Regulation of calpain B from Drosophila melanogaster by
phosphorylation
La
´
szlo
´
Kova
´
cs
1,
*, Anita Alexa
2,
*, Eva Klement
3
, Endre Ko
´
kai
1
,A
´
gnes Tantos
2
, Gergo
¨
Go
´
gl
2
,
Tama
´

s Sperka
4
, Katalin F. Medzihradszky
3,5
,Jo
´
zsef To
¨
zse
´
r
4
, Viktor Dombra
´
di
1,6
and
Pe
´
ter Friedrich
2
1 Department of Medical Chemistry, Faculty of Medicine, University of Debrecen, Hungary
2 Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Budapest, Hungary
3 Proteomics Research Group, Biological Research Center, Hungarian Academy of Sciences, Szeged, Hungary
4 Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Debrecen, Hungary
5 Department of Pharmaceutical Chemistry, University of California at San Francisco, CA, USA
6 HAS-DU Cell Biology and Signaling Research Group, Department of Medical Chemistry, Research Center for Molecular Medicine,
University of Debrecen, Hungary
Keywords
calcium-dependent protease; Drosophila

melanogaster ; enzyme kinetics; epidermal
growth factor; protein kinase
Correspondence
V. Dombra
´
di, Department of Medical
Chemistry, Faculty of Medicine, University
of Debrecen, 98 Nagyerdei krt, Debrecen,
H-4032, Hungary
Fax: +36 52 412 566
Tel: +36 52 412 345
E-mail:
*These authors contributed equally to this
work
(Received 23 April 2008, revised 15 June
2009, accepted 6 July 2009)
doi:10.1111/j.1742-4658.2009.07198.x
Calpain B is one of the two catalytically competent calpain (calcium-acti-
vated papain) isoenzymes in Drosophila melanogaster. Because structural
predictions hinted at the presence of several potential phosphorylation sites
in this enzyme, we investigated the in vitro phosphorylation of the recombi-
nant protein by protein kinase A as well as by the extracellular signal-regu-
lated protein kinases (ERK) 1 and 2. By MS, we identified Ser845 in the
Ca
2+
binding region of an EF-hand motif, and Ser240 close to the autocat-
alytic activation site of calpain B, as being the residues phosphorylated by
protein kinase A. In the transducer region of the protease, Thr747 was
shown to be the target of the ERK phosphorylation. Based on the results
of three different assays, we concluded that the treatment of calpain B with

protein kinase A and ERK1 and ERK2 kinases increases the rate of the
autoproteolytic activation of the enzyme, together with the rate of the
digestion of external peptide or protein substrates. Phosphorylation also
elevates the Ca
2+
sensitivity of the protease. The kinetic analysis of phos-
phorylation mimicking Thr747Glu and Ser845Glu calpain B mutants con-
firmed the above conclusions. Out of the three phosphorylation events
tested in vitro, we verified the in vivo phosphorylation of Thr747 in epi-
dermal growth factor-stimulated Drosophila S2 cells. The data obtained
suggest that the activation of the ERK pathway by extracellular signals
results in the phosphorylation and activation of calpain B in fruit flies.
Structured digital abstract
l
MINT-7214239: ERK1 (uniprotkb:P40417) phosphorylates (MI:0217) CalpainB (uniprotkb:
Q9VT65)byprotein kinase assay (MI:0424)
l
MINT-7214216, MINT-7214228: PKA (uniprotkb:P12370) phosphorylates (MI:0217) CalpainB
(uniprotkb:
Q9VT65)byprotein kinase assay (MI:0424)
l
MINT-7214325: CalpainB (uniprotkb:Q9VT65) cleaves (MI:0194) MAP2C (uniprotkb:
P11137)byprotease assay (MI:0435)
Abbreviations
CaMKII, calcium ⁄ calmodulin-dependent protein kinase II; CID, collision-induced dissociation; EGF, epidermal growth factor; ERK, extracellular
signal-regulated protein kinase; LY-AMC, N-succinyl-Leu-Tyr-7-amino-4-methyl-coumarin; MAP, microtubule-associated protein; Ni-NTA,
nickel–nitrilotriacetic acid; PKA, protein kinase A.
FEBS Journal 276 (2009) 4959–4972 ª 2009 The Authors Journal compilation ª 2009 FEBS 4959
Introduction
The regulation of intracellular signaling pathways

involves an intricate interplay of various enzyme sys-
tems. One intriguing example is the interaction
between calpains (i.e. the Ca
2+
-dependent SH-prote-
ases) and protein kinases (i.e. the enzymes of protein
phosphorylation). Regarding their modes of action,
calpains catalyze the irreversible, limited proteolysis of
their substrate proteins, whereas protein phosphoryla-
tion can be reversed by the protein phosphatases via
the hydrolytic elimination of the phosphate group
from Ser, Thr or Tyr residues. Calpains play crucial
roles in controlling various cellular processes, such as
cytoskeletal remodeling, cell cycle, apoptosis and cell
motility [1,2]. The ubiquitous mammalian l- and
m-calpains (calpain-1: EC 3.4.22.52; calpain-2: EC
3.4.22.53) are the best-characterized members of the
family. The regulation of these essential proteases still
remains an open question. The micromolar to millimo-
lar Ca
2+
concentrations required for the effective acti-
vation of calpains in vitro is not in the physiological
range. It has been suggested that the presence of addi-
tional regulatory substances (e.g. phospholipids) or
post-translational protein modifications (e.g. phosphor-
ylation) can modulate the Ca
2+
-sensitivity of these
proteases [3,4]. Phosphorylation of mammalian

calpains has been intensively studied. Because recombi-
nant calpains, which are devoid of phosphate groups,
are fully active, it can be concluded that phosphoryla-
tion is not essential for their activity. On the other
hand, l- and m-calpain extracted from different tissues
contain two to four phosphate group ⁄ molecule, which
are distributed over eight or nine different Ser, Thr
and Tyr residues [1]. The main phosphorylation sites
in m-calpain are Ser50 and Ser369⁄ Thr370. In vitro
and in vivo studies show that phosphorylation of Ser50
by extracellular signal-regulated protein kinases
(ERKs) enhances calpain activity, as well as calpain-
mediated physiological processes [5], whereas phos-
phorylation of Ser369 ⁄ Thr370 by protein kinase A
(PKA) inhibits calpain action [6]. Nicotine-induced
phosphorylation by an isoform of protein kinase
C, PKCi, enhances both the activity and secretion of
l- and m-calpain in human lung cancer cells [7].
In the present study, we examined calpain B from
Drosophila melanogaster. We chose a fly enzyme
because Drosophila is a handy model organism and,
out of its four calpains, three (calpain A, B and C)
have been characterized in our laboratory [2]. As far
as we are aware, only calpain A and B exhibit protease
activity in the fruit flies. Both of them are activated at
millimolar free Ca
2+
concentration. The active Dro-
sophila calpains consist of a single polypeptide chain,
for which the domain structure shows strong similarity

to the catalytic subunits of mammalian l- and m-cal-
pain. They are composed of an N-terminal regulatory
domain (I), a catalytic domain (II), a C2-like domain
(III) and a calmodulin-like calcium binding domain
(IV). The main difference between mammalian and
Drosophila calpains is represented by the length of the
N-terminal domains; for example, calpain B has a 240
amino acid long N-terminal region with an extended,
disordered structure [8]. Upon Ca
2+
-dependent activa-
tion, this N-terminal inhibitory region is first clipped
off in an autoproteolytic process. Because the bioinfor-
matic analysis of the primary structure of calpain B
suggested a number of potential phosphorylation sites
that correspond to consensus recognition motifs of sev-
eral protein kinases, we initiated the investigation of
the phosphorylation of this protein under both in vitro
and in vivo conditions. In the present study, we report
on the identification of cAMP-dependent protein
kinase (protein kinase A, PKA; EC 2.7.11.11) and
mitogen-activated protein kinase (ERK1 and ERK2;
EC 2.7.11.24) phosphorylation sites in calpain B and
describe the effects of phosphorylation on the proteo-
lytic activation and activity of the enzyme.
Results and discussion
Phosphorylation of calpain B in vitro
The phosphorylation of any of the Drosophila calpains
has not been reported yet. According to motif scan
( prediction,

there are five putative PKA consensus sites and several
ERK target sites in calpain B, when screened at a low
stringency level (data not shown). The feasibility of
ERK action was further supported by the presence of
three ERK1 binding sites and three ERK2 binding
sites in the protein. To confirm the structural predic-
l
MINT-7214275: ERK2 (uniprotkb:P40417-2) phosphorylates (MI:0217) CalpainB (uni-
protkb:
Q9VT65)byprotein kinase assay (MI:0424)
l
MINT-7214319: CalpainB (uniprotkb:Q9VT65) and CalpainB (uniprotkb:Q9VT65) cleave
(
MI:0194)byprotease assay (MI:0435)
Phosphorylation of calpain B L. Kova
´
cs et al.
4960 FEBS Journal 276 (2009) 4959–4972 ª 2009 The Authors Journal compilation ª 2009 FEBS
tions, purified recombinant calpain B was phosphory-
lated with PKA, as well as with the two isoforms of
the extracellular signal-regulated protein kinase, ERK1
and ERK2, in the presence of [
32
P]ATP[cP] in vitro
(Fig. 1). PKA incorporated 0.20 ± 0.09 (n = 5) mol
PÆmol protein
)1
. The phosphorylation with ERK1 was
more effective, and 0.62 ± 0.27 (n = 6) mol PÆmol
protein

)1
was achieved within 2 h, whereas ERK2
built in 0.73 ± 0.17 (n = 7) mol PÆmol protein
)1
under the same conditions (Fig. 1B). In all of the
experiments, more than 95% of the total protein
bound radioactivity resided in the band corresponding
to the apparent molecular mass of the recombinant
calpain B (Fig. 1A).
For the identification of the phosphorylated amino
acid residues, both the wild-type active protease and
an inactive calpain B mutant were phosphorylated as
described above; with the exception that nonradioac-
tive ATP was used instead of [
32
P]ATP[cP]. The inac-
tive mutant was generated (aiming to avoid unwanted
autoproteolytic degradation during sample handling)
by replacing Cys314 with Ala in the active center of
the enzyme. The active calpain B and the inactive
C314A mutant were purified by SDS ⁄ PAGE after
in vitro phosphorylation. The proteins were in-gel
digested with trypsin and analyzed by MS. The phos-
phopeptides were identified from the digests using
precursor ion scanning and affinity enrichment.
Phosphopeptides yield diagnostic m ⁄ z 79 (PO
3
)
) ions
in negative ion mode. The precursors of this fragment

were identified in nanoLC ⁄ MS ⁄ MS experiment on a
QTRAP mass spectrometer (Applied Biosystems, Fos-
ter City, CA, USA). Collision-induced dissociation
(CID) data acquired in positive ion mode from these
precursor ions provided sufficient information for
sequence and modification site assignment. These
A
B
Fig. 1. Phosphorylation of calpain B in vitro. (A) Recombinant calpain B was phosphorylated with PKA, ERK1 and ERK2 protein kinases in
the presence of [
32
P]ATP[cP]. Samples were analyzed by SDS ⁄ PAGE followed by autoradiography. The left-hand lanes show the molecular
mass standards (St) and 2 lg of unphosphorylated calpain B protein (P) stained with Coomassie brilliant blue. The molecular mass of the
standards is given in kDa. The right-hand lanes present the autoradiorams of the samples taken at the indicated time points after the initia-
tion of phosphorylation. (B) The phosphorylation reaction by PKA (
), ERK1 (.) and ERK2 ( ) was also monitored by counting the radioactiv-
ity incorporated into the TCA insoluble protein. The mean ± SD of five to seven independent experiments is shown.
L. Kova
´
cs et al. Phosphorylation of calpain B
FEBS Journal 276 (2009) 4959–4972 ª 2009 The Authors Journal compilation ª 2009 FEBS 4961
assignments were confirmed by phosphopeptide enrich-
ment using TiO
2
affinity chromatography followed by
LC ⁄ MS ⁄ MS analysis in information-dependent acqui-
sition mode on an ion trap mass spectrometer. In the
wild-type calpain B enzyme, two PKA-phosphorylation
sites at Ser240 and Ser845 were revealed by the
MS ⁄ MS spectra of the precursors at m ⁄ z 764.8 (2+)

and m⁄ z 591.7 (2+), respectively. The MS ⁄ MS spec-
trum of the precursor at m ⁄ z 764.8 (2+) represents
the phosphopeptide
238
qNS(p)VSKGDFQSLR
250
. The
phosphorylation site assignment is based on fragments
observed at m ⁄ z 393.3 (b
3
), 519.3 (y
9
2+
), 1037.0 (y
9
)
and 569.0 (y
10
2+
)(Fig. 2A). The phosphorylation site
at Ser845 was unambiguously identified from the
fragment observed at m ⁄ z 512.7 (y
8
) in the MS ⁄ MS
spectrum of the precursor at m ⁄ z 591.7 (2+) corre-
sponding to
842
TGS(p)IDGFHLR
852
(Fig. 2B). For

the ERK2 kinase, phosphorylation at Thr747 was
determined from the MS ⁄ MS spectrum of the pre-
cursor at m ⁄ z 689.9 (3+) corresponding to
739
IA-
PSLPPPT(p)PKEEDDPQR
756
; the fragments at m ⁄ z
482.0 (b
5
) and 793.5 (y
13
) clearly indicate phosphoryla-
tion at Thr747 (Fig. 2C). The same phosphorylation
sites were identified in the inactive calpain B mutant,
with the exception that Ser240 phosphorylation was
not found. In addition, we demonstrated that ERK1
phosphorylated the same Thr747 residue as the ERK2
isoenzyme (data not shown). The sites of in vitro phos-
phorylation in calpain B are summarized in Fig. 2D.
Although the 3D structure of calpain B has not been
solved yet, from the available atomic coordinates of
m-calpain [9], the sites of phosphorylation can easily
be assigned to the structural domains of the enzyme
by homologous modeling. In agreement with the motif
scan prediction, the PKA consensus site, Ser845, lies
in domain IV within the second EF-hand motif (resi-
dues 831–859). It is preceded by two basic residues,
Arg841 and Arg842, that create a favorable environ-
ment for PKA recognition. The second PKA site at

Ser240 is at the end of domain I, close to the activat-
365.7 a
3
400.3 y
3
464.0 b
5
#
557.3 y
9
2+
622.7 y
16
#3+
652.0 y
17
3+
683.3 MH
3
#3+
744.3 y
13
-98
2+
482.0 b
5
628.3 y
16
3+
793.3 y

13
2+
942.3 y
16
2+
850.0 y
14
2+
1114.0 y
9
393.3 b
3
297.7 PSL
503.0 y
4
374.7 y
6
*2+
519.3 y
9
2+
569.0 y
10
2+
755.7 MH
2
#2+
533.7 MH
2
-98

#2+
463.7 y
8
-98
2+
542.3 MH
2
-98
2+
629.3 y
5
583.0 MH
2
#2+
744.7 y
6
857.0 y
7
895.0 b
8
1009.0 b
9
797.0 b
8
-98
660.0 b
7
-98
579.0 b
5

650.7 y
5
x 5
A
B
CD
x 10
x 10
x 2 x 5
x 5 x 5
822.3 y
7
879.3 b
8
1073.0 y
9
781.0 b
8
-98
288.0 y
2
425.0 y
3
512.7 y
8
2+
227.7 b
3
-98
341.0 b

4
-98
716.0 MH
2
-98
2+
Fig. 2. Identification of the in vitro phosphorylation sites in calpain B by MS. (A) CID spectrum of the precursor at m ⁄ z 764.8 (2+) represent-
ing qNS(p)VSKGDFQSLR and (B) CID spectrum of the precursor at m ⁄ z 591.7 (2+) corresponding to TGS(p)IDGFHLR, both observed in the
TiO
2
enrichment of the PKA phosphorylated calpain B digest. (C) CID spectrum of the precursor at m ⁄ z 689.9 (3+) representing
IAPSLPPPT(p)PKEEDDPQR detected in the TiO
2
enrichment of the ERK2 phosphorylated calpain B digest. Water loss is marked with a hash
symbol (#); )98 indicates phosphoric acid loss. Nomenclature is used in accordance with Biemann [22]. The peaks used for the identification
of the phosphorylation sites are marked with an arrow. (D) Summary of the in vitro phosphorylation sites (shown in bold) in calpain B.
Phosphorylation of calpain B L. Kova
´
cs et al.
4962 FEBS Journal 276 (2009) 4959–4972 ª 2009 The Authors Journal compilation ª 2009 FEBS
ing scission site located between amino acids 224–225.
Because its environment (Fig. 2D) is less preferred by
the kinase, we consider this residue as a secondary site
of phosphorylation. Although Ser845 appears to be
the preferential PKA site within the molecule, it should
be noted that both the rate and the extent of PKA
phosphorylation are rather low (Fig. 1B). Thr747 was
identified as the site of phosphorylation of either
ERK1 or ERK2. In agreement with the consensus
sequence of the two kinase isoforms, there are three

consecutive Pro residues immediately at the N-terminal
side of Thr747. Indeed, this site is recognized by all of
the so called Pro-directed protein kinases. Thr747 is
situated at the surface of the molecule between
domains III and IV in an extended structural element
termed ‘transducer’ [10,11]. This region of the polypep-
tide was suggested to transmit the Ca
2+
signal from
the Ca
2+
binding EF-hands of domain IV to the active
site cleft that is situated between the IIa and IIb sub-
domains. The phosphorylation of additional potential
ERK consensus sites was not supported by the experi-
ments.
In comparison with the mammalian counterparts,
none of these phosphorylation sites of calpain B have
been conserved in calpain l or m and, vice versa, the
known phosphorylation sites of mammalian calpains
are missing from Drosophila calpain B. From the first
part of our studies, we conclude that calpain B can be
phosphorylated by three kinases at three different resi-
dues, although the sites of phosphorylations are dis-
tinct from those reported for the mammalian enzymes
[5,6]. Consequently, the regulation of the Drosophila
protease can be different from the well-known mam-
malian calpains.
Effects of phosphorylation on the activation and
activity of calpain B

Because the location of the phosphorylation sites
suggested an effect of phosphate incorporation on the
Ca
2+
regulation of calpain B, we compared the kinetic
properties of the nonphosphorylated (control) and
phosphorylated enzymes using three independent
methods.
Fluorimetric assay with a peptide substrate
At high Ca
2+
concentration, a continuous assay was
applied using the fluorescent dipeptide substrate N-suc-
cinyl-Leu-Tyr-7-amino-4-methyl-coumarin (LY-AMC).
In an earlier study [10], we determined that a
8.6 ± 0.8 mm free [Ca
2+
] concentration was required
for the half maximal activation of calpain B. Accord-
ingly, at 9 and 19 mm free Ca
2+
concentrations, the
enzyme works at 50% and 90% of its full activity,
respectively. Under these conditions, the reaction is
fast enough to reach the maximal velocity (v
max
), a
parameter that can be used to compare the phosphory-
lated forms with the nonphosphorylated one. As an
example, two calpain progress curves are presented in

Fig. 3A, demonstrating the effect of PKA-treatment.
Both progress curves start with a lag-phase, corre-
sponding to the autoproteolytic activation; later, the
sigmoid-like curves reach maximal activity. In the pres-
ent study, the progress curves were fitted with a log-
istic curve (see Experimental procedures), which
provided the k
act
and v
max
values. The phosphorylated
calpain B forms were found to be activated faster
(Fig. 3B), and had a greater activity (v
max
)ata9mm
free Ca
2+
concentration (Fig. 3C). Similar results were
obtained at a 19 mm free Ca
2+
concentration (data
not shown). In all of our experiments, ERK2 exerted a
more pronounced effect than ERK1, in accordance
with the fact that the stoichiometry of phosphorylation
was somewhat higher with the former kinase. The rela-
tively small effect of PKA can be attributed either to
the different sites of modification or, more likely, to
the lower level of phosphorylation.
Activity assay with a protein substrate
Microtubule-associated protein (MAP) 2c is readily

digested by mammalian m-calpain as well as calpain B,
even at lower Ca
2+
concentrations. The proteolytic
reaction was carried out at a 350 lm free Ca
2+
con-
centration and monitored by SDS ⁄ PAGE followed by
the densitometric scanning of the 62 kDa intact
MAP2c band ( Fig. 4A). Simple visual inspection of the
results demonstrates that calpain B phosphorylated
with PKA digested MAP2c faster than the nonphosph-
orylated form. For quantitative evaluation of the data,
we plotted the logarithm of the optical density of the
MAP2c band at a given time divided by the optical
density measured at the beginning of the reaction [i.e.
ln(A ⁄ A
0
)] against the reaction time and determined the
slope of the linear curve (Fig. 4B). Assuming that the
quantity of the active enzyme is constant during the
reaction, the slope gives the rate constant of the first-
order reaction. We used this constant for the charac-
terization of the enzyme activity and expressed the
effect of phosphorylation as described above (Fig. 4C).
In this independent assay, again, ERK2 increased cal-
pain B activity more vigorously than the other kinases.
The Ca
2+
dependence of PKA action on calpain B

activity was investigated in more detail (Fig. 4D). The
results obtained clearly demonstrate that the effect of
L. Kova
´
cs et al. Phosphorylation of calpain B
FEBS Journal 276 (2009) 4959–4972 ª 2009 The Authors Journal compilation ª 2009 FEBS 4963
PKA is larger at lower Ca
2+
concentrations. Because,
using this method, we were unable to collect data at
the very beginning of the reaction, the plots were not
suited to analyze the effect of phosphorylation on the
activation of calpain B.
Autolysis assay
To circumvent the above limitation of MAP2c diges-
tion assay, we designed an alternative approach for the
determination of the effects of phosphorylation on
autolysis. The approach was based on our observation
of a slight autocatalytic processing of calpain B in the
MAP2c based assay (Fig. 4A). Under modified condi-
tions, and in the absence of MAP2c, we monitored the
disappearance of the 104 kDa intact calpain B band as
a function of time (Fig. 5A) and used the same kinetic
approach as applied before for the determination of
the apparent first-order rate constant of the reaction
(Fig. 5B). According to our assumption, the first auto-
catalytic cleavage between amino acid residues 224–
225 is sufficient for the activation of calpain B; thus,
the rate of the elimination of the 104 kDa inactive
form is approximately proportional to the rate of acti-

vation. Figure 5C shows that phosphorylation by
either ERK2 or PKA slightly elevated the rates of
autolysis in the presence of a 19 mm free Ca
2+
concen-
tration. The effect was negligible when the free Ca
2+
concentration was set at 1 mm (data not shown).
ERK1 was not studied in this test because it phospho-
rylates the same site as ERK2, but with slightly lower
efficiency. When calpain B was phosphorylated with
[
32
P]ATP[cP], the distribution of the radioactive label
was monitored by autoradiography during autolysis
(Fig. 5D). Although the overall rate of autolysis was
comparable (i.e. the rate constants were k
ERK2
=
0.039 s
)1
and k
PKA
= 0.034 s
)1
, respectively), the gen-
eral picture was quite different for the two kinases
tested. The radioactive phosphate incorporated by
PKA was eliminated very quickly from the 104 kDa
band, and most of the total radioactivity (more than

85%) accumulated in the 75 kDa fragment after the
first steps of autolysis. According to the activation
model of calpain B [12], this fragment represents the
C-terminal portion of the protein. Thus, the distribu-
tion of radioactivity is in agreement with our previous
result indicating that the PKA phosphorylation sites of
calpain B reside inside the 75 kDa fragment. Regard-
ing the ERK2 phosphorylation, approximately 75% of
the radioactivity incorporated by the kinase dis-
appeared within less than 30 s from the gel. Only
21–26% of
32
P remained in the 75 kDa C-terminal
proteolytic fragment. The most likely explanation is
A
B
C
Fig. 3. Effect of phosphorylation on calpain B activity as measured
with a peptide substrate. The progress curve of calpain activity
measurement with the fluorimetric assay (see Experimental proce-
dures) in the presence of a 9 m
M free Ca
2+
concentration is shown
as an example (A). Calpain B was phosphorylated by PKA (broken
line) for 60 min to the stoichiometry of 0.21 mol PÆmol protein
)1
,or
treated under the same conditions without the kinase (solid line) in
a control experiment. The fluorescent signal was recorded after the

addition of the enzyme to the reaction mixture. From the progress
curves, the rate of activation (k
act
) as well as the maximal activity
(v
max
) of calpain B was calculated. The average k
act
for the non-
phosphorylated form was 8.5 · 10
3
M
)1
Æs
)1
at a 9 mM free Ca
2+
concentration, whereas the average v
max
was 10
)8
M
)1
Æs
)1
, and the
k
cat
, which can be determined from the v
max

, was 1.5 · 10
)2
s
)1
.
The increase in the rate of activation (B) and in the activity (C) upon
phosphorylation with PKA, ERK1 and ERK2 is given as the percent-
age of the unphosphorylated controls. The extent of phosphoryla-
tion was 0.19 ± 0.03, 0.63 ± 0.18 and 0.75 ± 0.18 mol PÆmol
protein
)1
for the three kinases, respectively. The mean ± SD of
three or four independent experiments is shown.
Phosphorylation of calpain B L. Kova
´
cs et al.
4964 FEBS Journal 276 (2009) 4959–4972 ª 2009 The Authors Journal compilation ª 2009 FEBS
that, beside Thr747, ERK2 effectively phosphorylated
another residue(s) in the unstructured N-terminal regu-
latory domain that was very quickly degraded during
the autoproteolytic activation process.
Site-directed mutagenesis mimicking
phosphorylation of calpain B
Although the direct phosphorylation of calpain B with
the selected protein kinases provides realistic results,
the explanation of the data is complicated by the low
stoichiometry of phosphorylation in the case of PKA,
and by the existence of multiple phosphorylation sites
in all cases. To assess the contribution of a well estab-
lished site to the regulation of calpain B, we adopted

the approach of Smith et al. [13] and replaced the
target sites of the kinases with Glu by site-directed
mutagenesis. The negative Glu side-chain mimics the
effect of phosphorylation. In addition to being specific,
site-directed mutagenesis has two additional advanta-
ges: the modifications are stoichiometric and perma-
nent. The mutated forms of calpain B carrying the
point-mutations T747E and S845E were expressed and
purified in exactly the same way as the wild-type
recombinant protein. The yield and the purity of the
three calpain B variants was the same (Fig. 6A). The
effect of the phosphorylation mimicking mutations on
calpain B activity was first analyzed by the fluorimetric
assay (Fig. 6B). Both mutations activated the protease
in a wide range of free Ca
2+
concentrations. The
mathematical analysis of the Ca
2+
response curves is
given in Table 1. The data can be fitted well to sig-
moid curves and the parameters of the curves provide
an excellent tool for the characterization of the Ca
2+
dependence of the three enzyme forms. On the basis of
the [Ca
2+
]
1 ⁄ 2
and dx parameters, we conclude that the

mutants are activated at lower Ca
2+
concentrations,
but are less sensitive to changes in the Ca
2+
concentra-
tion than the wild-type enzyme (Table 1). Although
phosphorylation-mimicking mutations can enhance the
Ca
2+
sensitivity of calpain B, the values of [Ca
2+
]
1 ⁄ 2
are still far above the physiological range. Both muta-
tions raise the maximal activity of calpain B, and this
activity enhancement is greater at low Ca
2+
(Table 1);
thus, the effect of phosphorylation could be more pro-
nounced at Ca
2+
concentrations that are closer to the
physiological conditions.
The effects of the mutations on the autocatalysis of
calpain B were tested using two independent methods.
The elimination of the intact 104 kDa protein band
A
B
C

D
Fig. 4. Effect of phosphorylation on calpain B activity as measured with MAP2c substrate. The digestion of MAP2c by calpain B (see Experi-
mental procedures) at a 50 l
M free Ca
2+
concentration is shown as an example (A). The time-course of proteolysis with calpain B that had
been either phosphorylated by PKA (0.21 mol PÆmol protein
)1
), or not phosphorylated (control) was monitored by SDS ⁄ PAGE. The arrow
points towards the calpain B bands. The densities of the intact MAP2c bands (indicated by an arrowhead) were estimated by densitometry
and the apparent first-order rate constants of MAP2c digestion were determined for both the phosphorylated (
) and nonphosphorylated (h)
protease (B). The percentage increase in the reaction rate upon phosphorylation by PKA, ERK1 and ERK2 was calculated as in Fig. 3. The
proteolytic reactions were carried out in the presence of a 350 l
M free Ca
2+
concentration, and the average first-order rate constant was
2.2 · 10
)3
Æs
)1
for the nonphosphorylated protease. The average stoichiometry of phosphorylation was the same as that in Fig. 3. The
mean ± SD of seven to eleven independent experiments is shown (C). The effect of PKA-mediated phosphorylation on calpain B activity
was also measured as a function of Ca
2+
concentration (D). The free Ca
2+
concentration is given in molÆdm
)3
, and the mean ± SD for three

to five experiments is shown. The stoichiometry of calpain B phosphorylation was 0.19 ± 0.03 mol PÆmol protein
)1
in this experiment.
L. Kova
´
cs et al. Phosphorylation of calpain B
FEBS Journal 276 (2009) 4959–4972 ª 2009 The Authors Journal compilation ª 2009 FEBS 4965
was determined by SDS ⁄ PAGE and densitometry
(Fig. 6C) as described above (Figs 5A,B). The data
obtained by this approach are in agreement with the
k
act
values calculated from the fluorimetric progress
curves. Both approaches resulted in comparable data
indicating that the T747E mutation causes a larger
(and the S845E mutation a small but reproducible)
increase in the activation of calpain B (Fig. 6D). It is
readily apparent that the Glu mutations and the
incorporation of a phosphate into the Thr or Ser side-
chains are not fully equivalent modification; neverthe-
less, the mutations tested here confirm our previous
results obtained with the phosphorylated proteins, at
least in qualitative terms.
In vivo phosphorylation of calpain B
To determine the possible physiological significance of
our in vitro findings, we investigated whether calpain B
was phosphorylated in the S2 Drosophila cell-line.
First, we isolated the protein by immunoprecipitation
and SDS⁄ PAGE from the untreated cells (Fig. 7A)
and analyzed the putative phosphorylation sites by

MS. None of the phosphopeptides presented in Fig. 2
D were detected; thus, we concluded that calpain B
was not phosphorylated in resting cells. The same
result was obtained when the cells were treated with
the phosphatase inhibitor calyculin A. Next, we inves-
tigated whether calpain B become phosphorylated
upon the stimulation of the cells. When epidermal
growth factor (EGF) was used to activate the MAP
kinase ⁄ ERK pathway and the dephosphorylation of
proteins was prevented by calyculin A, we noted that,
on SDS ⁄ PAGE, the calpain B band was split into a
doublet of two closely migrating bands, suggesting a
postsynthetic modification of the protein (Fig. 7A).
MS revealed that two residues (Thr747 and Ser240)
were phosphorylated in the EGF-treated cells. The ion
chromatograms corresponding to the m ⁄ z 690 and 765
values are shown in Fig. 7B. The identity of the two
phosphopeptides was confirmed by MS ⁄ MS experi-
ments, which gave mass spectra very similar to those
shown in Figs 2A,C. The phosphopeptide peaks were
missing from the ion chromatograms of the untreated
sample, despite the fact that, in the tryptic digest,
many unphosphorylated calpain B peptides were
detected with higher relative abundance than in the
+EGF sample. Thus, we demonstrated that the main
ERK1 ⁄ ERK2 site, Thr747, was phosphorylated in vivo
upon EGF stimulation. No additional ERK sites
expected from the motif scan prediction and from the
in vitro phosphorylation experiment (Fig. 5D) were
verified in vivo. Surprisingly, Ser240, a putative PKA

A
B
C
D
Fig. 5. Effect of phosphorylation on the autolysis of calpain B. The
autolysis of recombinant calpain B without phosphorylation (h)or
after treatment with ERK2 (m) was tested at a 19 m
M free Ca
2+
concentration. The process was monitored by SDS ⁄ PAGE (A) and
the rate constants for the disappearance of the 104 kDa band were
determined (B). The increase of autolysis rate caused by phosphor-
ylation with ERK2 and PKA is shown in (C) as the mean ± SD of
three to four independent experiments. The average first-order rate
constant of autolysis was 0.028 s
)1
for the nonphosphorylated cal-
pain B. The stoichiometry of phosphorylation was 0.77 and
0.13 mol PÆmol protein
)1
for ERK2 and PKA, respectively. (D) After
phosphorylation of the recombinant protein with [
32
P]ATP[cP], the
autolysis of radioactive calpain B was analyzed by SDS ⁄ PAGE and
Coomassie staining (upper panel) followed by autoradiography
(lower panel). The arrows point towards the intact calpain B bands.
Phosphorylation of calpain B L. Kova
´
cs et al.

4966 FEBS Journal 276 (2009) 4959–4972 ª 2009 The Authors Journal compilation ª 2009 FEBS
site, was also phosphorylated in the same experiment,
despite the fact that the treatment of S2 cell with fors-
kolin and calyculin A was not sufficient to induce in
vivo calpain B phosphorylation (data not shown).
Obviously, the activation of the MAP kinase signaling
was more effective than the stimulation of the PKA
pathway alone. A positive interaction between the two
signaling pathways cannot be excluded, but it is not
clear why the other, more potent in vitro PKA site (i.e.
Ser845) remained unaffected. One likely explanation is
that Ser240 was phosphorylated not by PKA, but by
another kinase in the living cells. In this respect, it is
important to note that the stoichiometry of in vitro
PKA phosphorylation has always been rather low and,
according to the results of the motif scan, the peptide
sequence
233
ATSARQNSVSKGDFQ
247
, containing
Ser240, is a preferred target of the calcium ⁄ calmodu-
lin-dependent protein kinase II (CaMKII kinase; EC
2.7.11.17) as well. Thus, it is possible that, after EGF
treatment, the EGF induced Ca
2+
influx activated the
latter kinase [14], which in turn incorporated the phos-
phate into Ser240 in a cAMP-independent manner. To
make things even more complicated, a motif scan test

indicates that CaMKII can also phosphorylate the
PKA site Ser845, but with lower efficiency. However,
modification at the latter site was not observed in any
Table 1. Parameters characterizing the Ca
2+
concentration dependence of enzyme activity. [Ca
2+
]
1 ⁄ 2
denotes the Ca
2+
concentration which
corresponds to half-maximal activity of the enzyme, whereas parameter dx is the width of the logistic function characterizing the depen-
dence of the activity on the logarithm of the Ca
2+
concentration, lg[Ca
2+
]. It is a dimensionless quantity and gives the sensitivity of enzyme
activity to small changes in the ion concentration around the value [Ca
2+
]
1 ⁄ 2
. Greater values of dx correspond to a lower sensitivity to
changes in Ca
2+
concentration.
Calpain B
[Ca
2+
]

1 ⁄ 2
(mM) A
max
dx
Activity ratio
at high [Ca
2+
]
Activity ratio
at low [Ca
2+
]
Wild-type 6.4 ± 0.4 110 ± 5 0.34 ± 0.02 – –
T747E 5.5 ± 0.8 150 ± 12 0.46 ± 0.03 1.44 ± 0.15 1.66 ± 0.2
S845E 5.5 ± 0.8 170 ± 10 0.44 ± 0.03 1.86 ± 0.25 2.3 ± 0.15
A
B
C
D
Fig. 6. The effects of phosphorylation mimicking mutations on calpain B. Thr747 (ERK site) and Ser845 (PKA site) were substituted with Glu
in calpain B by site-directed mutagenesis. The purified mutated proteins T747E and S845E behaved as the wild-type (Wt) recombinant protein
on SDS ⁄ PAGE (A). St, standards; molecular mass values are given in kDa. The Ca
2+
-dependent activity of the wild-type (h) as well as the
T747E (
) and S845E ( ) mutant calpain B was determined with the fluorescent LY-AMC substrate (B). The maximal activity of the wild-type
enzyme was taken as 100%. The mean ± SD of eight independent experiments is shown. The autolysis of the native (h) and mutated (
), ( )
calpain B was monitored by SDS ⁄ PAGE (C). The effect of the mutations on the activation of the protease was estimated from the progress
curves, as in Fig. 3 (white columns), or from the SDS ⁄ PAGE patterns, as in Fig. 5 (black columns), in the presence of a 19 m

M free Ca
2+
concentration (D). The mean ± SD of three to seven experiments is shown.
L. Kova
´
cs et al. Phosphorylation of calpain B
FEBS Journal 276 (2009) 4959–4972 ª 2009 The Authors Journal compilation ª 2009 FEBS 4967
of our in vivo experiments. Taking these arguments
together, the phosphorylation of Ser240 can take place
in living cells and may contribute to the regulation of
the enzyme, although, most probably, this reaction is
not catalyzed by PKA. Additional experiments are
required for the clarification of the role for this site.
On the other hand, it is clear that Thr747 can be phos-
phorylated in vitro and in vivo by the ERK enzymes.
The extracellular signal regulated modification of cal-
pain B, as identified in the present study, represents a
physiologically relevant regulatory tool.
Conclusions
The identification of the in vitro phosphorylation sites
comprised the first step towards a better understanding
of the regulation of calpain B. According to homolo-
gous modeling, the amino acid residues phosphory-
lated by PKA and the ERK isoforms are situated in
sensitive regions of the molecule and can be involved,
either directly or indirectly, in the regulation of enzyme
activity. We demonstrated that the postsynthetic modi-
fication of these sites increases the rate of autocatalytic
activation, as well as the activity and Ca
2+

sensitivity
of recombinant calpain B. Although the changes were
moderate, they could contribute to the modulation of
regulatory networks in a more significant way. Struc-
tural predictions and in vitro experiments with recom-
binant proteins reveal biochemically feasible
mechanisms that are not necessarily operating in a
living organism. We found that the phosphorylation of
Thr747 in the transducer region of the protease occurs
in EGF-stimulated S2 cells. The results obtained in the
present study suggest that the activation of the MAP
kinase ⁄ ERK pathway by extracellular signals, among
many diverse changes, results in the phosphorylation
and activation of calpain B in D. melanogaster.We
suggest that the regulation of calpain B in fruit flies
must be different from that in mammalian counter-
parts. Although calpains catalyze the same proteolytic
reaction, the position and function of the critical phos-
phorylation sites have not been conserved. Indeed,
instead of the Thr747 residue that is phosphorylated in
Drosophila calpain B, Asp524 or Glu524 are found in
the corresponding position in different mammalian
m-calpain enzymes [1], suggesting that a natural drift
in the protein sequence mimics the effect of phosphor-
ylation in mammals. This example confirms that evolu-
tion operates at the level of regulation. Without
understanding the small structural alterations affecting
the regulatory potential of a protein, it would be pre-
mature to suggest functional equivalence based on
overall structural similarities.

A
B
Fig. 7. Phosphorylation of calpain B in vivo.S2Drosophila cells
were incubated in the presence of 10 n
M EGF and 100 nM calyculin
A (+EGF) or in the absence of these additions (–EGF). Calpain B
was partially purified from the treated and the untreated cells and
was analyzed by SDS ⁄ PAGE (A). Two hundred nanograms of
recombinant calpain B was used as a control (C). The mass of the
standards (St) is given in kDa on the left-hand side. The excessive
band at around 52 kDa indicates the presence of immunoglobulins.
Arrows indicate a single band (–EGF) or a doublet of bands (+EGF)
that corresponds to the molecular mass of calpain B. These bands
were excised, digested with trypsin and analyzed by MS. The
extracted ion chromatograms of calpain B phosphopeptides after
TiO
2
enrichment are shown in (B). m ⁄ z 690 corresponds to the
IAPSLPPPT(p)PKEEDDPQR peptide, whereas m ⁄ z 765 represents
the qNS(p)VSKGDFQSLR peptide. The retention time (RT) is
given (min) above the peaks.
Phosphorylation of calpain B L. Kova
´
cs et al.
4968 FEBS Journal 276 (2009) 4959–4972 ª 2009 The Authors Journal compilation ª 2009 FEBS
Experimental procedures
Materials
The catalytic subunit of cAMP-dependent protein kinase
(PKA) either expressed in Escherichia coli (Upstate Bio-
chemicals, Lake Placid, NY, USA) or isolated from bovine

heart (Calbiochem, San Diego, CA, USA), as well as the
recombinant His-tagged, activated human ERKs 1 and 2
from Calbiochem were used. [
32
P]ATP[cP] was purchased
from the Institute of Isotopes (Budapest, Hungary). The
protease inhibitor mix was purchased from Roche (Basel,
Switzerland), nickel–nitrilotriacetic acid (Ni-NTA)-agarose
was from Qiagen (Valencia, CA, USA), and TiO
2
was from
SunChrom GmbH (Friedrichsdorf, Germany). Materials
for cell culture experiments were purchased from Greiner
Bio-One (Frickenhausen, Germany). All other chemicals
were obtained from Sigma (St Louis, MO, USA).
The expression vectors coding for the desired recombi-
nant proteins were transformed into E. coli strain
BL21(DE3) from Novagen (Madison, WI, USA) using
standard techniques. The expression vector encompassing
rat MAP2c was kindly provided by Professor A. Matus
(Friedrich Miescher Institute, Basel, Switzerland). This pro-
tein was expressed and purified as described previously [15].
Expression and purification of active and
mutated calpain B
The pET-21c-Calpain B-CHis6 expression vector encoding
active calpain B was constructed as described previously
[10]. Site-directed mutagenesis was performed using the
QuikChange mutagenesis kit (Stratagene, La Jolla, CA,
USA) according to the manufacturer’s instructions. The
template for the mutagenesis was the wild-type expression

vector, and the forward (5¢) primers were: C314A inactive
calpain B: 5¢-GGAGAACTTGGCGAAGCCTGGCTAC
TGGCTGCA-3¢; T747E mutant: 5¢ -CGTCTCTGCCGCC
ACCGGAGCCAAAGGAGGAGGATG-3¢; and S845E
mutant: 5¢-GACACCCGTCGCACTGGCGAGATTGATG
GATTCCATCTGC-3¢.
The complementary strands of the forward primers were
used as reverse (3¢) primers. In each case, the nucleotide
sequence around the mutated site was confirmed by DNA
sequencing at MWG Biotech AG (Ebersberg, Germany).
Expression of the wild-type and mutant proteins was
induced by 0.4 mm isopropyl thio-b-d-galactoside at 30 °C
for 3 h. The culture was cooled on ice and centrifuged at
3000 g for 20 min at 4 °C. Cells were suspended in ‘calpain
buffer’ (50 mm Tris, 0.15 m NaCl, 1 mm EDTA; pH 7.5)
containing 10 mm benzamidine, 1 mm phenylmethanesul-
fonyl fluoride, 10 mm b-mercaptoethanol, 10 lm aprotinin,
10 lm leupeptin, 1 lm pepstatin, and sonicated six times
for 15 s on ice. The lysate was centrifuged at 50 000 g for
30 min at 4 °C. The supernatant was applied to a 5 mL
Ni-NTA column equilibrated with Ni-NTA buffer (‘calpain
buffer’ without EDTA). The column was washed with Ni-
NTA buffer containing 0.3 mm NaCl and 10 mm imidazole.
Calpain B and its mutant forms were eluted with Ni-NTA
buffer containing 250 mm imidazole. Then the proteins
were dialyzed against ‘calpain buffer’ containing 1 mm
EGTA instead of EDTA, and 1 mm benzamidine, 0.25 mm
phenylmethanesulfonyl fluoride, 10 mm b-mercaptoethanol
at 4 °C. After overnight dialysis, the buffer was replaced
with the same buffer but containing 60% glycerol. The dial-

ysis in 60% glycerol was used for the concentration and
safe storage of the proteins. Calpain B stock solutions
(2–4 mgÆmL
)1
) were stored at )20 °C. The enzyme prepara-
tions were sufficiently pure for kinetic analysis according to
SDS ⁄ PAGE. Protein concentration was determined by the
Bradford assay [16].
Protein phosphorylation
In vitro phosphorylation of recombinant calpain B
Eighty micrograms of recombinant, wild-type or mutated,
calpain B was phosphorylated in vitro. The protein phos-
phorylation mixture contained 50 mm Tris-HCl (pH 7.5),
1mm benzamidine, 1 mm phenylmethanesulfonyl fluoride,
1mm EGTA, 2 mm dithiothreitol, 10 mm NaF, 0.05 mm
sodium vanadate, 25 mm MgCl
2
and 0.5 mm ATP in a final
volume of 200 lL. The reaction was initiated by the addi-
tion of either 0.08 lg (90 U) of PKA, 0.6 lg (3.6 U) of
ERK1 or 0.6 lg (3.3 U) of ERK2 kinase, and was termi-
nated with 50 mm EDTA. In the radioactive assays,
approximately 10
7
c.p.m. [
32
P]ATP[cP] was mixed with the
ATP solution and 5 lL samples were withdrawn at regular
time intervals for the quantitative analysis of phosphate
incorporation [17]. The radioactivity was counted by Cher-

enkov radiation. Other aliquots of the samples were sepa-
rated by SDS ⁄ PAGE in 10% polyacrylamide gels according
to Laemmli [18]. The gels were stained with Coomassie bril-
liant blue R250. Dried gels were investigated by autoradi-
ography using RX Fuji medical X-ray films (Fuji, Tokyo,
Japan). Radioactive and nonradioactive phosphorylation
reactions were run parallel under identical conditions.
In vivo phosphorylation of calpain B in S2 cells
Drosophila S2 cells were maintained in 25 cm
2
filtered cap
flasks (Techno Plastic Products, Trasadingen, Switzerland)
in Schneider’s insect medium (Sigma) supplemented with
50 UÆmL
)1
penicillin and 50 lgÆmL
)1
streptomycin (both
from Gibco, Gaithersburg, MD, USA), at 23 °C. The cells
were treated with forskolin (Sigma) or EGF (Sigma) as fol-
lows: after washing with NaCl ⁄ Pi, either 20 lm forskolin
and 100 nm calyculin A (Sigma) was added to confluent S2
cells in 5 mL of culture medium, which was incubated
for 15 min, or 10 nm EGF was added to 10 mL of cell
culture, which was incubated at 23 °C for 1 h, and than
L. Kova
´
cs et al. Phosphorylation of calpain B
FEBS Journal 276 (2009) 4959–4972 ª 2009 The Authors Journal compilation ª 2009 FEBS 4969
supplemented with 100 nm calyculin A for an additional

10 min. Treated and untreated (control) cells were washed
three times with ice-cold NaCl ⁄ Pi and suspended in 0.7 mL
of ‘lysis buffer’ [20 mm Tris-HCl (pH 7.5), 50 mm NaCl,
2mm EDTA, 1 mm NaF, 1 mm sodium vanadate, 1%
Triton X-100, 0.01% b-mercaptoethanol, and EDTA-free
protease inhibitor mix]. Cells were then sonicated four
times for 15 s with 1 min breaks. After sonication, the
lysate was centrifuged at 15 800 g for 10 min at 4 °C and
the supernatant was used for immunoprecipitation based
on a previously described procedure [19]. Briefly, to avoid
nonspecific binding, the supernatant was pre-cleared with
100 lL of Protein A Sepharose (Sigma) with gentle rotation
of the mixture for 3–4 h at 4 °C followed by centrifugation
at 15 800 g for 10 min. Meanwhile, 50 lL of Protein A
Sepharose was incubated with 40 lg of affinity-purified
calpain B antibody [12] in ‘lysis buffer’ for 3–4 h at 4 °C
and the calpain B antibody-coupled Protein A Sepharose
was collected by centrifugation at 15 800 g for 5 min at
4 °C. The pre-cleared supernatant was mixed with the
calpain B antibody-coupled Protein A Sepharose and was
incubated overnight at 4 °C with gentle rotation. The resin
was washed three times with 200 lL of ‘lysis buffer’ and
then the immune complex was released by boiling in
200 lL of SDS sample buffer. After clearing by centrifu-
gation at 15 800 g for 10 min, the sample was analyzed by
SDS ⁄ PAGE [18] and western blotting. In the latter experi-
ment, anti-calpain B sera [12] was used together with an
anti-rabbit secondary sera (Sigma) for the unambiguous
identification of the calpain B band.
MS

The active, wild-type and the inactive, mutant calpain B
preparations were phosphorylated by PKA, ERK1 or
ERK2 (see above) and the samples were resolved by
SDS ⁄ PAGE [18]. The corresponding bands were cut and
digested in-gel either with side-chain protected porcine tryp-
sin (Promega, Madison, WI, USA) or with stabilized
porcine trypsin (Sigma) as detailed elsewhere (http://
ms-facility.ucsf.edu/ingel.html). The resulting peptides were
extracted and subjected to TiO
2
enrichment as described
previously [20].
LC-MS ⁄ MS
Samples were analyzed on an Agilent 1100 nanoLC system
(Agilent Technologies Inc., Santa Clara, CA, USA) online
coupled to an XCT Plus ion trap mass spectrometer in
information-dependent acquisition mode: MS acquisitions
were followed by three CID analyses on computer-selected
multiply charged ions. HPLC conditions were: pre-column:
C18, 0.3 · 5 mm; injection flow rate: 10 lLÆmin
)1
for 5 min
in solvent A, separation column: C18, 75 lm · 150 mm;
flow rate: 300 nLÆmin
)1
; gradient: 5–45% B for 16 min,
then up to 90% B for 3 min; solvent A comprised 0.1%
formic acid in water, solvent B comprised 0.1% formic acid
in acetonitrile. Alternatively, we used an Agilent 1100
Nanoflow LC connected to the Nanospray source of a 4000

QTRAP (Applied Biosystems) mass spectrometer with
information-dependent acquisition or targeted fragmenta-
tion on ERK1 and ERK2 phosphorylated samples that were
not enriched. Slightly different HPLC conditions were used
in this case: flow rate: 500 nLÆmin
)1
, solvent A comprised
2% acetonitrile in 0.1% formic acid, solvent B comprised
98% acetonitrile in 0.1% formic acid. The gradient was
0–30% B for 60 min or 0–50% B for 10 min (depending on
the acquisition method), and then up to 100% B for 5 min.
Database search
MS ⁄ MS data were searched against the NCBI 20060603
and SwissProt 51.5 protein databases using the mascot
(version 2.1, ) and proid
(version 1.4, Applied Biosystems, Foster City, CA, USA)
search engines. Only tryptic peptides were considered, and
two missed cleavages were permitted. Monoisotopic masses
with a peptide mass tolerance of ± 1.2 Da and a fragment
mass tolerance of ± 0.6 Da were considered. Fixed modifi-
cation: Cys carbamidomethylation; variable modifications:
acetylation of protein N-termini, methionine oxidation, py-
roglutamic acid formation from N-terminal Gln residues
and phosphorylation of Ser, Thr and Tyr residues. Data
were also manually inspected.
Calpain B activity measurements
After 1 h of incubation for PKA or 2 h of incubation for
ERK1 and ERK2, kinase reactions were terminated by gel
filtration. The reaction mixtures were loaded onto a 0.5 mL
Sephadex G-25 mini-column pre-equilibrated with ‘calpain

buffer’ containing 1 mm benzamidine, 0.25 mm phenylmeth-
anesulfonyl fluoride, 10 mm b-mercaptoethanol and 10 mm
NaF. The column was washed repeatedly with 100 lL aliqu-
ots of this buffer, and the first and the second fractions were
pooled and stored on ice until activity measurement. The
calpain B content of the samples was determined by SDS ⁄
PAGE and densitometry using BSA standards for compari-
son. The activity of nonphosphorylated, phosphorylated and
mutated calpain B preparations was compared by fluori-
metric and SDS ⁄ PAGE measurements.
Fluorimetric assay
Enzyme activity was measured with a Fluorolog-3 (Jobin
Yvon, Longjumeau, France) spectrofluorimeter at excita-
tion ⁄ emission wavelengths of 380 ⁄ 460 nm, by continuously
recording the increase in fluorescence as a result of cleavage
of the substrate LY-AMC (Sigma), in a 3 mm quartz
Phosphorylation of calpain B L. Kova
´
cs et al.
4970 FEBS Journal 276 (2009) 4959–4972 ª 2009 The Authors Journal compilation ª 2009 FEBS
cuvette at air conditioned room temperature (25 °C). The
reactions were set up in 50 lL of ‘calpain buffer’, which
contained 1 mm LY-AMC and 0.4–0.8 lm calpain B. The
reaction was initiated by the addition of CaCl
2
, adjusting
the final free Ca
2+
concentration either to 9 mm or 19 mm.
In separate calibrating experiments, we performed the com-

plete hydrolysis of LY-AMC (10–100 lm) and calculated
that the total change of 2 · 10
5
(in arbitrary units) in fluo-
rescence intensity corresponded to 1 lm substrate cleaved.
Activity assay with MAP2c substrate
MAP2c, a good substrate for calpains, was used to measure
calpain B activity at low Ca
2+
concentrations [21]. The free
Ca
2+
concentration was adjusted with EDTA and was
checked by a commercial calcium-selective electrode. The
reaction was carried out in 80 lL of ‘calpain buffer’ con-
taining 2 mm dithiothreitol, 10 mm NaF, 0.1 mgÆmL
)1
MAP2c and 0.1–0.25 lm calpain B. The reaction was
started by the addition of CaCl
2
, to give the free concentra-
tions of 50, 100, 350 and 2000 lm. The reaction was termi-
nated by adding 15 lL of the reaction mixture to 5 lLof
4 · concentrated SDS sample buffer containing 50 mm
EDTA at the time indicated. Samples were boiled for 3 min
and loaded onto 10% SDS ⁄ PAGE [18]. After staining with
Coomassie brilliant blue R250, the intensity of residual
intact MAP2c was determined by densitometry using a Bio-
Rad Fluor-SÔ densitometer (Bio-Rad, Hercules, CA, USA)
and multi-analyst software, version 1.1.

Autolysis assay
The autocatalytic cleavage of calpain B was monitored in a
similar setup but in the absence of MAP2c. Twenty-five
micrograms of calpain B was subjected to self-proteolysis at
37 °C in a buffer containing 50 mm Tris-HCl (pH 7.5),
150 mm NaCl, 2 mm dithiothreitol and 10 mm NaF in the
presence of a 9 mm free Ca
2+
concentration, in a final vol-
ume of 50 lL. The reaction was initiated by the addition of
CaCl
2
. Aliquots were withdrawn from the mixture at regu-
lar time intervals and were investigated by SDS ⁄ PAGE and
densitometry of the 104 kDa intact calpain B band, as
described above. In a few experiments, the autolysis of
radioactive [
32
P]-labeled calpain B was also investigated by
SDS ⁄ PAGE and the autoradiography of the dried gels.
Evaluation of the data
Kinetic analysis of the fluorimetric calpain B activity
assay
First of all, it is necessary to make some assumptions about
the activation process. Assuming that activation of a proen-
zyme happens by reaction with an already active enzyme
molecule [bimolecular (second-order) process], the concen-
tration of the active enzyme (A) satisfies the following
differential equation:
dA ⁄ dt = k

A
A(E ) A)
where E is the total enzyme concentration and k
A
is the rel-
evant rate constant. The solution of this differential equa-
tion is a sigmoidal function:
A(t)=E ⁄ (1 + Cexp[–(t ) t0) ⁄ s])
Here, s =1⁄ (k
A
E) is the characteristic time of activa-
tion, t
0
is the initial time and C is a constant that gives the
enzyme concentration at t
0
by the equation:
A
0
= A(t
0
)=E ⁄ [1 + Cexp(t0 ⁄ s)]
In fact, one can always eliminate C by putting C =1,in
which case t
0
is redefined to the center of the sigmoid curve
(i.e. when A = E ⁄ 2). The product concentration can be
obtained using the Michaelis–Menten equation:
dP ⁄ dt = kSA ⁄ (K
M

+S)
Assuming that the substrate is present in a very large
concentration, and therefore S can be taken to be constant,
this equation can easily be integrated after substituting the
sigmoidal dependence of A (where we have put C = 1):
P(t)=P
0
+(kEs)ln(1 + exp[(t – t
0
) ⁄ s])
One can see that the curve reaches its maximum slope
(kE) at longer times. Using this function, we determined
the activation time (s) and the maximum rate (v
max
) of sub-
strate hydrolysis ( kE) from our data. From the length of
the lag-phase (s), the rate constant of activation (k
act
) can
be calculated ( k =1⁄ s [E ]).
origin 6.0 (Bio-Rad, Hercules, CA, USA) and SR2
(OriginLab Corporation, Northampton, MA, USA) soft-
ware was used for fitting the data. For the comparison of
the k
act
and v
max
parameters of the phosphorylated and
nonphosphorylated enzymes, we calculated the quotients of
k

act
(phosphorylated) ⁄ k
act
(nonphosphorylated) and v
max
(phosphorylated) ⁄ v
max
(nonphosphorylated), and corrected
these values for the calpain B content of reaction mixtures.
The corrected quotients of k
act
and v
max
are presented as
a percentage increase in activation or in activity [i.e.
(quotient ) 1) · 100].
Statistical analysis
The mean ± SD of the results was determined using excel
software from Microsoft Corp. (Redmond, WA, USA).
Acknowledgements
We thanks Professor Andrew Matus (Friedrich Mie-
scher Institute, Basel, Switzerland) for the rat MAP2c
expression vector. We are grateful to Dr Pe
´
ter Nagy
L. Kova
´
cs et al. Phosphorylation of calpain B
FEBS Journal 276 (2009) 4959–4972 ª 2009 The Authors Journal compilation ª 2009 FEBS 4971
and Dr Ja

´
nos Szo
¨
ll
}
osi (Section of Biophysics, Depart-
ment of Cell Biology and Biophysics, Research Center
for Molecular Medicine, University of Debrecen, Deb-
recen, Hungary) for access to their spectrofluorimeter.
Kromat Ltd is acknowledged for providing access to
an Agilent 1100 nanoLC-XCT Plus IonTrap system.
4000 QTRAP nanoLC ⁄ MS ⁄ MS measurements were
supported in part by GVOP 3.2.1. 0149 ⁄ 3.0. The
authors thank Dr Ga
´
bor Taka
´
cs (Department of The-
oretical Physics, Eo
¨
tvo
¨
s Lora
´
nd University, Budapest,
Hungary) for his help with the mathematical modeling
of the enzyme kinetic data. A.A. and E.K. were the
recipients of a Ja
´
nos Bolyai research scholarship. This

work was supported by the Hungarian Research Fund
grants OTKA 60723 (to P.F.) and 68754 (to V.D.).
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