Báo cáo khoa học: Calpain-mediated degradation of reversibly oxidized protein-tyrosine phosphatase 1B docx

Calpain-mediated degradation of reversibly oxidized
protein-tyrosine phosphatase 1B
Antje Tru
¨
mpler
1
, Bernhard Schlott
2
, Peter Herrlich
2
, Peter A. Greer
3
and Frank-D. Bo
¨
hmer
1
1 Institute of Molecular Cell Biology, Friedrich Schiller University, Jena, Germany
2 Leibniz Institute for Age Research – Fritz Lipmann Institute, Jena, Germany
3 Division of Cancer Biology and Genetics, Queen’s University Cancer Research Institute, Kingston, Canada
Introduction
Protein-tyrosine phosphatases (PTPs) are important
regulators of cell proliferation, migration and survival
[1,2]. Regulation by PTPs depends on modulation of
their activity. PTPs are subject to several post-transla-
tional modifications, e.g. phosphorylation, sumoy-
lation, subcellular localization caused by different
interacting partners and, in transmembrane PTPs,
ligand binding [2–6]. In addition, the chemical proper-
ties of the catalytic center cysteine of classical PTPs
allow for yet another mode of regulation, reversible
inactivation by oxidation. The chemical environment

of this cysteine residue results in a low pK
a
of  5.4,
and thus deprotonation of the sulfhydryl group under
physiological conditions [7]. Although this property is
a known prerequisite for catalytic activity, it also ren-
ders PTPs highly susceptible to oxidation. Sensitivity
to oxidation differs from phosphatase to phosphatase,
and, possibly, within cells even among different cell
types analyzed [8–10]. Interestingly, oxidation of PTPs
has been observed upon growth factor stimulation of
cells and the subsequent generation of hydrogen perox-
ide (H
2
O
2
) [11], and appears to be required for efficient
signal transduction [12]. Insulin induces the oxidation
of PTP1B (PTPN1), a physiological negative regulator
of insulin receptor signaling [13]. Oxidation of PTP1B
by H
2
O
2
converts the catalytic cysteine thiol group
Keywords
calpain; degradation; insulin signaling;
oxidation; protein-tyrosine phosphatase
Correspondence
F D. Bo

¨
hmer, Institute of Molecular Cell
Biology, Hans-Kno
¨
ll-Strasse 2, D-07745
Jena, Germany
Fax: +49 3641 939 5602
Tel: +49 3641 939 5631
E-mail:
(Received 20 January 2009, revised 22 July
2009, accepted 3 August 2009)
doi:10.1111/j.1742-4658.2009.07255.x
Protein-tyrosine phosphatases (PTPs) are regulated by reversible inactivat-
ing oxidation of the catalytic-site cysteine. We have previously shown that
reversible oxidation upon UVA irradiation is followed by calpain-mediated
PTP degradation. Here, we address the mechanism of regulated cleavage
and the physiological function of PTP degradation. Reversible oxidation of
PTP1B in vitro strongly facilitated the association with calpain and led to
greatly increased calpain-dependent inactivating cleavage. Both oxidation-
induced association and cleavage depended exclusively on the presence of
the catalytic (reversibly oxidized) cysteine residue 215. A major cleavage
site was identified preceding amino acid position Ala77. In calpain-deficient
cells, insulin signaling was apparently diminished, consistent with a possible
role for calpain in removing a negative regulator of insulin signaling.
Reversibly oxidized PTP1B may be a target of calpain in this context.
Structured digital abstract
l
MINT-7234580: Calpain 4 (uniprotkb:P04632) and Calpain 1 (uniprotkb:P07384) physically
interact (
MI:0915) with PTP1B (uniprotkb:P18031)byenzyme linked immunosorbent assay

(
MI:0411)
l
MINT-7234562, MINT-7234481: Calpain 4 (uniprotkb:P04632) and Calpain 1 (uniprotkb:
P07384) cleave (MI:0194) PTP1B (uniprotkb:P18031)byprotease assay (MI:0435)
Abbreviations
MEF, murine embryonic fibroblasts; PKB, protein kinase B (or AKT); PTP, protein-tyrosine phosphatase.
5622 FEBS Journal 276 (2009) 5622–5633 ª 2009 The Authors Journal compilation ª 2009 FEBS
initially into sulfenic acid, which then undergoes an
intramolecular reaction into a cyclic sulfenyl-amide
structure, causing a conformational change in the cata-
lytic center [14,15]. Oxidative PTP1B inactivation thus
appears to function as a mechanism of positive feed-
forward regulation of insulin signaling.
We have previously discovered yet another level of
PTP regulation: oxidation-dependent cleavage [16].
The requirement for Ca
2+
and inhibitor experiments
point to an involvement of calpains in the oxidation-
induced PTP degradation. Calpains are known to
require Ca
2+
for their activation and exhibit rather
broad substrate specificity with some preference for
PEST-rich domains [17]. The ubiquitously expressed
classical calpains 1 and 2 are heterodimers consisting
of a common regulatory subunit (encoded by capn4)
and a catalytic subunit (encoded by capn1 or capn2,
respectively) [18]. In this study, we have explored the

mechanism of oxidation-dependent cleavage of PTP1B
in vitro and its possible physiological significance. We
show that oxidation of the catalytic cysteine causes an
increased association of PTP1B with calpain and trig-
gers an inactivating cleavage, predominantly behind
amino acid position 76.
Results
Enhanced calpain-mediated cleavage of PTP1B
oxidized at the catalytic cysteine 215
PTP oxidation, including oxidation of PTP1B, upon
UVA irradiation of cells or treatment with H
2
O
2
trig-
gers proteolytic cleavage [16]. In an early study, C-ter-
minal cleavage of PTP1B by calpain in platelets was
described, leading to subcellular relocation without a
change in activity [19]. To avoid this putative compli-
cation, we made use of an engineered PTP1B lacking
the C-terminal cleavage site in our in vitro experiments
(scheme in Fig. 1A) [20]. Both oxidized and reduced
recombinant PTP1B were incubated with increasing
amounts of calpain, and degradation was analyzed by
SDS ⁄ PAGE followed by Coomassie Brilliant Blue
staining. As shown in Fig. 1B, oxidized PTP1B was
readily cleaved by calpain. The amount of intact pro-
tein decreased progressively with higher amounts of
calpain (Fig. 1B), and in a time-dependent manner
(Fig. 2D). Distinct proteolytic fragments appeared at

intermediate calpain concentrations, notably fragments
of  28–30 kDa. Longer incubation with calpain led
to complete loss of detectable PTP1B (data not
shown). By contrast, reduced PTP1B was less vulnera-
ble to cleavage. In particular, the 28 kDa fragment
was not produced, although larger fragments were
generated from both oxidized and nonoxidized PTP1B
(Fig. 1B and Coomassie Brilliant Blue stain of
Fig. 2D). In vitro cleavage was not complete, but could
be enhanced by increasing the amounts of calpain.
We conclude that oxidized PTP1B exhibits enhanced
susceptibility to calpain-mediated degradation.
We then investigated which oxidation was relevant
for the calpain-dependent cleavage. Previous studies
using MS had shown that reversible oxidation of
PTP1B under similar conditions as used here resulted
in nearly exclusive oxidation of the catalytic cysteine,
A
B
C
Fig. 1. Calpain cleavage of PTP1B is strongly enhanced upon oxida-
tion and inactivates the phosphatase. (A) Schematic drawing of
full-length PTP1B (upper) indicating the known C-terminal calpain
cleavage site, and of the truncated 37 kDa PTP1B (aa 1-321) used
for the in-vitro studies. (B) Recombinant 37 kDa PTP1B (10 lg) was
pretreated with or without 300 l
M H
2
O
2

, excess of H
2
O
2
was inac-
tivated by catalase and the samples were subjected to increasing
amounts of calpain and subsequently analyzed by SDS ⁄ PAGE and
Coomassie Brilliant Blue-staining. Arrows indicate oxidation-specific
fragments (28–30 kDa) or undegraded PTP1B, respectively. (C)
Aliquots of in vitro cleavage reactions of wild-type (WT) and cata-
lytic center mutant (C215S) were analyzed with immunoblotting
against the catalytic domain-epitope of PTP1B. Representative
examples of at least three independent experiments are shown.
A. Tru
¨
mpler et al. Calpain-mediated degradation of reversibly oxidized protein-tyrosine phosphatase 1B
FEBS Journal 276 (2009) 5622–5633 ª 2009 The Authors Journal compilation ª 2009 FEBS 5623
Cys215 [9,14]. In order to test whether oxidation of
Cys215 is important for the enhanced cleavage by
calpain, we compared cleavage of wild-type PTP1B
and a PTP1B Cys215Ser mutant upon oxidative treat-
ment. Fragments were detected by immunoblotting
with an antibody directed against the PTP1B catalytic
domain. Calpain treatment of oxidized wild-type
PTP1B led to the formation of small fragments,
including the major 28 kDa peptide (Fig. 1C). Oxi-
dant-treated PTP1B Cys215Ser mutant protein was not
cleaved, however. Thus, oxidation at Cys215 is indeed
necessary to trigger calpain-dependent degradation.
A

C
B
D
+ H
2
O
2
(µM)
300010003001003010
Phosphatase activity (% of untreated)
0%
20%
40%
60%
80%
100%
+ H
2
O
2
+ H
2
O
2
+ DTT
3000
1000
300
100
30

10
–
–
H
2
O
2
(µM)
Reversibly
oxidized PTP1B
DTT
10
30
100
300
H
2
O
2
(µM)
–
1000
3000
300
–+ Calpain
IB: anti PTP1B (N-terminus)
IB: anti PTP1B (catalytic domain)
Undegraded PTP1B
28–30 kDa fragments
Undegraded PTP1B

>30 kDa fragments
IB: anti oxPTP
Undegraded PTP1B
28–30 kDa fragments
Fig. 2. Irreversible inactivation of PTP1B by oxidation-specific cleavage. (A) Recombinant PTP1B was incubated with increasing amounts of
hydrogen peroxide (H
2
O
2
), and subjected to phosphatase assay (black bars, upper) using p-nitrophenyl phosphate (pNPP) as substrate. Aliqu-
ots were back-reduced with 5 m
M dithiothreitol (gray bars) before pNPP assay. The activity of mock-treated PTP1B was set to 100%.
Measurements were performed in triplicate, and the experiment was repeated three times with consistent results. In parallel, aliquots of
H
2
O
2
-treated PTP1B were alkylated using 15 mM sodium iodoacetate and subjected to radioactive in-gel assay (lower). (B) Recombinant
PTP1B (10 lg) was treated with increasing concentrations of H
2
O
2,
excess H
2
O
2
was inactivated by catalase and the samples were subse-
quently subjected to calpain cleavage (1 lg). Aliquots (corresponding to 0.5 lg PTP1B input) were analysed by immunoblotting using PTP1B
antibodies against different epitopes: the N-terminus (upper), the catalytic domain (middle) and irreversibly oxidized PTPs (‘oxPTP antibody’,
lower). (C) Recombinant PTP1B was treated with 300 l

M H
2
O
2
followed by catalase, as under (B), and was then either directly subjected to
calpain digestion, or was first treated with dithiothreitol (DTT; 5 m
M for 30 min), subsequently catalase and then subjected to calpain diges-
tion, as indicated. Controls were exposed to calpain alone or to calpain in presence of dithiothreitol. (D) Recombinant 37 kDa PTP1B (10 lg)
was pretreated with or without 300 l
M H
2
O
2
, excess of H
2
O
2
was inactivated by catalase, and the samples were subjected to digestion
with 1 lg calpain for different times as indicated. Samples were then subjected to SDS ⁄ PAGE and Coomassie Brilliant Blue staining (upper),
and aliquots to radioactive in-gel phosphatase assay (lower). Representative examples of at least three independent experiments are shown.
Note that no PTP activity is detectable for the 28–30 kDa fragment, consistent with the lack of a part of the catalytic core (see Fig. 3).
Calpain-mediated degradation of reversibly oxidized protein-tyrosine phosphatase 1B A. Tru
¨
mpler et al.
5624 FEBS Journal 276 (2009) 5622–5633 ª 2009 The Authors Journal compilation ª 2009 FEBS
Identification of the cleavage site in PTP1B:
a major cleavage site in the N-terminus
To analyze the fragments for residual PTP activity and
determine the site of fragmentation, we oxidized
recombinant PTP1B by treatment with H

2
O
2
and
obtained calpain-dependent fragments for in vitro anal-
ysis. Using dose-titration with H
2
O
2
, the concentration
for optimal reversible oxidation was identified. Revers-
ibility was assessed by two different types of PTP
assay: enzymatic activity after reduction by dithiothrei-
tol and in-gel phosphatase assay after prior incubation
with the alkylating agent sodium iodoacetate. In the
latter assay, only the reversibly oxidized PTP species is
detected because it is protected from alkylation and
subsequently reactivated by dithiothreitol [21]. Both
assays yielded similar results indicating complete and
up to 80% reversible inactivation at 300 lm H
2
O
2
(Fig. 2A). Higher H
2
O
2
concentrations caused substan-
tial irreversible oxidation, although reversibly oxidized
species could be readily detected even at 3000 lm

H
2
O
2
. The modified in-gel assay resulted in an appar-
ently better recovery of PTP activity at 1000–3000 lm
H
2
O
2
, possibly caused by more extensive reduction.
Subsequent to H
2
O
2
pretreatment and removal of
H
2
O
2
by catalase treatment, PTP1B was subjected to
calpain digestion and fragment formation was moni-
tored with different antibodies. Pronounced cleavage
of PTP1B and formation of the 28 kDa fragment
could be seen upon pretreatment with 300–3000 lm
H
2
O
2
using detection with an antibody recognizing the

PTP1B catalytic domain. Under these conditions,
PTP1B is both reversibly and irreversibly oxidized as
described above. Either of the two species or both
could potentially exhibit enhanced susceptibility to cal-
pain. To distinguish between these possibilities, we first
used an antibody detecting only irreversibly oxidized
PTPs (‘oxPTP antibody’) [10]. This antibody reacted
readily with non-degraded PTP1B which was pretreat-
ed with 300–3000 lm H
2
O
2
. However, it gave only
very weak signals with comparable amounts of the
28 kDa fragment (based on anti-PTP1B reactivity),
indicating that this fragment contained only minor
amounts of irreversibly oxidized PTP1B (Fig. 2B, low-
est panel), and suggesting that it was derived from
reversibly oxidized PTP1B. To further address this
question, PTP1B was first oxidized with H
2
O
2
under
conditions which result in the formation of reversibly
and irreversibly oxidized species, and was subsequently
treated with dithiothreitol to back-reduce the reversibly
oxidized fraction. Upon calpain digestion under these
conditions, degradation was greatly impaired and only
the > 30 kDa species were formed, identical to the

case of reduced PTP1B (Fig. 2C). These data clearly
indicate that reversibly oxidized PTP1B, but not irre-
versibly oxidized PTP1B, exhibits enhanced calpain
sensitivity.
An important question was how the observed oxida-
tion-dependent fragmentation would affect PTP activ-
ity. This was assessed by PTP in-gel assays, omitting
prior alkylation. Under the reducing conditions of the
in-gel-phosphatase assay only the full-length non-
degraded PTP1B and fragments > 30 kDa retained
abundant activity, whereas the 28 kDa fragment, char-
acteristic of the oxidation-dependent cleavage, was inac-
tive (Fig. 2D). Semiquantitative evaluation of the in-gel
assay yielded relative activities of 100, 84, and 0.1 (arbi-
trary units) of the undegraded PTP1B, fragments
> 30 kDa and the 28 kDa fragment, respectively.
Immunoblot experiments revealed that the 28 kDa
fragment resulted from an N-terminal cleavage: the
28 kDa fragment did not react with an antibody recog-
nizing an N-terminal epitope, although well recognized
by an antibody specific for the Cys215-carrying catalytic
domain (Fig. 2B), despite the fact that the fragment was
inactive. The exact cleavage site was determined by N-
terminal sequencing of the 28 kDa fragment. The first
amino acid of the fragment was Ala77. The calculated
size of the peptide corresponding to amino acids 77–321
is 28.2 kDa, which matches the observed size. Sequenc-
ing of some of the minor fragments of lower molecular
mass gave the same N-terminus (not shown). Thus, cal-
pain-mediated cleavage of oxidized PTP1B occurs, at

least predominantly, within the N-terminus, possibly in a
step-wise fashion. The PTP fragment of 28 kDa lacks
part of the PTP catalytic core including the invariant resi-
dues Asn44, Arg45, Tyr46, Tyr66 and Asn68 (Fig. 3A)
[20], consistent with its lack of activity. The chemical con-
stitution of the > 30 kDa fragments, which occur in the
absence of oxidation, was analyzed by MS. Consistent
with the immunoblotting experiments (Fig. 2B) and with
retained PTP activity (Fig. 2D), these fragments possess
an intact N-terminus and lack peptides from the C-termi-
nus downstream of amino acid 279 (details in Fig. S1).
Augmented association of oxidized PTP1B with
calpain in vitro
We considered two possibilities for the mechanism
of oxidation-enhanced, calpain-mediated cleavage.
Enhanced cleavage could be either caused by better
accessibility of the cleavage site in oxidized PTP
because of an alteration in the PTP1B structure, or
result from enhanced interaction of PTP with calpain.
The first possibility is unlikely, however, based on the
overlay of the crystal structures of both reduced and
A. Tru
¨
mpler et al. Calpain-mediated degradation of reversibly oxidized protein-tyrosine phosphatase 1B
FEBS Journal 276 (2009) 5622–5633 ª 2009 The Authors Journal compilation ª 2009 FEBS 5625
reversibly oxidized PTP1B: this comparison showed no
difference in the area of the cleavage site which is
located in the loop connecting b-sheets b2 and b3as
indicated in Fig. 3B [14,20].
The alternative explanation for enhanced degra-

dation by calpain could be an increased affinity of
calpain to oxidized PTP1B. Oxidation creates confor-
mational changes in PTP1B in the vicinity of the cata-
lytic site (Fig. 3B) [14]. The altered conformation may
affect an interacting domain, although not the cleavage
site directly, and may facilitate association with
calpain, and in turn cleavage of PTP1B. This possibil-
ity was experimentally assessed. We analyzed binding
of oxidized versus nonoxidized PTP1B to inactive
calpain in an ELISA-based assay. To this end, calpain
was immobilized in 96-well plates in the presence of an
irreversible calpain inhibitor. Thereafter, it was incu-
bated with oxidized or untreated PTP1B, and the
amount of bound PTP1B was detected by an immune
reaction. As shown in Fig. 4A, pretreatment of PTP1B
with increasing concentrations of H
2
O
2
strongly
increased the amount bound to calpain. Importantly,
the nonoxidizable Cys215Ser mutant of PTP1B showed
considerably less association. Thus, Cys215 oxidation
induced both calpain–PTP association (Fig. 4A) and,
as shown above, PTP cleavage. Further, the lack of
H
2
O
2
-induced association of the Cys215Ser mutant

also excludes that the binding is merely caused by
some nonspecific effect of H
2
O
2
such as increased
charges. We considered that generation of a PTP1B
mutant which was resistant to oxidation-dependent
cleavage by calpain would be an excellent tool with
which to investigate the role of this degradation for
signal transduction. Mutation of Ala77 itself was
excluded from this approach because this residue is
buried in the structure and mutation was expected to
destabilize the protein (D. Barford, personal communi-
cation). Instead, mutation of amino acid residues sur-
rounding the identified cleavage site (Lys73Ala,
Glu75Ala, Glu76Gln, Glu76Ala, Gln78Ala and the
double mutant Lys73Ala ⁄ Gln78Ala) was carried out
and the corresponding recombinant proteins were ana-
lyzed. These mutations had no impact on the associa-
tion of PTP1B or on cleavage: oxidized PTP1B
mutated in these positions was efficiently cleaved (data
not shown). This is consistent with the notion that the
interaction site and cleavage site are not identical.
We also attempted, but failed, to consistently detect
complexes of reversibly oxidized PTP1B and calpain in
intact cells by co-immunoprecipitation (data not shown).
Although complexes of calpain-regulatory subunits
with target proteins have previously been shown
[22,23], calpain–substrate interactions appear gener-

ally difficult to monitor by co-immunoprecipitation.
Calpain activity requires Ca
2+
and conformational
changes upon Ca
2+
binding are necessary for the rec-
ognition of some substrates [24–26]. We therefore
tested whether Ca
2+
is important for binding to
PTP1B. As shown in Fig. 4B, the H
2
O
2
-induced asso-
ciation of PTP1B with calpain was Ca
2+
independent.
Insulin signaling to protein kinase B (PKB/AKT)
and p70-S6 kinase appears attenuated in
calpain-deficient cells
PTP1B is a major negative regulator of insulin signal-
ing [11,13,27,28]. Insulin receptor stimulation is also
Reduced conformation
Oxidized conformation
Cys215 /
sulphenyl amide
Identified
cleavage

site
β3
β2
A
B
Fig. 3. Calpain removes an N-terminal fragment from oxidized
PTP1B. (A) Amino acid sequence of the 37 kDa PTP1B (aa 1-321).
Amino acids of the phosphatase domain are underlined, and invari-
ant residues within the N-terminal part are in bold and boxed. The
identified cleavage site between Glu76 and Ala77 is indicated by a
gray arrowhead. The removed N-terminus upon cleavage is in gray
letters, and the remaining C-terminus is black. Asterisks indicate
the amino acids that have been substituted with the aim of achiev-
ing calpain resistance. (B) Overlay of crystal structures of reduced
PTP1B (2HNQ), and of the sulfenyl amide, reversibly oxidized
PTP1B (1OEM) has been carried out using
PYMOL 0.99rc6 (DeLano
Scientific LLC, San Francisco, CA, USA). The cleavage site loop
located between b-sheets b2 and b3, as well as regions showing
conformational differences among the two structures are coloured
in yellow (reduced form) and green (sulfenyl amide form). Other
areas are kept in light and dark gray, respectively.
Calpain-mediated degradation of reversibly oxidized protein-tyrosine phosphatase 1B A. Tru
¨
mpler et al.
5626 FEBS Journal 276 (2009) 5622–5633 ª 2009 The Authors Journal compilation ª 2009 FEBS
known to cause reversible oxidation of PTP1B, which
is considered to be an important mechanism of signal
enhancement [11]. According to our data, reversibly
oxidized PTP1B should be prone to calpain-mediated

degradation. Because insulin-dependent oxidation pre-
sumably reaches only a small fraction of PTP1B in
proximity to activated insulin receptors, bulk degrada-
tion is unlikely to occur. Absence of calpain should,
however, cause a reduction in insulin signaling pro-
vided the PTP1B cleavage was relevant for enhanced
signal transduction. To address this question we used
immortalized fibroblasts from wild-type or capn4
knockout mice, which have no detectable calpain 1 or
2 activity [29]. The cells were stimulated with insulin
and analyzed with respect to signal transduction. As
shown in Fig. 5, insulin stimulation did not signifi-
cantly alter total PTP1B levels in these cells, and had
no obvious effects on cellular distribution of PTP1B
detected by immunocytochemistry (Fig. S2). Also, we
could not detect effects of insulin stimulation on total
levels of T-cell PTP, a close relative of PTP1B which
likewise negatively controls insulin signaling [30] (not
shown). Interestingly, however, the activation of two
critical downstream signaling mediators, AKT and
p70-S6 kinase, was significantly reduced in capn4
knockout fibroblasts, compared with wild-type fibro-
blasts. Importantly, lentiviral rescue by Capn4 of the
capn4 knockout cells was able to almost fully rescue
insulin-induced AKT and S6 kinase activation. It
should be noted that in unstimulated wild-type cells,
P-AKT and P-p70-S6Kinase levels were higher than in
calpain 4 knockout or rescued cells, possibly reflecting
a role for calpain in the regulation of basal activity of
these pathways. Phosphorylation of a protein of

 120 kDa, which reacted with antibodies against
phosphorylated insulin receptor, but is apparently not
representing the insulin receptor itself, was also
strongly diminished in the calpain negative cells
(Fig. 5), whereas using the same antibody no obvious
differences could be detected in phosphorylation of im-
munoprecipitated insulin receptor b subunit in absence
or presence of Capn4 (Fig. S3). Activation of ERK1 ⁄ 2
by insulin stimulation was very low under the
employed conditions, and not reduced or even some-
what higher in the absence of calpain (Fig. 5). Appar-
ently, calpain affects the strength of insulin signaling
to AKT, and p70-S6 kinase. The detailed mechanism
and the further biological consequences of Capn4-
deficiency for insulin signaling require further investi-
gation.
Discussion
We have shown previously that PTPs are subject to
calpain-dependent degradation under conditions which
cause PTP oxidation and calpain activation, such as
UVA irradiation [16]. In this study, we reconstituted
the oxidation-dependent degradation of PTP1B by cal-
pain in vitro in order to prove that calpain is responsi-
ble for the cleavage reaction, to identify the site of
cleavage and the mechanism of the reaction. Oxidation
of PTP1B at the catalytic Cys215 is required for both
AB
Fig. 4. Oxidation enhances binding of PTP1B to calpain. (A) Preoxidized recombinant PTP1B wild-type (black bars) and the PTP1B C215S
mutant (gray bars) were allowed to bind to calpain-coated 96-well plates in absence of Ca
2+

. Bound PTP1B was detected with a PTP1B anti-
body, and horseradish peroxidase-coupled secondary antibody. Calpain was preincubated with an irreversible inhibitor to prevent degradation
of bound PTP1B. The values were calculated relative to the association of wild-type PTP1B in the absence of H
2
O
2
treatment (100%). (B)
Association of PTP1B pretreated with (white bars) or without (black bars) 300 l
M H
2
O
2
to calpain was assessed as in (A). Experiments were
performed either in the absence of Ca
2+
(EGTA) or in the presence of 1 mM CaCl
2
(Ca
2+
). Data present means of triplicate measures and
consistent results have been obtained in at least three independent experiments.
A. Tru
¨
mpler et al. Calpain-mediated degradation of reversibly oxidized protein-tyrosine phosphatase 1B
FEBS Journal 276 (2009) 5622–5633 ª 2009 The Authors Journal compilation ª 2009 FEBS 5627
the association of calpain with its substrate and for the
cleavage reaction. Cleavage is induced by reversible
but apparently not by irreversible oxidation, and a
major cleavage occurs at amino acid position 77. The
resulting enzyme fragments are inactive, consistent

with the fact that important positions of the catalytic
center are removed.
What determines calpain-substrate association? In
addition to modulation of the activity of an enzyme,
altered interaction affinity is frequently used in cellu-
lar regulation. For example, Jun N-terminal kinase
finds its substrate Jun through a domain remote
from the phosphorylation sites [31]. The serine phos-
phatase MYPT-1-PP1 associates with and acts on its
substrate merlin only after its own dephosphoryla-
tion, and again substrate binding and dephosphoryla-
tion of the substrate occur at different sites [32].
Often the substrate appears to be made susceptible
for interaction with an enzyme. In our report, the
PTP is modified by oxidation to become an inter-
action partner for calpain, although we still lack
evidence that this complex formation can occur in
intact cells. Another substrate of calpain, a-actinin,
is influenced by binding to phosphoinositides: phos-
phatidylinositol-3,4,5-trisphosphate enhances and
phosphatidylinositol-4,5-diphosphate inhibits the
cleavage [33]. The subcellular localization or specific
cellular conditions may determine the cleavage, as
with PTP1B in platelets [19]. The interaction in vitro
is apparently independent of Ca
2+
(Fig. 4B) suggest-
ing that it may occur through a calpain region
which is distant from the active center.
The interaction domain in PTP1B or the conforma-

tional determinants have not yet been mapped. They
may define the cleavage site. Reversible Cys215 oxida-
tion generates a conformational change [14,15], which
apparently catalyzes the association with calpain and
leads to proteolytic action around amino acid position
77. For example, oxidation causes exposure of the
amino acid residues Tyr46 of the phosphoTyr loop
and Ile219 of the PTP loop, which could be important
for calpain binding [14]. Oxidation-specific cleavage
does not occur with nonoxidized PTP1B. Nonoxidized
PTP1B is instead cleaved C-terminally and activity is
retained in the fragments occurring under these condi-
tions (see Figs 1B, 2D and S1). Similarly, in activated
platelets, calpain splits an 8 kDa fragment off the
PTP1B C-terminus [19], and the truncated PTP1B
retains the enzymatic activity. The lost C-terminal
fragment carries an endoplasmic reticulum localization
signal causing relocalization of truncated PTP1B to
the cytosol. It thus appears that the state of the sub-
strate defines the association with calpain, the cleavage
site and the resulting enzymatic activity of the
substrate. In capn1-negative mouse platelets, PTP1B
protein levels and enzyme activity were increased,
resulting in reduced overall protein tyrosine phosphor-
ylation levels and impaired platelet aggregation. This
phenotype was rescued in capn1 and PTP1B doubly
deficient mice [34]. With respect to this platelet pheno-
type, calpain thus seems to negatively regulate PTP1B
activity. It is not currently known whether PTP1B
oxidation is involved in this context.

P-AKT
P-p70-S6 kinase
AKT
p70-S6 kinase
Ø 5 10 15 30
Wild-typecapn4 neg. Lenti-rescued
Ø 5 10 15 30Ø 5 1015 30
(min) Insulin
Vinculin
PTP1B
72
95
130
P-InsR
InsR-β
72
95
130
Calpain 4
26
panERK
P-ERK
Fig. 5. Calpain-deficiency results in reduced activation of AKT upon
insulin stimulation of cells. Immortalized murine fibroblasts, either
calpain 4 knockout (capn4 neg.), wild-type or calpain 4 knockout
with rescued calpain 4 expression by lentiviral transduction (lenti-
rescued), were starved in 0.5% serum overnight, and stimulated
with 10 n
M insulin for the indicated times. Lysates (100 lg) were
subjected to immunoblotting and analyzed for levels of PTP1B as

well as for the phosphorylation state of AKT (Ser473), p70-S6Kinase
(Thr389), the insulin receptor (Tyr1162,1163) and ERK1 ⁄ 2. Note
that pEKR1 ⁄ 2 phosphorylation became detectable only after
prolonged exposure. The antibody against phosphorylated insulin
receptor detects an obviously unrelated phosphoprotein in the
lysates. Detection of insulin receptor phosphorylation requires prior
immunopreciptitation (see Fig. S3). Controls for total amounts of
the corresponding proteins, and for calpain 4 expression are also
shown. Note that re-expression of calpain 4 was performed with a
construct encoding a somewhat truncated form. Total levels of
PTP1B were consistently somewhat higher in the Capn4-rescued
cells and total ERK1 ⁄ 2 levels in calpain 4 knockout cells from
unknown reasons, presumably reflecting an unrelated property of
the independently immortalized cell pools. The blots in panels 1–6,
11 (from top) are representative of 5, in panels 7–10 (from top) for
three independent experiments.
Calpain-mediated degradation of reversibly oxidized protein-tyrosine phosphatase 1B A. Tru
¨
mpler et al.
5628 FEBS Journal 276 (2009) 5622–5633 ª 2009 The Authors Journal compilation ª 2009 FEBS
PTP1B is not the only PTP accessible to calpain.
For example, SHP-1 and LAR-PTP were targets upon
UVA irradiation of cells [16]. Accordingly, oxidation-
dependent degradation of PTPs by calpain may be of
more general importance. The sensitivity to oxidation
may vary with the PTP. For example, the lower sensi-
tivity of SHP-1 for UVA-induced degradation than
that of PTP1B may reflect its comparatively lower sen-
sitivity to reversible oxidation [8,9]. The sequence of
the loop harboring the oxidation-induced cleavage site

identified in PTP1B is quite different among different
PTPs. Cleavage may nevertheless occur at correspond-
ing positions, given the relatively low sequence require-
ments for calpain cleavage evidenced by efficient
proteolysis of our PTP1B mutants around position 77.
Mutants of Lys73, Glu75, Glu76 and Gln78 are not
expected to exhibit grossly altered PTP structure [17].
They were similarly active, similarly reversibly oxidized
and, when oxidized, showed in vitro similar calpain-
sensitivity as wild-type PTP1B (data not shown).
It has recently been shown by Groen et al. [4] that
oxidation leads to enhanced and altered degradation
of four different receptor-like PTPs by trypsin. In
addition to conformational changes in the catalytic
domains upon oxidation, the authors link changes in
PTP dimerization to the altered susceptibility to prote-
olysis. It is tempting to speculate that susceptibility to
cell-endogenous proteinases such as calpain may also
be affected by oxidation-associated dimerization.
Calpain is a cysteine-proteinase and has been
reported to be prone to inactivating oxidation [35,36].
Under the conditions used here for PTP1B oxidation,
calpain activity appeared, however, unaffected. Also,
PTP1B cleavage occurred irrespective of the addition of
catalase to the in vitro experiments (not shown). Thus,
calpain seems to be less sensitive to H
2
O
2
than PTP1B.

Is regulated proteolysis by calpain a physiologically
relevant process? The association of calpain with
a-actinin appears to be important for actin dynamics
in tumor cells [33]. Calpain has been reported to par-
ticipate in integrin b3 inside-out signaling [37,38]. As
alluded to above, reduced levels of tyrosine phosphory-
lation and reduced platelet aggregation have been
observed in calpain 1-negative platelets and have been
attributed to a specific PTP1B cleavage by calpain
[34]. Furthermore, calpain-negative primary murine
embryonic fibroblasts (MEF) exerted an altered
response towards a variety of cell death stimuli (e.g.
tumour necrosis factor a, staurosporine, puromycin)
when compared with wild-type cells [39]. In the
absence of calpain, AKT phosphorylation was reduced
and the apoptosis rate was increased, indicating an
involvement of calpain in AKT activation possibly by
removing a phosphatase. Very recently, reduced AKT
activity in calpain 1-negative fibroblasts was attributed
to enhanced stability of the B56alpha regulatory sub-
unit of PP2A, leading to enhanced association of
PP2A with AKT [40].
The data obtained suggest a possible role of calpain
in insulin signaling. Target phosphorylation of insulin
was reduced in calpain-negative cells (capn4 knockout
MEFs) and restored upon lentiviral introduction of
calpain. Because insulin stimulation has been shown to
induce oxidation of PTP1B [41], one could speculate
that the here-described increased susceptibility of
oxidized PTP1B for an inactivating degradation by

calpain may play a role in this context. However, the
currently available data do not allow us to establish a
causal link between insulin signaling and potential
PTP1B degradation yet. For this, it will be necessary
to show inactivating fragmentation of PTP1B upon
insulin stimulation, and interference with insulin sig-
naling by PTP1B mutants which are less susceptible to
degradation. It is possible that the above described
mechanism of calpain-mediated positive control of
AKT [40] also contributes to the effects we have
observed in our setting of insulin stimulation.
Further, it is tempting to speculate that the require-
ment of calpain for insulin signaling described here
may have implications for pathology. There have been
a number of reports linking calpain dysfunction to
type II diabetes mellitus and type II diabetes mellitus-
related phenotypes [42–48]. We have obtained
indications for a previously unrecognized positive role
of calpain for insulin signaling, prompting further
investigation of the underlying mechanisms.
Materials and methods
Materials
Hydrogen peroxide (#H1009), catalase (#C9322), p-nitro-
phenyl phosphate (#S0942), sodium iodoacetate (#I9148),
3,3¢,5,5¢-tetramethylbenzidine (#T2885), poly(Glu,Tyr)
(#P0275), E64 (#E3132), insulin (#I0516) and poly-l-lysine
(#P2636) were purchased from Sigma-Aldrich (Taufkirchen,
Germany). Calpain (#208713) and PD150606 (#513022)
were from Merck Biosciences (Nottingham, UK). Complete
protease inhibitor cocktail (#11836170001) was from Roche

Diagnostics (Mannheim, Germany). Anti-PTP1B against
the catalytic domain (#PH01) was from Merck Biosciences;
against the N-terminus of PTP1B (sc1718) and anti-insulin
Rb (#sc711) were from Santa Cruz (Santa Cruz, CA,
USA); anti-PTP1B against the N-terminus of PTP1B
(AP8411c) was from Abgent (San Diego, CA, USA); and
anti-calpain (C0355) was from Sigma-Aldrich. Antibodies
A. Tru
¨
mpler et al. Calpain-mediated degradation of reversibly oxidized protein-tyrosine phosphatase 1B
FEBS Journal 276 (2009) 5622–5633 ª 2009 The Authors Journal compilation ª 2009 FEBS 5629
against phospho-Ser473-AKT (#4058S), panAKT (#9272),
phospho-Thr389-S6 kinase (#9202), S6-kinase (#2217) and
phospho-p44 ⁄ 42 MAPK (Erk1 ⁄ 2) (Thr202 ⁄ Tyr204)
(#9106S) were from Cell Signaling (Danvers, MA, USA);
against murine PTP1B (#07-088) was from Millipore (Sch-
walbach, Germany); anti-insulin ⁄ insulin-like growth factor-
1 receptor (IR ⁄ IGF1R) [pYpY1162 ⁄ 1163] (#44804G) and
Alexa Fluor 488 goat anti-(rabbit IgG) (#A11034) were
from Invitrogen (Carlsbad, CA, USA). Anti-vinculin
(V248) was from Upstate (Biomol, Hamburg, Germany),
horseradish peroxidase-coupled secondary antibodies [anti-
mouse and anti-(rabbit IgG)] were from KPL (Gaithers-
burg, MD, USA) or Pierce (#1858413; Rockford, IL,
USA). Polyclonal oxPTP antibody was a kind gift from
Arne O
¨
stman (Stockholm, Sweden) [10].
Wild-type (PZ ⁄ PZ), capn4 knockout (P ⁄ P), and lentivirally
rescued P ⁄ P immortalized mouse embryonic fibroblasts

have been described [29]. Cells were routinely kept in DMEM
supplied with 10% fetal bovine serum and passaged every
2–3 days by trypsinization. Immortalized murine fibroblasts
derived from mice with disruption of the PTP1B gene were
kindly provided by Ben Neel and Fawaz Haj and have been
described earlier [49]. These cells were routinely kept in
DMEM supplied with 10% newborn bovine serum.
Plasmids and mutants preparation
A plasmid encoding the C-terminally truncated variant of
wild-type PTP1B (aa 1-321) for recombinant expression was
a kind gift from D. Barford (Institute of Cancer Research,
London, UK) [20]. Mutants were created by site-directed
mutagenesis using Pfu turbo polymerase (Stratagene, La Jo-
lla, CA, USA). The forward primer for C215S mutant was:
5¢-CCCGTTGTGGTGCACTCC AGTGCAGGCATCG GC
A-3¢ (other primer sequences can be obtained on request).
Expression was performed in Escherichia coli Rosetta, and
purification to homogeneity by chromatography was carried
out according to a protocol slightly modified from the
previously published procedure [20]. Briefly, separation was
initially performed using a HiTrap Q anion exchange column
(GE-Amersham, Freiburg, Germany), and the pooled
fractions were directly subjected to separation with a phenyl-
superose column (GE-Amersham).
In vitro degradation assay
Recombinant PTP1B (10 lg in reaction volume of 50 lL)
was pretreated with or without 300 lm H
2
O
2

at room tem-
perature in the dark for 1 h. Afterwards, catalase was
added to a concentration of 0.3 lg Æ mL
)1
and incubation
continued for 30 min. The cleavage reaction was started
with addition of 1 mm CaCl
2
and 3 lg calpain achieving
final volume of 70 lL per reaction (for alternate amounts
of calpain see figure legends), and allowed to proceed for
another 30 min at 20 °C. The reaction was stopped by
addition of SDS ⁄ PAGE sample buffer and boiling at 95 °C
for 5 min. Aliquots were separated on 13% SDS ⁄ PAGE
and either Coomassie Brilliant Blue-stained or transferred
to a poly(vinylidene difluoride) membrane. Membranes
were either probed with antibodies against different PTP1B
epitopes or Coomassie Brilliant Blue-stained. For identifica-
tion of fragments, the Coomassie Brilliant Blue-stained oxi-
dation-specific bands on the poly(vinylidene difluoride)
membrane were cut out and sequenced by EDMAN
degradation with 494A Procise protein sequencer (Applied
Biosystems, Foster City, CA, USA).
In vitro association assay
Assays were performed according to a published protocol
[50]. Briefly, 96-well plates were coated with 0.1 lg calpain
per well in the presence of the irreversible inhibitor 1 lm
E64, either with 1 mm Ca
2+
or with 1 mm EGTA overnight.

All washings were carried out using NaCl ⁄ P
i
⁄ Tween (0.5 m
NaCl, 2.7 mm KCl, 4.3 mm Na
2
HPO
4
, 1.4 m m KH
2
PO
4
,
pH 7.4, 0.2% Tween-20). Plates were blocked against unspe-
cific binding of proteins with blocking solution consisting of
2.5% milk and 1% BSA in NaCl ⁄ P
i
for 3 h. PTP1B was
preincubated with or without H
2
O
2
as described above and,
after washing the plates twice, allowed to bind to immobi-
lized calpain for 2 h. Washing was repeated three times and
primary PTP1B antibody as well as secondary antibody
(PH01 1 : 1000 and anti-mouse IgG–horseradish peroxidase
1 : 10 000, respectively) diluted in blocking solution were
incubated 2 h each. Detection was done using 3,3¢,5,5¢-tetra-
methylbenzidine as substrate and 1 m HCl to stop reaction
after  30 min. Quantification was carried out by measuring

A at 405 nm.
Insulin stimulation
Cells were starved overnight in 0.5% serum, washed twice
with serum-free DMEM, and stimulated for the indicated
times with 10 nm insulin at 37 °C. After washing twice with
ice-cold NaCl ⁄ P
i
, cells were lysed in ice-cold lysis buffer
(50 mm Hepes pH 7.5, 150 mm NaCl, 0.5% NP-40, 10 mm
NaPP, 10 mm b-glycerophosphate, 1 mm NaVO
4
,50mm
NaF and protease inhibitors). Protein concentration was
determined using BCA kit (Pierce). Adjusted volumes were
boiled in SDS ⁄ PAGE sample buffer, separated on 7.5%
SDS ⁄ PAGE, transferred onto poly(vinylidene difluoride)
membrane and probed with respective antibodies. Stripping
of membranes was performed in 62.5 mm Tris pH 6.8, 2%
SDS, and 100 mm b-mercaptoethanol for 30 min at 50 °C.
Immunhistochemistry
Cells were plated on polylysine-coated cover slips in 24-well
plates and starved overnight in 0.5% serum. After washing
Calpain-mediated degradation of reversibly oxidized protein-tyrosine phosphatase 1B A. Tru
¨
mpler et al.
5630 FEBS Journal 276 (2009) 5622–5633 ª 2009 The Authors Journal compilation ª 2009 FEBS
twice with serum-free DMEM cells were incubated for
30 min with or without 10 nm insulin in DMEM, washed
twice with ice-cold NaCl ⁄ P
i

and fixed with 3.7% formalde-
hyde in NaCl ⁄ P
i
for 10 min at room temperature. Permea-
bilization was carried out after washing with NaCl ⁄ P
i
with
5 min incubation with 0.2% Triton X-100 in NaCl ⁄ P
i
and
followed by blocking using 1.5% BSA in NaCl ⁄ P
i
. Incuba-
tion with primary antibody against murine PTP1B (1 : 200
dilution in BSA ⁄ NaCl ⁄ P
i
) was performed overnight at
4 °C. After washing with BSA ⁄ NaCl ⁄ P
i
, slides were incu-
bated with secondary antibody against rabbit IgG (1 : 500
dilution in BSA ⁄ NaCl ⁄ P
i
, Alexa Fluor 488 coupled IgG)
for 20 min in the dark. Slides were washed, mounted
and kept in the dark until further analysis. Images
were acquired using a LSM510 Meta from Carl Zeiss (Jena,
Germany).
Mass spectrometry
Appropriate gel sections were subjected to in gel digestion

with trypsin according to Shevchenko et al. [51] with one
modification: reduction and oxidation of thiol goups was
performed with a mixture of 0.1 mm tributylphosphine
(Sigma-Aldrich) and 0.82 mm 4-vinylpyridine (Sigma-
Aldrich). Sequencing grade modified trypsin was purchased
from Promega (Mannheim, Germany). The tryptic in gel
protein digests were analyzed with a LC-ESI-MS equipment
consisting an Ettan MDLC
Ô
-HPLC (GE Healthcare, Frei-
burg, Germany) coupled online to a Finnigan LTQ mass
spectrometer (Thermo Electron Corp., Dreieich, Germany).
The HPLC was equipped with a Zorbax 300SB, 5 lm,
5 · 0.3 mm trapping column and a Zorbax 300SB, 5 lm,
150 · 0.075 mm separation column (Agilent, Boeblingen,
Germany). The separation of peptides on the HPLC
occurred by applying a linear gradient running from 0% to
47% acetonitrile, followed by a stepwise elution with 84%
acetontrile in 0.1% formic acid each under control of the
unicornÔ software (GE Healthcare). The LTQ was oper-
ated under control of the xcalibur 1.4Ô software (Thermo
Electron Corp.). For processing of the CID-spectra
(MS ⁄ MS-spectra spectra) with final protein identification
the bioworks 3.2Ô software (Thermo Electron Corp.) and
the NCBI human protein database were used.
Acknowledgements
This work was supported by grants DFG He 551⁄ 11
(to PH) and DFG Bo 1043 ⁄ 6-2 (to FDB) and from the
European Community (MRTN-CT-2006-035830) (to
FDB). We thank David Barford for discussions and

provision of PTP1B expression constructs, and Benja-
min Neel, Fawaz Haj and Arne O
¨
stman for reagents.
Furthermore, we thank Andrea Uecker for technical
assistance with a part of the experiments.
References
1 Hendriks WJ, Elson A, Harroch S & Stoker AW (2008)
Protein tyrosine phosphatases: functional inferences
from mouse models and human diseases. FEBS J 275,
816–830.
2 Tonks NK (2006) Protein tyrosine phosphatases: from
genes, to function, to disease. Nat Rev Mol Cell Biol 7,
833–846.
3 Flint AJ, Gebbink MF, Franza BR Jr, Hill DE & Ton-
ks NK (1993) Multi-site phosphorylation of the protein
tyrosine phosphatase, PTP1B: identification of cell cycle
regulated and phorbol ester stimulated sites of phos-
phorylation. EMBO J 12, 1937–1946.
4 Groen A, Overvoorde J, van der Wijk T & den Hertog
J (2008) Redox regulation of dimerization of the recep-
tor protein-tyrosine phosphatases RPTPalpha, LAR,
RPTPmu and CD45. FEBS J 275, 2597–2604.
5 Brautigan DL & Pinault FM (1993) Serine phosphory-
lation of protein tyrosine phosphatase (PTP1B) in HeLa
cells in response to analogues of cAMP or diacylglyc-
erol plus okadaic acid. Mol Cell Biochem 127–128,
121–129.
6 Dadke S, Cotteret S, Yip SC, Jaffer ZM, Haj F, Ivanov
A, Rauscher F III, Shuai K, Ng T, Neel BG et al.

(2007) Regulation of protein tyrosine phosphatase 1B
by sumoylation. Nat Cell Biol 9, 80–85.
7 Lohse DL, Denu JM, Santoro N & Dixon JE (1997)
Roles of aspartic acid-181 and serine-222 in intermedi-
ate formation and hydrolysis of the mammalian
protein-tyrosine-phosphatase PTP1. Biochemistry 36,
4568–4575.
8 Weibrecht I, Bo
¨
hmer SA, Dagnell M, Kappert K,
O
¨
stman A & Bo
¨
hmer FD (2007) Oxidation sensitivity of
the catalytic cysteine of the protein-tyrosine phosphatases
SHP-1 and SHP-2. Free Radic Biol Med 43, 100–110.
9 Groen A, Lemeer S, van der Wijk T, Overvoorde J,
Heck AJ, O
¨
stman A, Barford D, Slijper M & den Her-
tog J (2005) Differential oxidation of protein-tyrosine
phosphatases. J Biol Chem 280, 10298–10304.
10 Persson C, Sjo
¨
blom T, Groen A, Kappert K, Engstrom
U, Hellman U, Heldin CH, den Hertog J & O
¨
stman A
(2004) Preferential oxidation of the second phosphatase

domain of receptor-like PTP-alpha revealed by an anti-
body against oxidized protein tyrosine phosphatases.
Proc Natl Acad Sci USA 101, 1886–1891.
11 Meng TC, Buckley DA, Galic S, Tiganis T & Tonks
NK (2004) Regulation of insulin signaling through
reversible oxidation of the protein-tyrosine phosphata-
ses TC45 and PTP1B. J Biol Chem 279, 37716–
37725.
12 Sundaresan M, Yu ZX, Ferrans VJ, Irani K & Finkel T
(1995) Requirement for generation of H
2
O
2
for platelet-
derived growth factor signal transduction. Science 270,
296–299.
A. Tru
¨
mpler et al. Calpain-mediated degradation of reversibly oxidized protein-tyrosine phosphatase 1B
FEBS Journal 276 (2009) 5622–5633 ª 2009 The Authors Journal compilation ª 2009 FEBS 5631
13 Elchebly M, Payette P, Michaliszyn E, Cromlish W,
Collins S, Loy AL, Normandin D, Cheng A, Himms-
Hagen J, Chan CC et al. (1999) Increased insulin sensi-
tivity and obesity resistance in mice lacking the protein
tyrosine phosphatase-1B gene. Science 283, 1544–1548.
14 Salmeen A, Andersen JN, Myers MP, Meng TC, Hinks
JA, Tonks NK & Barford D (2003) Redox regulation
of protein tyrosine phosphatase 1B involves a sulphe-
nyl-amide intermediate. Nature 423, 769–773.
15 van Montfort RL, Congreve M, Tisi D, Carr R & Jhoti

H (2003) Oxidation state of the active-site cysteine in
protein tyrosine phosphatase 1B. Nature 423, 773–777.
16 Gulati P, Markova B, Go
¨
ttlicher M, Bo
¨
hmer FD &
Herrlich PA (2004) UVA inactivates protein tyrosine
phosphatases by calpain-mediated degradation. EMBO
Rep 5, 812–817.
17 Tompa P, Buzder-Lantos P, Tantos A, Farkas A,
Szilagyi A, Banoczi Z, Hudecz F & Friedrich P (2004)
On the sequential determinants of calpain cleavage.
J Biol Chem 279, 20775–20785.
18 Goll DE, Thompson VF, Li H, Wei W & Cong J
(2003) The calpain system. Physiol Rev 83, 731–801.
19 Frangioni JV, Oda A, Smith M, Salzman EW & Neel
BG (1993) Calpain-catalyzed cleavage and subcellular
relocation of protein phosphotyrosine phosphatase 1B
(PTP-1B) in human platelets. EMBO J 12, 4843–4856.
20 Barford D, Flint AJ & Tonks NK (1994) Crystal struc-
ture of human protein tyrosine phosphatase 1B. Science
263, 1397–1404.
21 Markova B, Gulati P, Herrlich PA & Bo
¨
hmer FD
(2005) Investigation of protein-tyrosine phosphatases by
in-gel assays. Methods 35, 22–27.
22 Rosenberger G, Gal A & Kutsche K (2005) AlphaPIX
associates with calpain 4, the small subunit of calpain,

and has a dual role in integrin-mediated cell spreading.
J Biol Chem 280, 6879–6889.
23 Shimada M, Mahon MJ, Greer PA & Segre GV (2005)
The receptor for parathyroid hormone and parathyroid
hormone-related peptide is hydrolyzed and its signaling
properties are altered by directly binding the calpain
small subunit. Endocrinology 146, 2336–2344.
24 Strobl S, Fernandez-Catalan C, Braun M, Huber R,
Masumoto H, Nakagawa K, Irie A, Sorimachi H,
Bourenkow G, Bartunik H et al. (2000) The crystal
structure of calcium-free human m-calpain suggests an
electrostatic switch mechanism for activation by
calcium. Proc Natl Acad Sci USA 97, 588–592.
25 Moldoveanu T, Hosfield CM, Lim D, Elce JS, Jia Z &
Davies PL (2002) A Ca(2+) switch aligns the active site
of calpain. Cell 108, 649–660.
26 Hanna RA, Campbell RL & Davies PL (2008)
Calcium-bound structure of calpain and its mechanism
of inhibition by calpastatin. Nature 456, 409–412.
27 Ahmad F, Li PM, Meyerovitch J & Goldstein BJ
(1995) Osmotic loading of neutralizing antibodies
demonstrates a role for protein-tyrosine phosphatase 1B
in negative regulation of the insulin action pathway.
J Biol Chem 270, 20503–20508.
28 Kenner KA, Anyanwu E, Olefsky JM & Kusari J
(1996) Protein-tyrosine phosphatase 1B is a negative
regulator of insulin- and insulin-like growth factor-I-
stimulated signaling. J Biol Chem
271, 19810–19816.
29 Tan Y, Dourdin N, Wu C, De Veyra T, Elce JS &

Greer PA (2006) Conditional disruption of ubiquitous
calpains in the mouse. Genesis 44, 297–303.
30 Galic S, Hauser C, Kahn BB, Haj FG, Neel BG, Tonks
NK & Tiganis T (2005) Coordinated regulation of insu-
lin signaling by the protein tyrosine phosphatases
PTP1B and TCPTP. Mol Cell Biol 25, 819–829.
31 Karin M & Gallagher E (2005) From JNK to pay dirt:
jun kinases, their biochemistry, physiology and clinical
importance. IUBMB Life 57, 283–295.
32 Jin H, Sperka T, Herrlich P & Morrison H (2006)
Tumorigenic transformation by CPI-17 through inhibi-
tion of a merlin phosphatase. Nature 442, 576–579.
33 Sprague CR, Fraley TS, Jang HS, Lal S & Greenwood
JA (2008) Phosphoinositide binding to the substrate
regulates susceptibility to proteolysis by calpain. J Biol
Chem 283, 9217–9223.
34 Kuchay SM, Kim N, Grunz EA, Fay WP & Chishti
AH (2007) Double knockouts reveal that protein tyro-
sine phosphatase 1B is a physiological target of calpain-
1 in platelets. Mol Cell Biol 27, 6038–6052.
35 Guttmann RP, Elce JS, Bell PD, Isbell JC & Johnson
GV (1997) Oxidation inhibits substrate proteolysis by
calpain I but not autolysis. J Biol Chem 272, 2005–
2012.
36 Guttmann RP & Johnson GV (1998) Oxidative stress
inhibits calpain activity in situ. J Biol Chem 273, 13331–
13338.
37 Xi X, Bodnar RJ, Li Z, Lam SC & Du X (2003) Criti-
cal roles for the COOH-terminal NITY and RGT
sequences of the integrin beta3 cytoplasmic domain in

inside-out and outside-in signaling. J Cell Biol 162,
329–339.
38 Xi X, Flevaris P, Stojanovic A, Chishti A, Phillips DR,
Lam SC & Du X (2006) Tyrosine phosphorylation of
the integrin beta 3 subunit regulates beta 3 cleavage by
calpain. J Biol Chem 281 , 29426–29430.
39 Tan Y, Wu C, De Veyra T & Greer PA (2006) Ubiqui-
tous calpains promote both apoptosis and survival sig-
nals in response to different cell death stimuli. J Biol
Chem 281, 17689–17698.
40 Bertoli C, Copetti T, Lam EW, Demarchi F & Schnei-
der C (2008) Calpain small-1 modulates Akt ⁄ FoxO3A
signaling and apoptosis through PP2A. Oncogene 28,
721–733.
41 Mahadev K, Zilbering A, Zhu L & Goldstein BJ (2001)
Insulin-stimulated hydrogen peroxide reversibly inhibits
protein-tyrosine phosphatase 1b in vivo and enhances
Calpain-mediated degradation of reversibly oxidized protein-tyrosine phosphatase 1B A. Tru
¨
mpler et al.
5632 FEBS Journal 276 (2009) 5622–5633 ª 2009 The Authors Journal compilation ª 2009 FEBS
the early insulin action cascade. J Biol Chem 276,
21938–21942.
42 Brown AE, Yeaman SJ & Walker M (2007) Targeted
suppression of calpain-10 expression impairs insulin-
stimulated glucose uptake in cultured primary human
skeletal muscle cells. Mol Genet Metab 91, 318–324.
43 Sreenan SK, Zhou YP, Otani K, Hansen PA, Currie
KP, Pan CY, Lee JP, Ostrega DM, Pugh W, Horikawa
Y et al. (2001) Calpains play a role in insulin secretion

and action. Diabetes 50, 2013–2020.
44 Stalker TJ, Gong Y & Scalia R (2005) The calcium-
dependent protease calpain causes endothelial dysfunc-
tion in type 2 diabetes. Diabetes 54, 1132–1140.
45 Turner MD (2007) Coordinated control of both insulin
secretion and insulin action through calpain-10-medi-
ated regulation of exocytosis? Mol Genet Metab 91,
305–307.
46 Weedon MN, Schwarz PE, Horikawa Y, Iwasaki N,
Illig T, Holle R, Rathmann W, Selisko T, Schulze J,
Owen KR et al. (2003) Meta-analysis and a large asso-
ciation study confirm a role for calpain-10 variation in
type 2 diabetes susceptibility. Am J Hum Genet 73,
1208–1212.
47 Zhou YP, Sreenan S, Pan CY, Currie KP, Bindokas
VP, Horikawa Y, Lee JP, Ostrega D, Ahmed N, Bald-
win AC et al. (2003) A 48-hour exposure of pancreatic
islets to calpain inhibitors impairs mitochondrial fuel
metabolism and the exocytosis of insulin. Metabolism
52, 528–534.
48 Smith LK, Rice KM & Garner CW (1996) The insulin-
induced down-regulation of IRS-1 in 3T3-L1 adipocytes
is mediated by a calcium-dependent thiol protease. Mol
Cell Endocrinol 122, 81–92.
49 Haj FG, Markova B, Klaman LD, Bo
¨
hmer FD & Neel
BG (2003) Regulation of receptor tyrosine kinase sig-
naling by protein tyrosine phosphatase-1B. J Biol Chem
278, 739–744.

50 Raynaud F, Fernandez E, Coulis G, Aubry L, Vignon
X, Bleimling N, Gautel M, Benyamin Y & Ouali A
(2005) Calpain 1–titin interactions concentrate calpain 1
in the Z-band edges and in the N2-line region within
the skeletal myofibril. FEBS J 272, 2578–2590.
51 Shevchenko A, Wilm M, Vorm O & Mann M (1996)
Mass spectrometric sequencing of proteins silver-stained
polyacrylamide gels. Anal Chem 68, 850–858.
Supporting information
The following supplementary material is available:
Fig. S1. Chemical constitution of PTP1B fragments
obtained by calpain-mediated cleavage in the absence
of oxidation.
Fig. S2. Detection of PTP1B in wild-type and calpain-
deficient cells by immunocytochemistry.
Fig. S3. Insulin receptor phosphorylation in Capn4-
deficient cells.
This supplementary material can be found in the
online article.
Please note: As a service to our authors and readers,
this journal provides supporting information supplied
by the authors. Such materials are peer-reviewed and
may be re-organized for online delivery, but are not
copy-edited or typeset. Technical support issues arising
from supporting information (other than missing files)
should be addressed to the authors.
A. Tru
¨
mpler et al. Calpain-mediated degradation of reversibly oxidized protein-tyrosine phosphatase 1B
FEBS Journal 276 (2009) 5622–5633 ª 2009 The Authors Journal compilation ª 2009 FEBS 5633