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

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

(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

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

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

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

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

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

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.

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

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.

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

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

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

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

¨
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).
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