HSL phosphorylation and activation with variable ATP concentrations Because ATP interacted with both bis-ANS and SYPRO Orange, creating high levels of background fluorescence, we investig
Trang 1kinase A in vitro promotes an increase in its hydrophobic surface area
Christian Krintel1,2, Matthias Mo¨rgelin3, Derek T Logan2and Cecilia Holm1
1 Department of Experimental Medical Science, Division of Diabetes, Metabolism and Endocrinology, Lund University, Sweden
2 Department of Molecular Biophysics, Lund University, Sweden
3 Department of Clinical Sciences, Division of Infection Medicine, Lund University, Sweden
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]
Phosphoryla-tion 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
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 mobili-zation 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 Kdof 1 lm in binding assays Upon PKA phosphory-lation, the interactions of HSL with both bis-ANS and the alternative probe SYPRO Orange were increased By negative stain transmission elec-tron microscopy, phosphorylated HSL was found to have a closer interac-tion 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 )
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.
Trang 2levels 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
activa-tion of phosphodiesterase 3B, thus lowering cAMP
lev-els 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
molecu-lar 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
phosphory-lated 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¢-binaph-thyl-5,5¢-disulfonic acid (bis-ANS) [12] Another
hydrophobic probe, i.e SYPRO Orange, is now
rou-tinely used for differential scanning fluorimetry in
ther-mal denaturation experiments for buffer optimization
prior to crystallization trials [13] As these probes bind
to exposed hydrophobic patches in proteins, they were
used in this study to generate evidence that PKA-phos-phorylated HSL exhibits an increase in the solvent-exposed 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 fol-lowing 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 baculovi-rus⁄ insect cell expression system The protein was puri-fied 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 3 mg per litre of insect cell culture The specific activities of the purified protein against
TO, 1-mono-oleoyl-2-O-mono-oleylglycerol and choles-terol oleate were 2.6 UÆmg)1, 30 UÆmg)1, and 3.5 UÆmg)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 1 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.
Trang 3recombinant 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
per-formed with increasing amounts of bis-ANS using TO
as substrate Bis-ANS inhibited the activity of both
phosphorylated and nonphosphorylated HSL
Normal-ization of the two sets of data revealed differences
between the normalized activities of phosphorylated
and nonphosphorylated HSL at bis-ANS
concentra-tions above 5 lm, indicating that the bis-ANS
interac-tion with HSL is altered upon phosphorylainterac-tion of
HSL by PKA (Fig 2)
HSL interaction with bis-ANS
To evaluate the binding of bis-ANS to
nonphosphory-lated 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,
indicat-ing that there could be more than one bindindicat-ing site for
bis-ANS on HSL (Fig 3A) The maximum emission
intensity of the complex increased in an inverse
hyper-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
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 phosphoryla-tion to that obtained at an ATP concentraphosphoryla-tion of
200 lm (Fig 4A) The stoichiometry of phosphoryla-tion was 0.20 mol phosphate per mol HSL for the
200 lm ATP reaction, in accordance with results pub-lished for human HSL [11], and 0.16 mol phosphate
Fig 2 Inhibition of HSL lipase activity by bis-ANS HSL,
phosphor-ylated or nonphosphorphosphor-ylated, was preincubated for 10 min and
assayed in the presence of the given concentrations of the
hydro-phobic probe bis-ANS, using TO as substrate The activity of
phos-phorylated HSL in the TO assay was normalized to the activities of
nonphosphorylated HSL, and the activities were compared
(unpaired nonparametric t-test, n = 4).
Fig 3 Interaction of bis-ANS with HSL Spectra of bis-ANS obtained at concentrations ranging from 0.1 to 10 l M were sub-tracted from spectra of bis-ANS mixed with HSL in order to gener-ate 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 (r 2 = 0.998), which provided a K d of 1.0 l M for the HSL–bis-ANS complex (B) The spectra shown have been smoothed (using two neighbouring values).
Trang 4per mol HSL for the 15 lm ATP reaction In
accor-dance 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
interaction of phosphorylated HSL with bis-ANS in comparison with nonphosphorylated HSL, using fluo-rescence Even at ATP concentrations of 15 lm in the phosphorylation reaction mix, resulting in a final con-centration below 150 nm in the fluorescence measure-ments, the interaction between bis-ANS and ATP was too strong for reliable spectra to be obtained There-fore, 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 pro-vided only minor contributions to the total fluores-cence) 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 perfor-mance in a short range of the bis-ANS concentrations tested in Fig 2, and only 1 lm, 1.5 lm and 2 lm bis-ANS provided acceptable signal-to-noise ratios At low bis-ANS concentrations, the HSL–bis-ANS complex fluorescence was lost in noise, and at high concentra-tions, the absolute fluorescence signal was out of range for the instrumentation Thus, to further verify the increase in hydrophobicity of HSL after phosphoryla-tion by PKA, we also applied an alternative hydropho-bic probe, i.e SYPRO Orange HSL was incubated with or without PKA, and the fluorescence after exci-tation at 492 nm was recorded by scanning the emis-sion between 550 nm and 800 nm In order to create difference spectra illustrating solely the interaction between HSL and SYPRO Orange, spectra of phos-phorylation reaction mixes lacking both HSL and PKA or lacking only HSL were subtracted from the recorded spectra of the nonphosphorylated or phos-phorylated samples, respectively An advantage with this approach was that even though ATP also inter-fered 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 · 1 For all tested
concentra-Fig 4 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 l M or 200 l M ,
and analysed for incorporation of 32 P (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.
Trang 5tions of SYPRO Orange, the fluorescence of
phosphor-ylated HSL lay above the fluorescence of
nonphosph-orylated HSL, indicating that HSL gains hydrophobic
surface area upon phosphorylation by PKA (Fig 5
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
phos-pholipid vesicles using negative stain electron
micros-copy Phospholipid vesicles mimic the lipid droplets
found in vivo, which are covered by a single layer of
phospholipids, but avoid the problems of lipid
hydro-lysis during anahydro-lysis, 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
or PKA-phosphorylated HSL, stained with uranyl formate, and analysed by transmission electron micros-copy 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 6 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-Fig 5 Comparison of fluorescence from the hydrophobic probes bis-ANS and SYPRO Orange in complex with phosphorylated and non-phosphorylated HSL HSL was non-phosphorylated by PKA, dialysed, and mixed with bis-ANS, and spectra were recorded at an excitation wave-length 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 l M and 2.0 l M in (A) and (B), respectively HSL was phosphorylated with 15 l M 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 respec-tive probes.
Trang 6lated 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
con-formational 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]
Simi-larly, 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
bis-ANS 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
prein-cubation buffer, in accordance with the observation
that LPL was not inhibited by bis-ANS when BSA
was present in the assay This is also supported by the observation that even though the HSL–bis-ANS com-plex has a Kdof 1 lm (Fig 3), much higher concentra-tions 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 Kdof 1 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–bis-ANS 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 experi-ments 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
Fig 6 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
vesi-cles to a much higher extent, and is present
in higher numbers per vesicle for the
phos-phorylated enzyme than for the
nonphosph-orylated 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).
Trang 7degree 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 1 lm, 1.5 lm and 2 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
bis-ANS This enabled us to employ a simplified protocol
without the need for dialysis prior to fluorescence
mea-surements when using only 15 lm ATP in the
preced-ing 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
phos-phorylated HSL was higher than that from
non-phosphorylated 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
pro-nounced interaction with the vesicles for the
phosphor-ylated 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
possi-ble that phosphorylated HSL binds more avidly to the
polar head of the phospholipids and that this is
fol-lowed 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
phos-pholipase activity It is indeed possible that binding of
phosphorylated HSL to the phospholipid vesicles,
fol-lowed 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
explana-tion 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
PKA phsophorylation increases both maximum turn-over rate and S0.5[11]
In this study, we provide evidence that HSL gains accessible hydrophobic surface area upon PKA phos-phorylation 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 activ-ity assays involves several aspects of lipase activactiv-ity, i.e adsorption, entry and binding of individual lipid mole-cules 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 phosphoryla-tion of HSL by PKA induces a conformaphosphoryla-tional 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 phos-phorylated forms
Experimental procedures
Expression and purification of C-terminal His-tagged recombinant rat adipocyte HSL
To generate a recombinant baculovirus encoding C-termi-nally 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
Trang 8product 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
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
27C in suspension cultures (160 r.p.m.) in Sf-900 medium
supplemented with 4% fetal bovine serum and 1%
penicil-lin⁄ streptomycin (all from Gibco, through Invitrogen AB,
Lidingo¨, Sweden) Cell cultures (2 · 106cellsÆmL)1) were
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,
1 mm dithiothreitol, 1 mm EDTA, 1% C13E12, 10%
glyc-erol) The cell suspension was gently sonicated and
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), 1 mm dithiothreitol, 1 mm EDTA, 0.01% C8E4,
and 10% glycerol, and then eluted with approximately two
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,
dith-iothreitol, 1% Triton X-100, and 10% glycerol, and 15
vol-umes of 5 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
8.0), 1 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, 1 mm
dithiothrei-tol, 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
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 phospholipid-stabilized emulsions of TO, 1-mono-oleoyl-2-O-mono-oleyl-glycerol or cholesterol oleate [18,20] Briefly, labelled and nonlabelled lipid substrates and phosphatidylcholine⁄ phos-phatidylinositol (3 : 1) in cyclohexane solutions were dried under a stream of N2, and this was followed by emulsi-fication by sonication and addition of 2% BSA (1-mono-oleoyl-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), 1 mm EDTA, 1 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 min at
37C before the reaction was quenched by the addition of 3.25 mL of methanol⁄ chloroform ⁄ heptane (10 : 9 : 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 min incubation period
HSL inhibition by bis-ANS
8), 300 mm NaCl, 10% glycerol, 1 mm dithiothreitol,
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 5 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 min of incubation at 37C
HSL phosphorylation using32P-labelled ATP
HSL (9 lg) was phosphorylated at room temperature in
300 mm NaCl, 10% glycerol, 1 mm dithiothreitol, 0.02%
C8E4, 10 mm MgCl2, 25 U of PKA (New England Biolabs), protease inhibitor cocktail (Roche Complete; Roche Diag-nostics, Mannheim, Germany), and either 200 lm ATP
0.0225 lCiÆlL)1[32P]ATP[cP] Aliquots were taken after 4,
8, 16, 32 and 64 min of incubation, and quenched by the addition of Laemmli buffer [21] In control reactions,
Trang 9P-labelled HSL was detected as described above For
quantification of incorporated phosphate, HSL bands were
excised from the gel and placed in scintillation vials
con-taining 10 mL of scintillation liquid and quantified on a
scintillation counter (Wallac 1414 liquid scintillation
coun-ter; Perkin Elmer, Waltham, MA, USA) The original
reac-tion mixtures were included as standards
HSL phosphorylation by PKA for activity and
hydrophobicity measurements
HSL (4 lg) was phosphorylated in 50 lL volumes
1 mm dithiothreitol, 0.01% C8E4, 10 mm MgCl2, 15 lm or
protease inhibitor cocktail (Roche Complete) for 1 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, 8 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, 1 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
1 mm dithiothreitol, 0.01% C8E4 and 10 mm MgCl2 were
added to the cuvette, and fluorescence was recorded
simi-larly The first spectrum not containing HSL was
sub-tracted from the later spectra containing HSL, creating
difference spectra reflecting the interaction between
bis-ANS and HSL The amount of HSL used for each
spec-trum 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 2 h against 10 000 volumes of 50 mm Tris⁄ HCl
(pH 8), 300 mm NaCl, 10% glycerol, 1 mm dithiothreitol,
0.01% C8E4and 10 mm MgCl2to remove ATP and thereby
decrease background fluorescence After dialysis, samples
were centrifuged for 20 min 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-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 Invitro-gen AB) was used The measurements were performed in quartz cuvettes by adding samples to 1 mL of 50 mm Tris⁄ HCl (pH 8), 300 mm NaCl, 10% glycerol, 1 mm
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 phos-phorylation reaction, a mock phosphos-phorylation reaction without added PKA was used for measurements on non-phosphorylated HSL Similarly, a reaction in which HSL was replaced by dialysis buffer and without PKA was per-formed, and a spectrum of this reaction was subtracted from the spectrum recorded for the nonphosphorylated HSL sample The experiments were carried out at concen-trations of SYPRO Orange ranging from· 0.25 to · 1 The concentrations providing the best reproducibility for these
signal-to-noise 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 min at room temperature in 20 mm Tris⁄ HCl (pH 7.0), 0.1 m NaCl, 1 mm EDTA and 1 mm dithioerythritol at a
After swelling, the solution was sonicated with 0.5 s pulses for 45 min at 4C 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 min to remove multila-mellar vesicles and titanium from the microtip Vesicle sam-ples were mixed with HSL and immediately prepared for electron microscopy In some experiments, HSL containing vesicles were incubated for 30 min at room temperature with antibodies against HSL that were conjugated with
5 nm colloidal gold as described by Baschong and Wrigley
Trang 10[22] All protein concentrations were in the 10–20 nm range.
Subsequently, 5 lL aliquots of the solution were adsorbed
onto carbon-coated grids for 1 min, washed with two drops
of water, and stained on two drops of 0.75% uranyl
for-mate 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 Walle´n
and E Hallberg (Cell and Organism Biology, Lund
University) for help with electron microscopy
Finan-cial support was obtained from the Swedish Research
Council (project no 11284 to C Holm, and project
no 7480 to M Mo¨rgelin), the Swedish Diabetes
Asso-ciation, Faculty of Medicine at Lund University, and
the following foundations: Novo Nordisk, A
Pa˚hls-son, Salubrin⁄ Druvan, Johan och Greta Kock, Alfred
O¨sterlund, Crafoord, Konung Gustav V:s 80-a˚rsfond
and Torsten and Ragnar So¨derberg C Krintel was
supported by the Research School in Pharmaceutical
Sciences (FLA¨K)
References
1 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
2 Duncan RE, Ahmadian M, Jaworski K, Sarkadi-Nagy
E & Sul HS (2007) Regulation of lipolysis in adipocytes
Annu Rev Nutr 27, 79–101
3 Langin D & Arner P (2006) Importance of TNFalpha
and neutral lipases in human adipose tissue lipolysis
Trends Endocrinol Metab 17, 314–320
4 Greenberg AS, Egan JJ, Wek SA, Garty NB,
Blanch-ette-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
5 Granneman JG & Moore HP (2008) Location, location:
protein trafficking and lipolysis in adipocytes Trends
Endocrinol Metab 19, 3–9
6 Granneman JG, Moore HP, Granneman RL,
Green-berg AS, Obin MS & Zhu Z (2007) Analysis of lipolytic
protein trafficking and interactions in adipocytes J Biol
Chem 282, 5726–5735
7 Egan JJ, Greenberg AS, Chang MK, Wek SA, Moos
MC Jr & Londos C (1992) Mechanism of hormone-stimulated lipolysis in adipocytes: translocation of hormone-sensitive lipase to the lipid storage droplet Proc Natl Acad Sci USA 89, 8537–8541
8 Stralfors P & Belfrage P (1983) Phosphorylation of hormone-sensitive lipase by cyclic AMP-dependent protein kinase J Biol Chem 258, 15146–15152
9 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 inter-actions 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 phospha-tidylcholine vesicles Lipids 25, 254–259
16 Shi L, Palleros DR & Fink AL (1994) Protein confor-mational changes induced by 1,1¢-bis(4-anilino-5-naph-thalenesulfonic 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