various perilipins was confirmed with N or C terminus-specific antibodies (see Methods) (Fig. 3,  A and B). Transduced cells were incubated for 48 h with FA, Compared with cells transduced with Ad Control (control), expression of Peri A lowered basal lipolysis by ϳ60% (p Ͻ 0.001) (Fig. 3C). Expression of Peri B and Peri 1-300, however, had no significant effect on basal lipolysis (p Ͼ 0.05 versus control). In contrast Peri 142-517, Peri 201-517, Peri 233-517, Peri 251-517, and Peri 301-517 all lowered basal lipolysis to levels at or near that observed with full-length Peri A (p Ͻ 0.05 versus control) (Fig. 3C). These results demonstrate that the C-terminal region of Peri A (Peri 301-517) inhibits basal lipolysis by non-HSL(s).
In forskolin-treated cells, expression of Peri A enhanced (rather than blocked) lipolysis ϳ2-fold as compared with forskolin-treated control (p Ͻ 0.001) (Fig. 3C). Expression of Peri B and Peri 1-300 had no significant effect on lipolysis in forskolin-treated cells (p Ͼ 0.05 versus forskolin-treated control). In contrast, expression of Peri 142-517, Peri 201-517, Peri 233-517, Peri 251-517, or Peri 301-517 enhanced lipolysis in forskolin-treated cells ϳ2-fold as compared with forskolin-treated control (p Ͻ 0.001). These results identify the C-terminal region (aa 301-517) of Peri A as a domain that fully promotes PKA-stimulated lipolysis by non-HSL(s). Data obtained in the presence and absence of forskolin therefore support the conclusion that Peri 301-517 can regulate both basal and PKAstimulated lipolysis mediated by non-HSL(s).
As expected, Peri A enhanced lipolysis in forskolin-stimulated cells (ϳ2-fold) as compared with forskolin-stimulated (control) cells expressing ␤-gal or GFP (p Ͻ 0.001) (Fig. 4B). Surprisingly, expression of Peri A⌬123, Peri A⌬1-6, or Peri A⌬456 failed to enhance lipolysis in forskolin-treated cells (p Ͼ 0.05 versus forskolin-treated control). These data strongly suggest that the ability of Peri A to facilitate PKA-stimulated lipolysis by non-HSL requires phosphorylation of one or more of PKA sites 1-3 in conjunction with phosphorylation of one or more of PKA sites 4 -6. Mutation of PKA sites 1-3, 4 -6, or 1-6 did not alter the ability of Peri A to inhibit (basal) lipolysis in the absence of forskolin (p Ͼ 0.05 versus Peri A) (Fig. 4B).
Effects of Truncated and Mutated Peri A on HSL-mediated Lipolysis-Having identified the domains of Peri A that modulate non-HSL-mediated lipolysis, we examined the effects of truncated and mutated Peri A on HSL-mediated lipolysis. ACS1/FATP1 cells were co-transduced with either Ad control (␤-gal or GFP) or Ad HSL in combination with Peri A, Peri B, or Peri 1-300 (Fig. 5A). HSL migrated as an 82-kDa band on SDS-PAGE. HSL expression was not affected by co-expression with Peri A, Peri B, or Peri 1-300 as compared with control cells co-expressing HSL and either ␤-gal or GFP.
Expression of HSL alone increased basal lipolysis by ϳ50% as compared with cells lacking HSL (Fig. 5B). Co-expression of Peri A with HSL reduced lipolysis (ϳ40%) to levels observed in the absence of HSL (p Ͼ 0.05 versus cells lacking HSL), confirming the ability of Peri A to block basal lipolysis mediated by HSL. Peri B and Peri 1-300 also decreased basal lipolysis mediated by HSL (p Ͻ 0.05 versus HSL alone), although the magnitude of inhibition was approximately one-half that obtained with full-length Peri A (Fig. 5B). Reduced lipolysis in cells co-expressing HSL and either Peri 1-300 or Peri B was manifest as increased intracellular TAG storage as compared with cells expressing HSL alone (data not shown). These results suggest that the N-terminal region of Peri A and Peri B (aa 1-300) can significantly inhibit constitutive HSL lipolytic actions at the lipid droplet surface. Treatment with forskolin increased lipolysis by ϳ2.5-fold in cells expressing HSL alone (p Ͻ 0.001 versus cells lacking HSL). Co-expression of HSL with either Peri A, Peri B, or Peri 1-300 in forskolin-stimulated cells enhanced HSL-mediated lipolysis ϳ2-fold (p Ͻ 0.001 versus cells expressing HSL alone). These results suggest that aa 1-300 within Peri A and Peri B function to promote PKAstimulated lipolysis by HSL.
We next investigated modulation of HSL-mediated lipolysis by N-terminal Peri A truncations. The electrophoretic migration of HSL and N-terminal perilipin truncations on SDS-PAGE are presented in Fig. 6A. As above, HSL expression increased basal lipolysis (p Ͻ 0.001), and expression of Peri A abrogated this increase (p Ͻ 0.001) (Fig. 6B). Progressive Nterminal truncations of Peri A resulted in progressively reduced ability to inhibit HSL-mediated basal lipolysis, with no inhibition obtained with Peri 301-517 (p Ͼ 0.05 versus control). This result suggests that a domain within the first 300 amino acids of Peri A is required to block constitutive HSL lipolytic action, a conclusion consistent with our demonstration that Peri 1-300 can partially block basal lipolysis mediated by HSL (Fig. 5B).
We also investigated effects of perilipin N-terminal truncations on lipolysis in response to forskolin treatment. Forskolin dramatically increased lipolysis in cells expressing HSL and ␤-gal as compared with cells expressing only ␤-gal (control), presumably reflecting PKA-stimulated enhancement of HSL lipolytic activity (p Ͻ 0.001) (Fig. 6B). Peri A expression again resulted in enhanced lipolysis as compared with HSL alone. Similar increases in lipolysis were obtained with Peri 141-517, Peri 201-517, Peri 233-517, Peri 251-517, and Peri 301-517 (p Ͻ 0.001). Thus, progressive N-terminal truncation of Peri A resulted in reduced basal lipolysis but no decrease in PKAstimulated lipolysis. Considered together, our results indicate that the C-terminal region of Peri A (aa 301-517) has no significant effect on HSL-mediated basal lipolysis, but it fully promotes PKA-stimulated lipolysis mediated by HSL.
Our truncation analyses revealed that both the N-terminal region of Peri A (aa 1-300) containing PKA sites 1-3 and the C-terminal region of Peri A (aa 301-517) containing PKA sites 4 -6 can independently and fully promote PKA stimulated lipolysis by HSL. We therefore hypothesized that phosphorylation of one or more N-terminal PKA sites (sites 1-3) or one or more C-terminal PKA sites (sites 4 -6) in Peri A is sufficient to promote PKA-stimulated lipolysis by HSL. Previously we (22) have demonstrated that mutation of PKA sites 1-3 blocked hormone-stimulated, HSL-mediated lipolysis, but no data are available on whether or not Peri A ⌬456 or Peri A⌬1-6 differentially effect HSL-mediated lipolysis. To test this hypothesis, we measured lipolysis in ACS1/FATP1 cells co-expressing HSL and either Peri A, Peri A⌬123, Peri A⌬1-6, or Peri A⌬456. Expression of these proteins is shown in Fig. 7A.
As previously observed (Fig. 6B), HSL expression increased basal lipolysis (p Ͻ 0.001), and expression of Peri A blocked this increase (p Ͻ 0.001) (Fig. 7B). Forskolin increased lipolysis in cells expressing HSL as compared with cells expressing only ␤-gal or GFP (non-HSL control) (p Ͻ 0.001), and Peri A expression further enhanced forskolin-stimulated, HSL-mediated lipolysis (Ͼ 2-fold) as compared with HSL expression alone (p Ͻ 0.001) (Fig. 7B). Notably, mutation of PKA sites 1-3 (Peri A⌬123) and PKA sites 1-6 (Peri A⌬1-6) completely abrogated the lipolysis-enhancing effect of Peri A in forskolin-treated cells (Fig. 7B). However, mutation of PKA sites 4 -6 (Peri A⌬456) failed to diminish the ability of Peri A to promote PKA-stimulated lipolysis mediated by HSL. These results indicate that 1) phosphorylation of one or more of PKA sites 1-3 is required to facilitate PKA-mediated lipolysis by HSL, and phosphorylation of PKA sites 4 -6, which are present in Peri 301-517, is not required. PKA sites appear to play no role in regulating basal lipolysis mediated by HSL, because none of the three Peri A PKA site mutants tested exhibited impaired ability to block HSL-mediated lipolysis in the absence of forskolin (p Ͻ 0.001 versus HSL control) (Fig. 7B). DISCUSSION The present study provides a new framework for understanding how perilipins regulate lipid metabolism. In particu-  7. PKA sites 1-3 are the primary PKA sites in Peri A that allow PKA-stimulated lipolysis mediated by HSL. ACS1/FATP1 cells were transduced with either Ad control or Ad HSL in combination with adenoviruses expressing Peri A or various PKA site-mutated Peri A. The cells were incubated with FA and treated with/without forskolin as described under "Experimental Procedures." The cell extracts (10 g/sample) were immunoblotted for perilipin. The glycerol content in the conditioned medium was measured. A, ectopic expression of perilipins and HSL in ACS1/FATP1 cells. B, mutation of PKA sites 1-3 in Peri ⌬123 abrogated the enhancing effect on PKA-stimulated lipolysis. Mutation of PKA sites 4 -6 has no effect on the ability of Peri A⌬ 456 to enhance PKA-stimulated lipolysis. The results are the mean Ϯ S.E. of sextuplicate measurements and are representative of three independent experiments. lar, the use of engineered ACS/FATP1 cells enabled us to identify a surprising degree of lipase selectivity among multiple, lipolysis-regulating domains of Peri A. An important strength of our approach was our use of Peri A truncations as well as full-length Peri A PKA site mutants. This approach allowed us to integrate how Peri A regulatory domains and PKA sites functioned together to regulate lipolysis. Our data suggest a model (Fig. 8) in which 1) specific Peri A protein domains selectively act as barriers against HSL and non-HSLs, respectively; 2) phosphorylation of different PKA sites is required for PKA-stimulated lipolysis mediated by HSL and non-HSL(s); and 3) additional domains facilitate PKA-stimulated lipolysis, again with lipase selectivity.
The barrier function of Peri A maps to two lipase-specific domains; Peri 1-300 blocks HSL, and Peri 301-517 blocks non-HSL(s) (Fig. 8). Thus, it was not totally unexpected that differential patterns of Peri A phosphorylation are required for PKA-stimulated lipolysis mediated by HSL versus non-HSL(s). However, whereas phosphorylation of one or more of PKA sites 1-3 is required to stimulate HSL-mediated lipolysis, phosphorylation of one or more of PKA sites 4 -6 and 1-3 in full-length Peri A is required to stimulate non-HSL-mediated lipolysis (Fig. 8). This suggests that HSL and non-HSL(s) interact with the lipid droplet surface and gain access to stored TAG by different means. Intriguingly, Peri 301-517 does not contain sites 1-3, yet it promotes PKA-stimulated lipolysis by non-HSL(s). This observation suggests that 1) non-HSL-mediated, PKA-stimulated lipolysis requires a conformational change in the C terminus of Peri A that is induced by phosphorylation of N-terminal PKA sites 1-3, and 2) truncation of aa 1-300 results in that conformational change.
Our data indicate that the domains of Peri A that facilitate PKA-stimulated lipolysis can be separate from the domains that function as barriers to lipases. For example a domain within Peri 301-517 distinct from PKA sites 4 -6 can facilitate PKA-stimulated lipolysis by HSL (Fig. 7B), yet Peri 301-517 fails to block HSL-mediated basal lipolysis (Fig. 6B). One potential explanation for this observation is that Peri 301-517 contains a "docking site" or other facilitory domain for HSL that in full-length Peri A becomes accessible to the lipase only after phosphorylation of PKA site 1, 2, or 3. In contrast to Peri 301-517, Peri 1-300 functions as both a barrier to and facilitator of HSL actions (Fig. 5B). It is plausible that Peri 1-300 also contains an additional domain that becomes accessible to HSL after phosphorylation of PKA sites 1-3.
The present study provides the first demonstration that Peri B is biologically active as a regulator of lipolysis. The HSL selectivity of Peri B reflects the HSL-directed barrier function of aa 1-300 (inhibition of basal lipolysis), the role of PKA sites 1-3 in PKA-stimulated lipolysis, and the lack of C-terminal domains and PKA sites (sites 4 -6) that regulate non-HSL(s). The identity of aa 301-405 in Peri A and Peri B argue that the unique C-terminal domain(s) of Peri A that regulates non-HSL(s) is contained within Peri 405-517.
While our manuscript was in preparation, Tansey et al. (23) reported that the C-terminal region of Peri A inhibits basal lipolysis and that PKA sites 1-3 promote PKA-stimulated lipolysis (PKA sites 4 -6 were not examined). They also reported that Peri B had no significant effect on either basal or PKAstimulated lipolysis. These observations into perilipin function were made in Chinese hamster ovary cells, which do not express HSL.
Perilipin function requires association with the lipid droplet. Our confocal studies (Fig. 2) demonstrate that neither the perilipin N terminus (aa 1-300), C terminus (aa 301-517), nor PKA sites are required to target Peri A to lipid droplets. How-ever, Peri 1-141 (data not shown) and Peri 1-200 failed to localize to the lipid droplet (Fig. 2). Peri 1-141 contains the conserved PAT-1 region found in other lipid droplet proteins such as adipose differentiation-related protein and TIP 47 (27). The inability of Peri 1-141 and Peri 1-200 to localize to the lipid droplet confirms that the PAT-1 region is insufficient to target perilipins to the lipid droplet (34). Considered together, our confocal data support the recent identification of three putative lipid droplet-binding regions of Peri A between aa 233 and 364 (34).
The present study elucidates the structural features of perilipin that regulate adipocyte lipolysis, with surprising lipase specificity. A complete understanding of how perilipin regulates the molecular interplay among HSL and non-HSLs remains a challenge for the future. The importance of this challenge is underscored by recent studies linking reduced perilipin expression to dysregulated lipolysis in obese subjects (38,39) and by the association of increased circulating free fatty acids with the development of insulin resistance and type 2 diabetes (9,20).