Tài liệu Báo cáo khoa học: Phosphorylation of hormone-sensitive lipase by protein kinase A in vitro promotes an increase in its hydrophobic surface area ppt

Phosphorylation of hormone-sensitive lipase by protein kinase A in vitro promotes an increase in its hydrophobic surface area Christian Krintel1,2, Matthias Morgelin3, Derek T Logan2 and Cecilia Holm1 ă Department of Experimental Medical Science, Division of Diabetes, Metabolism and Endocrinology, Lund University, Sweden Department of Molecular Biophysics, Lund University, Sweden Department of Clinical Sciences, Division of Infection Medicine, Lund University, Sweden Keywords cholesterol ester hydrolase; electron microscopy; fluorescence spectroscopy; phospholipid vesicles Correspondence C Holm, Department of Experimental Medical Science, BMC, C11, SE-221 84 Lund, Sweden Fax: +46 462224022 Tel: +46 462228581 E-mail: cecilia.holm@med.lu.se (Received 10 March 2009, revised 17 May 2009, accepted 25 June 2009) doi:10.1111/j.1742-4658.2009.07172.x Hormone-sensitive lipase (EC 3.1.1.79; HSL) is a key enzyme in the mobilization of fatty acids from stored triacylglycerols HSL activity is controlled by phosphorylation of at least four serines In rat HSL, Ser563, Ser659 and Ser660 are phosphorylated by protein kinase A (PKA) in vitro as well as in vivo, and Ser660 and Ser659 have been shown to be the activity-controlling sites in vitro The exact molecular events of PKA-mediated activation of HSL in vitro are yet to be determined, but increases in both Vmax and S0.5 seem to be involved, as recently shown for human HSL In this study, the hydrophobic fluorescent probe 4,4¢-dianilino-1,1¢-binaphthyl-5,5¢-disulfonic acid (bis-ANS) was found to inhibit the hydrolysis of triolein by purified recombinant rat adipocyte HSL, with a decrease in the effect of bis-ANS upon PKA phosphorylation of HSL The interaction of HSL with bis-ANS was found to have a Kd of lm in binding assays Upon PKA phosphorylation, the interactions of HSL with both bis-ANS and the alternative probe SYPRO Orange were increased By negative stain transmission electron microscopy, phosphorylated HSL was found to have a closer interaction with phospholipid vesicles than unphosphorylated HSL Taken together, our results show that HSL increases its hydrophobic nature upon phosphorylation by PKA This suggests that PKA phosphorylation induces a conformational change that increases the exposed hydrophobic surface and thereby facilitates binding of HSL to the lipid substrate Structured digital abstract l MINT-7211789: PKA (uniprotkb:P05132) phosphorylates (MI:0217) HSL (uniprotkb:P15304) by protein kinase assay (MI:0424) Introduction In mammals, fatty acids are mobilized from stored triacylglycerols by the consecutive action of adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL), and monoacylglycerol lipase [1] Phosphorylation of HSL by protein kinase A (PKA) is central to the molecular control of lipolysis, but other events, notably phosphorylation of the lipid droplet protein perilipin, are also of key importance In adipocytes, stimulation of lipolysis by catecholamines results in activation of adenylate cyclase, leading to elevated Abbreviations ATGL, adipose triglyceride lipase; bis-ANS, 4,4¢-dianilino-1,1¢-binaphthyl-5,5¢-disulfonic acid; HSL, hormone-sensitive lipase; LPL, lipoprotein lipase; PKA, protein kinase A; TO, triolein 4752 FEBS Journal 276 (2009) 4752–4762 ª 2009 The Authors Journal compilation ª 2009 FEBS C Krintel et al levels of cAMP, which causes the catalytic subunits of PKA to dissociate from the regulatory subunits and thereby become active [2,3] On the other hand, insulin prevents lipolysis, an effect mainly executed via activation of phosphodiesterase 3B, thus lowering cAMP levels Both HSL and perilipin are phosphorylated directly by PKA, whereas ATGL and its cofactor CGI58 appear to be indirectly controlled by PKA [2,4] Nevertheless, these phosphorylation events appear to promote the interaction of both ATGL and HSL with the stored lipids, thus increasing hydrolysis of the latter Whereas CGI58 and perilipin form a complex under basal conditions, they dissociate after phosphorylation of perilipin by PKA ATGL then forms a new complex with CGI58, rendering ATGL enzymatically active [5,6] HSL is known to translocate to perilipin-containing lipid droplets after stimulation of lipolysis [7], but a direct interaction between the two proteins has never been proven The exact molecular events following PKA phosphorylation of HSL and perilipin leading to the activation of lipolysis remain to be elucidated Using HSL from several different species, it has been shown that its activity increases approximately 100% after in vitro phosphorylation by PKA [8–10] In rat HSL, Ser563, Ser659 and Ser660 are phosphorylated by PKA in vitro as well as in vivo [9] Of these, Ser659 and Ser660, corresponding to Ser649 and Ser650 in human HSL, have been shown to regulate activity in vitro [9,11] In contrast to the relatively large number of studies devoted to the elucidation of the phosphorylation sites in HSL and the regulation of its translocation, few studies have addressed the effects of PKA phosphorylation on the HSL molecule itself However, in a recent study, we showed that even though PKA phosphorylation increases the activity of human HSL in vitro, the affinity for triolein (TO) emulsions decreases [9,11] This may reflect the fact that PKA phosphorylation induces structural changes in the vicinity of the lipid-binding region of HSL Thus, it is possible that HSL adopts a more open and flexible conformation upon PKA phosphorylation, allowing for easier release of product molecules and leading to an increased turnover rate Previous work has shown that lipoprotein lipase (LPL) interacts strongly with, and in fact is inhibited by, the hydrophobic probe 4,4¢-dianilino-1,1¢-binaphthyl-5,5¢-disulfonic acid (bis-ANS) [12] Another hydrophobic probe, i.e SYPRO Orange, is now routinely used for differential scanning fluorimetry in thermal denaturation experiments for buffer optimization prior to crystallization trials [13] As these probes bind to exposed hydrophobic patches in proteins, they were PKA phosphorylation of hormone-sensitive lipase used in this study to generate evidence that PKA-phosphorylated HSL exhibits an increase in the solventexposed hydrophobic surface area as compared with the unphosphorylated enzyme Further proof was obtained from negative stain transmission electron microscopy studies, which demonstrated that HSL interacts more closely with phospholipid vesicles following PKA phosphorylation Results Expression and purification of C-terminally His-tagged rat adipocyte HSL C-terminally His-tagged rat HSL was successfully expressed in Sf9 insect cells using the baculovirus ⁄ insect cell expression system The protein was purified by anion exchange chromatography followed by nickel affinity chromatography and dialysis (Fig 1) Western blot analysis confirmed the identity of the purified protein as HSL (data not shown) The yield of pure protein was mg per litre of insect cell culture The specific activities of the purified protein against TO, 1-mono-oleoyl-2-O-mono-oleylglycerol and cholesterol oleate were 2.6 mg)1, 30 mg)1, and 3.5 mg)1, respectively These specific activities are lower than those previously reported for nontagged 250 kDa 150 kDa 100 kDa 50 kDa 37 kDa 25 kDa Fig Purity of recombinant HSL SDS ⁄ PAGE gel displaying the expression and purification of recombinant C-terminally His-tagged rat adipocyte HSL Lane 1: supernatant fraction of lysed Sf9 cells expressing HSL Lane 2: molecular mass marker Lane 3: purified rat adipocyte HSL FEBS Journal 276 (2009) 4752–4762 ª 2009 The Authors Journal compilation ª 2009 FEBS 4753 PKA phosphorylation of hormone-sensitive lipase C Krintel et al recombinant rat HSL, but are in accordance with the published activity of His-tagged human HSL [11,14] HSL inhibition by bis-ANS To evaluate whether bis-ANS had an effect on the enzymatic activity of HSL, lipase assays were performed with increasing amounts of bis-ANS using TO as substrate Bis-ANS inhibited the activity of both phosphorylated and nonphosphorylated HSL Normalization of the two sets of data revealed differences between the normalized activities of phosphorylated and nonphosphorylated HSL at bis-ANS concentrations above lm, indicating that the bis-ANS interaction with HSL is altered upon phosphorylation of HSL by PKA (Fig 2) HSL interaction with bis-ANS To evaluate the binding of bis-ANS to nonphosphorylated HSL, we measured the fluorescence of bis-ANS in complex with HSL With an excitation wavelength of 296 nm, the emission was scanned between 300 nm and 550 nm at bis-ANS concentrations ranging from 0.1 to 10 lm, both with and without added HSL The HSL–bis-ANS complex fluorescence was derived by subtracting spectra of bis-ANS alone from spectra obtained with added HSL The HSL–bis-ANS complex maximum emission wavelength ranged from 476 nm at 0.1 lm bis-ANS to 488 nm at 10 lm bis-ANS, indicating that there could be more than one binding site for bis-ANS on HSL (Fig 3A) The maximum emission intensity of the complex increased in an inverse hyper- Fig Interaction of bis-ANS with HSL Spectra of bis-ANS obtained at concentrations ranging from 0.1 to 10 lM were subtracted from spectra of bis-ANS mixed with HSL in order to generate difference spectra displaying the interaction between HSL and bis-ANS (A) The maximum fluorescence increased according to the concentration of bis-ANS, following a hyperbolic curve (r2 = 0.998), which provided a Kd of 1.0 lM for the HSL–bis-ANS complex (B) The spectra shown have been smoothed (using two neighbouring values) bolic fashion (r2 = 0.99 for an inverse hyperbolic curve fit), and Kd for the complex was determined to be 1.00 lm bis-ANS (Fig 3B) HSL phosphorylation and activation with variable ATP concentrations Fig Inhibition of HSL lipase activity by bis-ANS HSL, phosphorylated or nonphosphorylated, was preincubated for 10 and assayed in the presence of the given concentrations of the hydrophobic probe bis-ANS, using TO as substrate The activity of phosphorylated HSL in the TO assay was normalized to the activities of nonphosphorylated HSL, and the activities were compared (unpaired nonparametric t-test, n = 4) 4754 Because ATP interacted with both bis-ANS and SYPRO Orange, creating high levels of background fluorescence, we investigated the possibility of using lower amounts of ATP for the phosphorylation of HSL Using radiolabelled ATP, we showed that, by using only 15 lm ATP in the phosphorylation reaction mix, we could obtain a similar extent of phosphorylation to that obtained at an ATP concentration of 200 lm (Fig 4A) The stoichiometry of phosphorylation was 0.20 mol phosphate per mol HSL for the 200 lm ATP reaction, in accordance with results published for human HSL [11], and 0.16 mol phosphate FEBS Journal 276 (2009) 4752–4762 ª 2009 The Authors Journal compilation ª 2009 FEBS C Krintel et al Fig In vitro phosphorylation and activation of HSL HSL was phosphorylated in the presence of radiolabelled ATP, with total ATP concentrations in the phosphorylation reaction of 15 lM or 200 lM, and analysed for incorporation of 32P (A) and activity against the TO substrate (B) The results in (B) include two individual experiments (n = 6) Data represents means ± standard error of six assays *P < 0.05, ***P < 0.0005, unpaired, nonparametric t-test per mol HSL for the 15 lm ATP reaction In accordance with the similar degree of phosphorylation at the two ATP concentrations, there was no significant difference between the activation levels obtained at the two different ATP concentrations with TO as substrate (Fig 4B) Incubatation of the enzyme preparations with alkaline phosphatase did not affect the enzymatic activity (data not shown) This indicates that HSL purified from the baculovirus ⁄ insect cell system was obtained in a dephosphorylated form, at least with regard to activity-controlling sites This is in agreement with previous reports for His-tagged human HSL and non-tagged rat HSL [11,15] HSL interaction with bis-ANS and SYPRO Orange after phosphorylation To investigate whether HSL gains hydrophobic surface area upon phosphorylation by PKA, we analysed the PKA phosphorylation of hormone-sensitive lipase interaction of phosphorylated HSL with bis-ANS in comparison with nonphosphorylated HSL, using fluorescence Even at ATP concentrations of 15 lm in the phosphorylation reaction mix, resulting in a final concentration below 150 nm in the fluorescence measurements, the interaction between bis-ANS and ATP was too strong for reliable spectra to be obtained Therefore, HSL samples were dialysed after phosphorylation and then reanalysed for protein content before being used in fluorescence measurements Spectra of samples containing phosphorylated and dialysed HSL mixed with bis-ANS were recorded, and spectra of dialysed phosphorylation mixes (including PKA, which provided only minor contributions to the total fluorescence) lacking HSL were subtracted to eliminate the influence of interactions with buffers and PKA, thus providing difference spectra solely representing the interaction between HSL and bis-ANS (Fig 5A,B) The spectra for phosphorylated HSL were above the spectra for nonphosphorylated HSL for all three concentrations tested Owing to the interaction between bis-ANS and ATP, the binding assay provided only reliable performance in a short range of the bis-ANS concentrations tested in Fig 2, and only lm, 1.5 lm and lm bisANS provided acceptable signal-to-noise ratios At low bis-ANS concentrations, the HSL–bis-ANS complex fluorescence was lost in noise, and at high concentrations, the absolute fluorescence signal was out of range for the instrumentation Thus, to further verify the increase in hydrophobicity of HSL after phosphorylation by PKA, we also applied an alternative hydrophobic probe, i.e SYPRO Orange HSL was incubated with or without PKA, and the fluorescence after excitation at 492 nm was recorded by scanning the emission between 550 nm and 800 nm In order to create difference spectra illustrating solely the interaction between HSL and SYPRO Orange, spectra of phosphorylation reaction mixes lacking both HSL and PKA or lacking only HSL were subtracted from the recorded spectra of the nonphosphorylated or phosphorylated samples, respectively An advantage with this approach was that even though ATP also interfered with these measurements (the performance of the assay was reliable only within a limited range of probe concentrations), dialysis after phosphorylation was not necessary, probably because of a lower degree of interaction of ATP with SYPRO Orange than with bis-ANS The molar concentration of SYPRO Orange is impossible to calculate, as the molecular mass is not publicly available, but the relative concentrations of SYPRO Orange tested in the measurements were · 0.2, · 0.25, · 0.5, and · For all tested concentra- FEBS Journal 276 (2009) 4752–4762 ª 2009 The Authors Journal compilation ª 2009 FEBS 4755 PKA phosphorylation of hormone-sensitive lipase C Krintel et al Fig Comparison of fluorescence from the hydrophobic probes bis-ANS and SYPRO Orange in complex with phosphorylated and nonphosphorylated HSL HSL was phosphorylated by PKA, dialysed, and mixed with bis-ANS, and spectra were recorded at an excitation wavelength of 296 nm Spectra of reaction mixes containing no HSL were subtracted to generate the displayed difference spectra illustrating the interaction between bis-ANS and HSL The concentrations of bis-ANS used were 1.5 lM and 2.0 lM in (A) and (B), respectively HSL was phosphorylated with 15 lM ATP in the reaction mix, and mixed with SYPRO Orange, and spectra were recorded at an excitation wavelength of 492 nm Spectra of reaction mixes lacking HSL or both HSL and kinase were subtracted from the spectra of the nonphosphorylated and phosphorylated HSL samples, respectively, to generate the displayed difference spectra illustrating the interaction between SYPRO Orange and HSL The concentrations of SYPRO Orange used were · 0.25 and · 0.5 in (C) and (D), respectively The spectra shown are smoothed (using four neighbouring values) and normalized to the maximum fluorescence of the complex between phosphorylated HSL and the respective probes tions of SYPRO Orange, the fluorescence of phosphorylated HSL lay above the fluorescence of nonphosphorylated HSL, indicating that HSL gains hydrophobic surface area upon phosphorylation by PKA (Fig C,D) Electron microscopy of phosphorylated and nonphosphorylated HSL As a first attempt to investigate whether the increase in hydrophobic surface area is reflected by increased binding of phosphorylated HSL to lipid surfaces, we investigated the interaction between HSL and phospholipid vesicles using negative stain electron microscopy Phospholipid vesicles mimic the lipid droplets found in vivo, which are covered by a single layer of phospholipids, but avoid the problems of lipid hydrolysis during analysis, as HSL is known not to exhibit phospholipase activity Furthermore, we know from previous work that HSL associates with phospholipid vesicles [15] Thus, sonicated phosphatidylcholine vesicles were mixed with either nonphosphorylated 4756 or PKA-phosphorylated HSL, stained with uranyl formate, and analysed by transmission electron microscopy In the obtained micrographs, HSL appeared as light particles even when observed inside the vesicles When vesicles mixed with nonphosphorylated HSL (Fig 6A) were compared with vesicles mixed with phosphorylated HSL (Fig 6B), 12% of the imaged vesicles contained nonphosphorylated HSL, and 82% of the imaged vesicles contained phosphorylated HSL (based on observing 300 vesicles for each condition) In addition, the content of HSL in each vesicle was markedly increased for phosphorylated HSL (Fig 6D) when compared with nonphosphorylated HSL (Fig C), reflecting a stronger interaction with phospholipids and ⁄ or the fact that phosphorylated HSL more easily penetrates the phospholipid membrane to gain access to the underlying lipid substrate The presence of HSL in the vesicles was confirmed by immunogold electron microscopy (Fig 6E,F) When vesicles mixed with nonphosphorylated HSL (Fig 6E) were compared with vesicles mixed with phosphorylated HSL (Fig 6F), 23% of the imaged vesicles contained nonphosphory- FEBS Journal 276 (2009) 4752–4762 ª 2009 The Authors Journal compilation ª 2009 FEBS C Krintel et al PKA phosphorylation of hormone-sensitive lipase A Fig Negative stain electron microscopy analysis of the interaction between HSL and phospholipid vesicles Electron micrograph illustrating HSL integrated into phospholipid vesicles in either the nonphosphorylated form (A, C) or phosphorylated form (B, D) HSL appears as white shadows inside vesicles to a much higher extent, and is present in higher numbers per vesicle for the phosphorylated enzyme than for the nonphosphorylated one Immunogold labelling confirms the presence of HSL associated with and inside the vesicles [nonphosphorylated HSL in (E), and phosphorylated HSL in (F)] The size bar in (F) corresponds to 50 nm (A, B, E, F) and 25 nm (C, D) E B F C lated HSL, and 74% of the imaged vesicles contained phosphorylated HSL (based on observing 300 vesicles for each condition), which is in good agreement with the observations made without immunogold labelling (Fig 6A–D) Discussion In this study, we used recombinant rat adipocyte HSL produced in Sf9 cells using baculovirus-mediated expression to demonstrate that HSL undergoes a conformational change upon PKA phosphorylation, which increases the solvent-exposed hydrophobic surface area The fluorescent probe bis-ANS has previously been used to study the lipid-binding properties of another mammalian lipase, i.e LPL [12] Bis-ANS was found to bind tightly to LPL in the vicinity of the active site and also to inhibit the enzymatic activity [12] Similarly, we show here that bis-ANS inhibits the lipase activity of HSL Interestingly, there were only minor effects of bis-ANS concentrations up to 60 lm on HSL activity when preincubation of the enzyme with bisANS prior to assaying was omitted We believe that this is due to the presence of BSA in the assay buffer and the lipid substrate emulsion, but not in the preincubation buffer, in accordance with the observation that LPL was not inhibited by bis-ANS when BSA D was present in the assay This is also supported by the observation that even though the HSL–bis-ANS complex has a Kd of lm (Fig 3), much higher concentrations are needed to decrease HSL activity (Fig 2) Scavenging of bis-ANS by BSA is presumably the reason for a sigmoidal inhibition curve, rather than the expected hyperbolic one The binding of bis-ANS to HSL followed an inverse hyperbolic saturation curve The estimated Kd of lm is lower than what has been reported for most other proteins [16], indicating that HSL exhibits high affinity for bis-ANS, although not as high as that of LPL, for which the Kd was reported to be 0.10–0.26 lm [12] The maximum emission wavelength of the HSL–bisANS complex shifted 13 nm from the lowest to the highest concentrations of bis-ANS This shift suggests that there is more than one binding site for bis-ANS on HSL The change in solvent-exposed hydrophobic surface area of HSL following PKA phosphorylation was examined using both bis-ANS and SYPRO Orange Because of the interaction of both bis-ANS and SYPRO Orange with ATP, we were forced to use significantly lower ATP concentrations in these experiments than those normally used Thus, prior to the fluorescence experiments with these hydrophobic probes, we established that the use of 15 lm ATP in the phosphorylation reaction resulted in the same FEBS Journal 276 (2009) 4752–4762 ª 2009 The Authors Journal compilation ª 2009 FEBS 4757 PKA phosphorylation of hormone-sensitive lipase C Krintel et al degree of both phosphorylation and activation as the use of 200 lm ATP (Fig 4A,B) The interaction between bis-ANS and dialysed phosphorylated HSL was measured using lm, 1.5 lm and lm bis-ANS The fluorescence of the phosphorylated form of HSL was found to be substantially higher than that of the nonphosphorylated form at all three concentrations This result was verified using SYPRO Orange, which interacts to a lesser degree with ATP than does bisANS This enabled us to employ a simplified protocol without the need for dialysis prior to fluorescence measurements when using only 15 lm ATP in the preceding phosphorylation reactions Relative concentrations of · 0.2, · 0.25, · 0.5 and · 1.0 SYPRO Orange were used for the comparison of phosphorylated and nonphosphorylated HSL The fluorescence from phosphorylated HSL was higher than that from nonphosphorylated HSL for all four concentrations of SYPRO Orange, thus strengthening the argument that HSL gains solvent-exposed hydrophobic surface area upon phosphorylation Electron microscopy of phospholipid vesicles mixed with phosphorylated and nonphosphorylated HSL demonstrated a more pronounced interaction with the vesicles for the phosphorylated variant, in terms of both the number of vesicles invaded by HSL and the larger HSL content of the individual vesicles (Fig 6) This may be due to the increased hydrophobic nature of phosphorylated HSL as compared with nonphosphorylated HSL, although alternative explanations exist For instance, it is possible that phosphorylated HSL binds more avidly to the polar head of the phospholipids and that this is followed by an interaction between the apolar acyl chains of the phospholipids and side chains of particular amino acids, thus accounting for the increased capacity to penetrate to the interior of the vesicle Phospholipid vesicles mimic the lipid droplets found in vivo, but avoid the problem of hydrolysis, as HSL lacks phospholipase activity It is indeed possible that binding of phosphorylated HSL to the phospholipid vesicles, followed by penetration of the membrane, mimics what happens in vivo as HSL is anchored to the lipid droplet to hydrolyse acylglycerols Even though PKA-phosphorylated HSL was found to bind more bis-ANS than nonphosphorylated HSL, the inhibition of TO activity by bis-ANS was decreased upon phosphorylation A possible explanation for this apparent discrepancy could be that PKA phosphorylation induces a conformational change that increases the accessible hydrophobic surface area, enabling freer access to the lipid-binding site, in return for weaker binding This is well in line with our recent kinetic measurements on human HSL, showing that 4758 PKA phsophorylation increases both maximum turnover rate and S0.5 [11] In this study, we provide evidence that HSL gains accessible hydrophobic surface area upon PKA phosphorylation This gain in hydrophobic surface area presumably accounts for the increase in in vitro activity of HSL following PKA phosphorylation through increased binding between HSL and the lipid substrate emulsion It is possible that the gain in accessible hydrophobic surface area not only affects the ability of HSL to interact with the lipid droplet, but is also is involved in driving the translocation of HSL that occurs upon lipolytic stimulation of adipocytes [7] The exact molecular events involved in the translocation of HSL are, as yet, incompletely understood It seems clear that perilipin is required for translocation of HSL, although a direct interaction between the two proteins has been not been demonstrated [5,6,17] Data are emerging that point to perilipin as a key player in directing proteins involved in lipolysis to a subset of lipid droplets [5] Interestingly, we recently showed that the affinity of human HSL for TO decreased in in vitro asssays upon PKA phosphorylation [11] Taken together with the results presented here, this underscores the fact that the affinity measured in activity assays involves several aspects of lipase activity, i.e adsorption, entry and binding of individual lipid molecules to the enzyme Future studies will be needed to determine whether the phosphorylation-induced gain in hydrophobic surface area described here affects other properties of HSL than binding to lipids, e.g binding to lipid droplet-associated proteins In conclusion, our results demonstrate that HSL increases its hydrophobic nature upon phosphorylation by PKA Thus, it can be speculated that phosphorylation of HSL by PKA induces a conformational change that exposes and ⁄ or increases the lipid-binding area of the enzyme A direct demonstration of this presumed conformational change will have to await the solving of the atomic structure of HSL in its native and phosphorylated forms Experimental procedures Expression and purification of C-terminal His-tagged recombinant rat adipocyte HSL To generate a recombinant baculovirus encoding C-terminally tagged rat adipocyte HSL, full-length rat adipocyte HSL cDNA, including a sequence encoding one Pro residue and eight C-terminal His residues, was subcloned into the BamHI and XbaI sites of pVL1393, as follows The PCR FEBS Journal 276 (2009) 4752–4762 ª 2009 The Authors Journal compilation ª 2009 FEBS C Krintel et al product obtained using the sense primer 5¢-ATC ATC TCC ATC GAC TAC TCC CTG-3¢, the antisense primer 5¢-AAG AAT TCT AGA TTA ATG GTG ATG ATG GTG ATG ATG GTG TGG GGT CAG CGG TGC AGC AGG GGG GGT-3¢ (XbaI sites underlined; His-tag in italic) and pVL1393–HSL [18] as template was digested using XbaI and BssHII and subcloned into pVL1393–HSL PCR was performed using Vent polymerase (New England Biolabs, Ipswich, MA, USA), and the PCR product was sequenced using BigDye (Applied Biosystems, Foster City, CA, USA) upon subcloning Recombinant virus was generated by transfecting Sf9 cells using the BaculoGold Transfection Kit (BD Biosciences Pharmingen, San Diego, CA, USA), according to the manufacturer’s instructions but using the Sf-900 II medium instead of TMN-FH Plaque purification was performed and high-titre virus stocks were generated using standard procedures For protein expression, Sf9 insect cells were grown at 27 °C in suspension cultures (160 r.p.m.) in Sf-900 medium supplemented with 4% fetal bovine serum and 1% penicillin ⁄ streptomycin (all from Gibco, through Invitrogen AB, Lidingo, Sweden) Cell cultures (2 Ã 106 cellsặmL)1) were ă infected at a multiplicity of infection of 10 Infection was followed by a 72 h expression period Cells were harvested by centrifugation (1200 g, 10 min), and resuspended in five pellet volumes of lysis buffer (50 mm Tris ⁄ HCl, pH 8.0, mm dithiothreitol, mm EDTA, 1% C13E12, 10% glycerol) The cell suspension was gently sonicated and centrifuged for 45 at °C and 50 000 g The supernatant fraction was filtered through a 0.22 lm filter and loaded onto a Q-Sepharose Fast Flow anion exchange column (GE Healthcare, Uppsala, Sweden) The column was washed with 10 volumes of 50 mm NaCl, 20 mm Tris ⁄ HCl (pH 8.0), mm dithiothreitol, mm EDTA, 0.01% C8E4, and 10% glycerol, and then eluted with approximately two column volumes of m NaCl, 20 mm Tris ⁄ HCl (pH 8.0), 0.1 mm dithiothreitol, 0.01% C8E4, and 10% glycerol Protein eluted from the Q-Sepharose column was loaded directly onto a nickel affinity chromatography column (Ni2+–nitrilotriacetic acid Superflow; Qiagen, Valencia, CA, USA), washed with 10 volumes of 18 mm imidazole, 300 mm NaCl, 50 mm Tris ⁄ HCl (pH 8.0), 0.1 mm dithiothreitol, 1% Triton X-100, and 10% glycerol, and 15 volumes of mm imidazole, 300 mm NaCl, 50 mm Tris ⁄ HCl (pH 8.0), 0.1 mm dithiothreitol, 0.01% C8E4, and 10% glycerol, and eluted with a stepwise gradient towards 250 mm imidazole, 300 mm NaCl, 50 mm Tris ⁄ HCl (pH 8.0), mm dithiothreitol, 0.01% C8E4, and 10% glycerol The eluted protein was then dialysed overnight against 50 mm Tris ⁄ HCl (pH 8.0), 300 mm NaCl, mm dithiothreitol, 0.01% C8E4, and 10% glycerol, and stored at )80 °C Protein amounts were measured by the 2D Quant method (GE Healthcare) and the Bradford method [19] The latter underestimated HSL content by a factor of 1.5 relative to the 2D Quant method The C-terminally His-tagged rat PKA phosphorylation of hormone-sensitive lipase HSL was used for all analyses in this study except for the electron microscopy studies, where nontagged rat HSL, expressed and purified as described in [15], was used HSL activity assays HSL lipase activity was measured against phospholipidstabilized emulsions of TO, 1-mono-oleoyl-2-O-mono-oleylglycerol or cholesterol oleate [18,20] Briefly, labelled and nonlabelled lipid substrates and phosphatidylcholine ⁄ phosphatidylinositol (3 : 1) in cyclohexane solutions were dried under a stream of N2, and this was followed by emulsification by sonication and addition of 2% BSA (1-monooleoyl-2-O-mono-oleylglycerol assay) or 5% BSA (TO and cholesterol oleate assays) Enzymes were diluted to a suitable concentration in 100 lL of 20 mm potassium phosphate (pH 7.0), mm EDTA, mm dithiothreitol, and 0.02% BSA, and 100 lL of the emulsified substrate was added and mixed Reactions were typically incubated for a period of 30 at 37 °C before the reaction was quenched by the addition of 3.25 mL of methanol ⁄ chloroform ⁄ heptane (10 : : 7) and 1.1 mL of 0.1 m potassium carbonate and 0.1 m boric acid (pH 10.5) Samples were then vortexed and centrifuged (800 g, 20 min), and the content of released fatty acids in the upper phase was determined by scintillation counting For all assays, we confirmed that the reaction velocity was constant during the 30 incubation period HSL inhibition by bis-ANS HSL was incubated for 5–10 in 50 mm Tris ⁄ HCl (pH 8), 300 mm NaCl, 10% glycerol, mm dithiothreitol, 0.02% C8E4 and 10 mm MgCl2 containing either lm, 10 lm, 15 lm, 20 lm, 30 lm, 45 lm or 60 lm bis-ANS at room temperature and assayed against TO as previously described, with the exception that the resulting reaction mixtures contained either lm, 10 lm, 15 lm, 20 lm, 30 lm, 45 lm or 60 lm bis-ANS For all assays including bis-ANS, the reaction rates were constant for at least 30 of incubation at 37 °C HSL phosphorylation using 32 P-labelled ATP HSL (9 lg) was phosphorylated at room temperature in 110 lL volumes containing 50 mm Tris ⁄ HCl (pH 8), 300 mm NaCl, 10% glycerol, mm dithiothreitol, 0.02% C8E4, 10 mm MgCl2, 25 U of PKA (New England Biolabs), protease inhibitor cocktail (Roche Complete; Roche Diagnostics, Mannheim, Germany), and either 200 lm ATP and 0.3 lCiỈlL)1 [32P]ATP[cP], or 15 lm ATP and 0.0225 lCiỈlL)1 [32P]ATP[cP] Aliquots were taken after 4, 8, 16, 32 and 64 of incubation, and quenched by the addition of Laemmli buffer [21] In control reactions, PKA was omitted Samples were analysed by SDS ⁄ PAGE, stained with Coomassie, scanned, and slab dried FEBS Journal 276 (2009) 4752–4762 ª 2009 The Authors Journal compilation ª 2009 FEBS 4759 PKA phosphorylation of hormone-sensitive lipase C Krintel et al 32 P-labelled HSL was detected as described above For quantification of incorporated phosphate, HSL bands were excised from the gel and placed in scintillation vials containing 10 mL of scintillation liquid and quantified on a scintillation counter (Wallac 1414 liquid scintillation counter; Perkin Elmer, Waltham, MA, USA) The original reaction mixtures were included as standards HSL phosphorylation by PKA for activity and hydrophobicity measurements HSL (4 lg) was phosphorylated in 50 lL volumes containing 50 mm Tris ⁄ HCl (pH 8), 300 mm NaCl, 10% glycerol, mm dithiothreitol, 0.01% C8E4, 10 mm MgCl2, 15 lm or 200 lm ATP and 0.25 U of PKA ⁄ lL supplemented with a protease inhibitor cocktail (Roche Complete) for h at room temperature For activity measurements, the protein was diluted to suitable amounts and assayed in the TO assay For hydrophobicity measurements using SYPRO Orange, lL of the phosphorylation reaction mixture was used in measurements Fluorescence measurements including HSL and bis-ANS Fluorescence measurements were performed essentially as in [12] One millilitre of 50 mm Tris ⁄ HCl (pH 8), 300 mm NaCl, 10% glycerol, mm dithiothreitol, 0.01% C8E4, and 10 mm MgCl2, including the respective concentration of bis-ANS, was excited at 296 nm, and emission was scanned from 300 to 550 nm Subsequently, samples of HSL in 50 mm Tris ⁄ HCl (pH 8), 300 mm NaCl, 10% glycerol, mm dithiothreitol, 0.01% C8E4 and 10 mm MgCl2 were added to the cuvette, and fluorescence was recorded similarly The first spectrum not containing HSL was subtracted from the later spectra containing HSL, creating difference spectra reflecting the interaction between bisANS and HSL The amount of HSL used for each spectrum ranged between 0.8 lg and 2.4 lg, depending on the concentration of bis-ANS Owing to a strong interaction between bis-ANS and ATP, phosphorylated HSL samples had to be dialysed twice for h against 10 000 volumes of 50 mm Tris ⁄ HCl (pH 8), 300 mm NaCl, 10% glycerol, mm dithiothreitol, 0.01% C8E4 and 10 mm MgCl2 to remove ATP and thereby decrease background fluorescence After dialysis, samples were centrifuged for 20 at 25 000 g, and protein contents were remeasured using the Bradford method before analysis Spectra were recorded as described above Reactions without added HSL were used as controls for the interaction between bis-ANS and PKA: spectra recorded with samples containing only PKA were subtracted from spectra containing HSL Even after dialysis of samples, there was a considerable interaction with ATP, and signal- 4760 to-noise ratios decreased from 5.7 without ATP added to 1.4 in samples including ATP Fluorescence measurements including HSL and SYPRO Orange For the evaluation of the increase in hydrophobicity of HSL after phosphorylation by PKA, the commercial probe SYPRO Orange (from Molecular Probes through Invitrogen AB) was used The measurements were performed in quartz cuvettes by adding samples to mL of 50 mm Tris ⁄ HCl (pH 8), 300 mm NaCl, 10% glycerol, mm dithiothreitol, 0.01% C8E4, 10 mm MgCl2, and SYPRO Orange The sample was excited at 492 nm, and emission was scanned from 550 to 800 nm HSL was phosphorylated as described above, using 15 lm ATP in the reaction mix In parallel with the phosphorylation reaction, a mock phosphorylation reaction without added PKA was used for measurements on nonphosphorylated HSL Similarly, a reaction in which HSL was replaced by dialysis buffer and without PKA was performed, and a spectrum of this reaction was subtracted from the spectrum recorded for the nonphosphorylated HSL sample The experiments were carried out at concentrations of SYPRO Orange ranging from · 0.25 to · The concentrations providing the best reproducibility for these experiments were · 0.25 and · 0.5, where the signal-tonoise ratio was the highest, i.e 2.4 in the absence of PKA, and 3.2 in the presence of PKA Negative stain transmission electron microscopy analysis of the interaction between HSL and sonicated phospholipid vesicles Phosphatidylcholine vesicles were prepared as described in [15] In brief, phosphatidylcholine was evaporated under nitrogen to remove the solvent Evaporation was repeated twice after addition of 0.1 mL of freshly distilled, dried diethyl ether The resulting lipid film was placed under reduced pressure for 12 h, and then allowed to swell for 30 at room temperature in 20 mm Tris ⁄ HCl (pH 7.0), 0.1 m NaCl, mm EDTA and mm dithioerythritol at a final concentration of 25 mgỈmL)1 phosphatidylcholine After swelling, the solution was sonicated with 0.5 s pulses for 45 at °C under nitrogen with a microtip sonicator (model B-15P; Branson, Danbury, CT, USA) at a setting of 50% of maximum intensity The clear solution obtained was centrifuged at 100 000 g for 60 to remove multilamellar vesicles and titanium from the microtip Vesicle samples were mixed with HSL and immediately prepared for electron microscopy In some experiments, HSL containing vesicles were incubated for 30 at room temperature with antibodies against HSL that were conjugated with nm colloidal gold as described by Baschong and Wrigley FEBS Journal 276 (2009) 4752–4762 ª 2009 The Authors Journal compilation ª 2009 FEBS C Krintel et al [22] All protein concentrations were in the 10–20 nm range Subsequently, lL aliquots of the solution were adsorbed onto carbon-coated grids for min, washed with two drops of water, and stained on two drops of 0.75% uranyl formate Prior to this, the grids were rendered hydrophilic by glow discharge at low pressure in air Specimens were observed in a Jeol JEM 1230 electron microscope operated at 60 kV accelerating voltage (Jeol, Tokyo, Japan) Images were recorded with a Gatan Multiscan 791 CCD camera (Gatan UK, Abingdon, UK) [23] Acknowledgements We would like to thank B Danielsson and M Baum´ garten for excellent technical assistance, and R Wallen and E Hallberg (Cell and Organism Biology, Lund University) for help with electron microscopy Financial support was obtained from the Swedish Research Council (project no 11284 to C Holm, and project no 7480 to M Morgelin), the Swedish Diabetes Assoă ciation, Faculty of Medicine at Lund University, and ˚ the following foundations: Novo Nordisk, A Pahlsson, Salubrin Druvan, Johan och Greta Kock, Alfred ă Osterlund, Crafoord, Konung Gustav V:s 80-arsfond and Torsten and Ragnar Soderberg C Krintel was ă supported by the Research School in Pharmaceutical ă Sciences (FLAK) References Schweiger M, Schreiber R, Haemmerle G, Lass A, Fledelius C, Jacobsen P, Tornqvist H, Zechner R & Zimmermann R (2006) Adipose triglyceride lipase and hormone-sensitive lipase are the major enzymes in adipose tissue triacylglycerol catabolism J Biol Chem 281, 40236–40241 Duncan RE, Ahmadian M, Jaworski K, Sarkadi-Nagy E & Sul HS (2007) Regulation of lipolysis in adipocytes Annu Rev Nutr 27, 79–101 Langin D & Arner P (2006) Importance of TNFalpha and neutral lipases in human adipose tissue lipolysis Trends Endocrinol Metab 17, 314–320 Greenberg AS, Egan JJ, Wek SA, Garty NB, Blanchette-Mackie EJ & Londos C (1991) Perilipin, a major hormonally regulated adipocyte-specific phosphoprotein associated with the periphery of lipid storage droplets J Biol Chem 266, 11341–11346 Granneman JG & Moore HP (2008) Location, location: protein trafficking and lipolysis in adipocytes Trends Endocrinol Metab 19, 3–9 Granneman JG, Moore HP, Granneman RL, Greenberg AS, Obin MS & Zhu Z (2007) Analysis of lipolytic protein trafficking and interactions in adipocytes J Biol Chem 282, 5726–5735 PKA phosphorylation of hormone-sensitive lipase Egan JJ, Greenberg AS, Chang MK, Wek SA, Moos MC Jr & Londos C (1992) Mechanism of hormonestimulated lipolysis in adipocytes: translocation of hormone-sensitive lipase to the lipid storage droplet Proc Natl Acad Sci USA 89, 8537–8541 Stralfors P & Belfrage P (1983) Phosphorylation of hormone-sensitive lipase by cyclic AMP-dependent protein kinase J Biol Chem 258, 15146–15152 Anthonsen MW, Ronnstrand L, Wernstedt C, Degerman E & Holm C (1998) Identification of novel phosphorylation sites in hormone-sensitive lipase that are phosphorylated in response to isoproterenol and govern activation properties in vitro J Biol Chem 273, 215–221 10 Contreras JA, Danielsson B, Johansson C, Osterlund T, Langin D & Holm C (1998) Human hormone-sensitive lipase: expression and large-scale purification from a baculovirus ⁄ insect cell system Protein Expr Purif 12, 93–99 11 Krintel C, Osmark P, Larsen MR, Resjo S, Logan DT & Holm C (2008) Ser649 and Ser650 are the major determinants of protein kinase A-mediated activation of human hormone-sensitive lipase against lipid substrates PLoS ONE 3, e3756, doi:10.1371/journal.pone.0003756 12 Lookene A, Zhang L, Tougu V & Olivecrona G (2003) 1,1¢-bis(anilino)-4-,4¢-bis(naphtalene)-8,8¢-disulfonate acts as an inhibitor of lipoprotein lipase and competes for binding with apolipoprotein CII J Biol Chem 278, 37183–37194 13 Niesen FH, Berglund H & Vedadi M (2007) The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability Nat Protoc 2, 2212–2221 14 Ben Ali Y, Chahinian H, Petry S, Muller G, Carriere F, Verger R & Abousalham A (2004) Might the kinetic behavior of hormone-sensitive lipase reflect the absence of the lid domain? Biochemistry 43, 9298–9306 15 Holm C, Fredrikson G, Sundler R & Belfrage P (1990) Incorporation of hormone-sensitive lipase into phosphatidylcholine vesicles Lipids 25, 254–259 16 Shi L, Palleros DR & Fink AL (1994) Protein conformational changes induced by 1,1¢-bis(4-anilino-5-naphthalenesulfonic acid): preferential binding to the molten globule of DnaK Biochemistry 33, 7536–7546 17 Sztalryd C, Xu G, Dorward H, Tansey JT, Contreras JA, Kimmel AR & Londos C (2003) Perilipin A is essential for the translocation of hormone-sensitive lipase during lipolytic activation J Cell Biol 161, 1093– 1103 18 Osterlund T., Danielsson B., Degerman E., Contreras J A., Edgren G., Davis R C., Schotz M C & Holm C (1996) Domain-structure analysis of recombinant rat hormone-sensitive lipase Biochem J 319, 411–420 19 Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein FEBS Journal 276 (2009) 4752–4762 ª 2009 The Authors Journal compilation ª 2009 FEBS 4761 PKA phosphorylation of hormone-sensitive lipase C Krintel et al utilizing the principle of protein-dye binding Anal Biochem 72, 248–254 20 Tornqvist H, Krabisch L & Belfrage P (1972) Rapid assay for hormone-sensitive lipase activity of adipose tissue J Lipid Res 13, 424–426 21 Laemmli U K (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4 Nature 227, 680–685 22 Baschong W & Wrigley NG (1990) Small colloidal gold conjugated to Fab fragments or to immunoglob- 4762 ulin G as high-resolution labels for electron microscopy: a technical overview J Electron Microsc Tech 14, 313–323 23 Engel J & Furthmayr H (1987) Electron microscopy and other physical methods for the characterization of extracellular matrix components: laminin, fibronectin, collagen IV, collagen VI, and proteoglycans Methods Enzymol 145, 3–78 FEBS Journal 276 (2009) 4752–4762 ª 2009 The Authors Journal compilation ª 2009 FEBS ... that HSL gains accessible hydrophobic surface area upon PKA phosphorylation This gain in hydrophobic surface area presumably accounts for the increase in in vitro activity of HSL following PKA... solvent-exposed hydrophobic surface area of HSL following PKA phosphorylation was examined using both bis-ANS and SYPRO Orange Because of the interaction of both bis-ANS and SYPRO Orange with ATP, we... demonstrate that HSL increases its hydrophobic nature upon phosphorylation by PKA Thus, it can be speculated that phosphorylation of HSL by PKA induces a conformational change that exposes and ⁄ or increases