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 Krintel
1,2
, Matthias Mo
¨
rgelin
3
, Derek T. Logan
2
and Cecilia Holm
1
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:
(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 V
max
and S
0.5
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 K
d
of 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.
4752 FEBS Journal 276 (2009) 4752–4762 ª 2009 The Authors Journal compilation ª 2009 FEBS
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 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
,30UÆmg
)1
, and
3.5 UÆmg
)1
, respectively. These specific activities are
lower than those previously reported for nontagged
250 kDa
123
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.
C. Krintel et al. PKA phosphorylation of hormone-sensitive lipase
FEBS Journal 276 (2009) 4752–4762 ª 2009 The Authors Journal compilation ª 2009 FEBS 4753
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 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 phosphorylation 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 binding site for
bis-ANS on HSL (Fig. 3A). The maximum emission
intensity of the complex increased in an inverse hyper-
bolic fashion (r
2
= 0.99 for an inverse hyperbolic
curve fit), and K
d
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 concentration 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 nonphosphorylated, 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 lM for the HSL–bis-ANS complex (B).
The spectra shown have been smoothed (using two neighbouring
values).
PKA phosphorylation of hormone-sensitive lipase C. Krintel et al.
4754 FEBS Journal 276 (2009) 4752–4762 ª 2009 The Authors Journal compilation ª 2009 FEBS
per 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 lM,
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.
C. Krintel et al. PKA phosphorylation of hormone-sensitive lipase
FEBS Journal 276 (2009) 4752–4762 ª 2009 The Authors Journal compilation ª 2009 FEBS 4755
tions 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 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
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 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 lM 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.
PKA phosphorylation of hormone-sensitive lipase C. Krintel et al.
4756 FEBS Journal 276 (2009) 4752–4762 ª 2009 The Authors Journal compilation ª 2009 FEBS
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 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 K
d
of 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 K

d
of 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 K
d
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
A
E
B
F
C
D
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).
C. Krintel et al. PKA phosphorylation of hormone-sensitive lipase
FEBS Journal 276 (2009) 4752–4762 ª 2009 The Authors Journal compilation ª 2009 FEBS 4757
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 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 S
0.5
[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 activity, 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 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 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
PKA phosphorylation of hormone-sensitive lipase C. Krintel et al.
4758 FEBS Journal 276 (2009) 4752–4762 ª 2009 The Authors Journal compilation ª 2009 FEBS
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% penicil-
lin ⁄ streptomycin (all from Gibco, through Invitrogen AB,
Lidingo
¨
, Sweden). Cell cultures (2 · 10
6

cellsÆmL
)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,
1mm dithiothreitol, 1 mm EDTA, 1% C
13
E
12
, 10% glyc-
erol). The cell suspension was gently sonicated and centri-
fuged for 45 min at 4 °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), 1 mm dithiothreitol, 1 mm EDTA, 0.01% C
8
E
4
,
and 10% glycerol, and then eluted with approximately two
column volumes of 1 m NaCl, 20 mm Tris ⁄ HCl (pH 8.0),
0.1 mm dithiothreitol, 0.01% C
8
E
4
, and 10% glycerol.

Protein eluted from the Q-Sepharose column was loaded
directly onto a nickel affinity chromatography column
(Ni
2+
–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 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% C
8
E
4
, and 10%
glycerol, and eluted with a stepwise gradient towards
250 mm imidazole, 300 mm NaCl, 50 mm Tris⁄ HCl (pH
8.0), 1 mm dithiothreitol, 0.01% C
8
E
4
, 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% C
8
E
4
, 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 N
2
, 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 lLof20mm 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
37 °C 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

HSL was incubated for 5–10 min in 50 mm Tris ⁄ HCl (pH
8), 300 mm NaCl, 10% glycerol, 1 mm dithiothreitol,
0.02% C
8
E
4
and 10 mm MgCl
2
containing either 5 lm,
10 lm,15lm,20lm,30lm,45lm 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,10lm,15lm,20lm,
30 lm,45lm or 60 lm bis-ANS. For all assays including
bis-ANS, the reaction rates were constant for at least
30 min 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, 1 mm dithiothreitol, 0.02%
C
8
E
4
,10mm MgCl
2
, 25 U of PKA (New England Biolabs),
protease inhibitor cocktail (Roche Complete; Roche Diag-

nostics, Mannheim, Germany), and either 200 lm ATP
and 0.3 lCiÆlL
)1
[
32
P]ATP[cP], or 15 lm ATP and
0.0225 lCiÆlL
)1
[
32
P]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,
PKA was omitted. Samples were analysed by SDS ⁄ PAGE,
stained with Coomassie, scanned, and slab dried.
C. Krintel et al. PKA phosphorylation of hormone-sensitive lipase
FEBS Journal 276 (2009) 4752–4762 ª 2009 The Authors Journal compilation ª 2009 FEBS 4759
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 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 contain-
ing 50 mm Tris ⁄ HCl (pH 8), 300 mm NaCl, 10% glycerol,
1mm dithiothreitol, 0.01% C

8
E
4
,10mm MgCl
2
,15lm or
200 lm ATP and 0.25 U of PKA ⁄ lL supplemented with a
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 l L 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% C
8
E
4
, and
10 mm MgCl
2
, 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,
1mm dithiothreitol, 0.01% C
8

E
4
and 10 mm MgCl
2
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% C
8
E
4
and 10 mm MgCl
2
to 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 dith-
iothreitol, 0.01% C
8
E
4
,10mm MgCl
2
, 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 phos-
phorylation reaction, a mock phosphorylation 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
experiments were · 0.25 and · 0.5, where the 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
final concentration of 25 mgÆmL
)1
phosphatidylcholine.
After swelling, the solution was sonicated with 0.5 s pulses
for 45 min at 4 °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 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

PKA phosphorylation of hormone-sensitive lipase C. Krintel et al.
4760 FEBS Journal 276 (2009) 4752–4762 ª 2009 The Authors Journal compilation ª 2009 FEBS
[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

C. Krintel et al. PKA phosphorylation of hormone-sensitive lipase
FEBS Journal 276 (2009) 4752–4762 ª 2009 The Authors Journal compilation ª 2009 FEBS 4761
utilizing the principle of protein-dye binding. Anal Bio-
chem 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-
ulin G as high-resolution labels for electron micros-
copy: 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.
PKA phosphorylation of hormone-sensitive lipase C. Krintel et al.
4762 FEBS Journal 276 (2009) 4752–4762 ª 2009 The Authors Journal compilation ª 2009 FEBS