Redox-based probes as tools to monitor oxidized protein tyrosine phosphatases in living cells

Article abstract of DOI:10.1016/j.ejmech.2014.06.040

Reversible oxidation of protein tyrosine phosphatases (PTPs) has emerged as an important regulatory mechanism whereby reactive oxygen species (ROS) inactivates the PTP and promotes phosphorylation and induction of the signaling cascade. The lack of sensitive and robust methods to directly detect oxidized PTPs has made it difficult to understand the effects that PTP oxidative inactivation play in redox signaling. We report the use of redox-based probes to directly detect oxidized PTPs in a cellular context, which highlights the importance of direct approaches to assist in the study of physiological and pathophysiological PTP activity in redox regulation. We also demonstrate, as a proof-of-concept, that these redox-based probes serve as prototypes for the design and development of a new class of inhibitors for phosphatases. We envision a nucleophile reacting with the oxidized inactive catalytic cysteine to generate an irreversible thioether adduct which prevents the phosphatase from being reactivated and ultimately fortifies the signaling cascade. Our results reveal the potential of translation of our redox-based probes, which are used to understand redox cell circuitry and disease biology, to small-molecule nucleophile-based inhibitors, which may treat diseases associated with redox stress. This may have implications in the treatment of type 2 diabetes and cancer.

Full text of DOI:10.1016/j.ejmech.2014.06.040

European Journal of Medicinal Chemistry 88 (2014) 28e33
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Redox-based probes as tools to monitor oxidized protein tyrosine
phosphatases in living cells
Francisco J. Garcia, Kate S. Carroll*
Department of Chemistry, The Scripps Research Institute, 130 Scripps Way, Jupiter, FL 33458, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Reversible oxidation of protein tyrosine phosphatases (PTPs) has emerged as an important regulatory
mechanism whereby reactive oxygen species (ROS) inactivates the PTP and promotes phosphorylation
and induction of the signaling cascade. The lack of sensitive and robust methods to directly detect
oxidized PTPs has made it difficult to understand the effects that PTP oxidative inactivation play in redox
signaling. We report the use of redox-based probes to directly detect oxidized PTPs in a cellular context,
which highlights the importance of direct approaches to assist in the study of physiological and path-
ophysiological PTP activity in redox regulation. We also demonstrate, as a proof-of-concept, that these
redox-based probes serve as prototypes for the design and development of a new class of inhibitors for
phosphatases. We envision a nucleophile reacting with the oxidized inactive catalytic cysteine to
generate an irreversible thioether adduct which prevents the phosphatase from being reactivated and
ultimately fortifies the signaling cascade. Our results reveal the potential of translation of our redox-
based probes, which are used to understand redox cell circuitry and disease biology, to small-
molecule nucleophile-based inhibitors, which may treat diseases associated with redox stress. This
may have implications in the treatment of type 2 diabetes and cancer.
Received 20 February 2014
Received in revised form
15 June 2014
Accepted 18 June 2014
Available online 19 June 2014
Keywords:
Redox-based probes
Redox biology
Reactive oxygen species
Phosphatases
Bioconjugation
Nucleophile-based therapeutics
© 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
functions such as cellular growth, proliferation, differentiation,
survival, metabolism, and motility [6]. PTPs are tightly regulated by
Reactive oxygen species (ROS) such as superoxide (O2 ꢁ),
hydrogen peroxide (H2O2), and hydroxyl radicals (ꢀOH) are formed
by the partial reduction of oxygen. Cellular ROS can be generated
endogenously by univalent reduction of molecular oxygen to
several mechanisms ranging from differential expression, subcel-
lular localization, limited proteolysis, post-translational modifica-
tions, ligand binding, and dimerization [7]. The PTP catalytic
domain contains a unique microenvironment in which the catalytic
cysteine exhibits a depressed pKa and therefore exists as a thiolate
anion at physiological pH. This facilitates the phosphatases to carry
out their enzymatic function but also renders them sensitive to
oxidation [8]. Upon exposure of the phosphatase to H2O2, the cat-
alytic cysteine residue is converted to a sulfenic acid (RSOH), which
results in PTP inactivation. Reactions with cellular thiols can restore
the oxidized PTPs to the catalytically active form [9]. Therefore,
reversible PTP oxidation has emerged as an important cellular
regulatory mechanism whereby ROS, such as H2O2 generated from
physiological responses, promotes phosphorylation and induction
of the signal cascade by oxidizing and inactivating the PTP at its
catalytic site.
ꢀ
generate O2 ꢁ, which can dismutase spontaneously or thorough the
ꢀ
assistance of superoxide dismutase to give H2O2. Superoxide for-
mation typically results from the premature leakage of electrons
from the electron transport chain, byproducts of xanthine and
aldehyde oxidases, or induced through growth factor stimulation
[1e3]. Substantial evidence indicates ROS functions as a signaling
molecule during signal transduction in a diverse range of biological
processes to mediate distinct physiological responses such as pro-
liferation, differentiation, and apoptosis [4,5].
Protein tyrosine phosphatases (PTPs) are crucial regulators of
signal transduction. This class of enzymes function as antagonists
towards protein tyrosine kinases (PTKs) to control reversible tyro-
sine phosphorylation, which governs fundamental physiological
The lack of sensitive and robust methods for detecting oxidized
phosphatases, as opposed to abundant redox-sensitive enzymes,
has made understanding redox regulation of phosphatases in the
cellular context challenging. The majority of approaches available
to detect PTP oxidation are indirect and not apt for cell based
* Corresponding author.
E-mail address: KCarroll@scripps.edu (K.S. Carroll).
http://dx.doi.org/10.1016/j.ejmech.2014.06.040
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

F.J. Garcia, K.S. Carroll / European Journal of Medicinal Chemistry 88 (2014) 28e33
29
studies [10]. In-gel activity assays use radioactively labeled sub-
strate to monitor reversible PTP oxidation [11]. The in-gel phos-
phatase assay is biased towards non-membrane bound
phosphatases and is not quantifiable. Since cysteine thiols are
known to react with electrophiles, a modified cysteinyl-labeling
assay was developed that relies on biotinylated thiol-alkylating
agents to expose reversibly oxidized phosphatases [12]. The
modified cysteinyl-labeling assay is not a targeted approach and
suffers from high background of non-PTP proteins (Fig. 1a). An
alternative strategy to survey oxidized PTPs is to use an antibody
generated against the conserved signature motif of phosphatases
harboring the terminally oxidized (RSO3H) active-site cysteine
[13,14]. This immunochemical approach does not differentiate be-
tween phosphatases regulated by reversible oxidation (RSOH) from
those that are inherently hyperoxidized (RSO2H, RSO3H).
conformational changes associated with oxidized PTP1B [18].
Though the conformation sensing antibodies provides a direct
approach to monitor PTP oxidation, they are specific for a single
protein and may not be used to monitor oxidation of the entire
classical PTP family.
The low cellular abundance of signaling proteins has made the
detection of oxidized PTPs difficult. Herein, we report the use of the
RBPs to detect oxidized phosphatases in cells and to investigate PTP
regulation in redox signaling (Fig. 1c). Literature has reported that
the bioorthogonal reaction is enhanced when the chemical reporter
harbors an alkyne handle and is used in combination with an azide
bearing detection tag [19]. In an effort to circumvent detection
limitations of the low abundant phosphatases, we synthesized
alkyne analogues of our previously reported RBPs to give the parent
compound (DYn-0), biphenyl (BiPhYn-1), and naphthyl (NaphYn-1)
probes (Fig. 1d). We've also adopted a more robust ligand for
the Huisgen [3 þ 2] cycloaddition reaction (click chemistry). We
report the use of a more reactive tris(triazolylmethyl)amine-based
ligand e BTTP as our ligand of choice for the bioorthogonal
chemical reaction, as opposed to TBTA, to append reporter tags to
the low abundant probe-modified proteins [20].
Direct approaches to monitor protein oxidation currently rely on
nucleophiles that exploit the selective reaction between RSOH
(Fig. 1b). The most common example is 5,5-dimethyl-1,3-cyclo-
hexanedione (dimedone) which generates
a stable thioether
adduct upon reacting with the RSOH [15]. The chemoselective re-
action of several dimedone-based probes has been applied to
detect oxidized proteins in vitro and in situ [16]. Based on the
success of this critical reaction, several probes to exclusively
monitor PTP oxidation have been developed. These PTP redox-
based probes (RBPs) are composed of: 1) a dimedone-based
warhead that forms a covalent adduct with the oxidized active-
site cysteine; 2) a module that directs binding to the PTP catalytic
site; and 3) a reporter tag used for the identification, purification, or
direct visualization of the labeled protein [17]. Additionally, single-
chain variable fragment (ScFv) antibodies directly detect unique
2. Results
The catalytic cysteine thiolate of PTP1B reacts with H2O2 to yield
the RSOH, which rapidly condenses with the main-chain nitrogen
of an adjacent serine residue to give the cyclic sulfenamide [21,22].
To determine whether dimedone could trap the PTP1B-SOH inter-
mediate, we performed in vitro experiments with dimedone and
the resulting protein S-dimedone adduct was detected using an
Fig. 1. (a) Electrophiles are promiscuous and may react with varying nucleophilic species found within a proteome. (b) Nucleophiles react selectively with the RSOH to generate a
stable thioether bond. Figure adapted from Ref. [38]. (c) A direct approach to monitor the extent of PTP oxidation within a cell. Cells are treated with redox-based probes (RBPs) to
selectively label the endogenously oxidized phosphatases. The cells are lysed and proteins of interest are immunoprecipitated. Bioorthogonal ligation is performed on the
immunocomplex to append a reporter tag. Samples are resolved by SDS-PAGE and visualized by avidin blotting or fluorescence. (d) Structures of alkyne based RBPs used to directly
monitor PTP oxidation.

30
F.J. Garcia, K.S. Carroll / European Journal of Medicinal Chemistry 88 (2014) 28e33
immunochemical approach previously reported in our laboratory
[23]. We treated recombinant PTP1B (aa 1-321) with increasing
concentrations of dimedone in the presence of H2O2. A stable
adduct between dimedone and oxidized PTP1B was generated and
detected by the antibody (Supplementary Fig. 1). In order to eval-
uate the capacity of RBPs to react towards the oxidized phospha-
tase, we treated PTP1B with increasing concentrations of the RBPs
in the presence of H2O2 followed by the conjugation of a biotin tag
via bioorthogonal ligation and visualization by avidin blotting. The
data demonstrates that RBPs have increased sensitivity towards the
oxidized phosphatase as opposed to the parent compound (Fig. 2a).
Carbon acids, such as dimedone, can be oxidized by H2O2 to
generate a trione species, which could act as an electrophile and
form an adduct with the thiol form of PTP1B. It is important to note
that the concentrations of H2O2 required to effect such a chemical
reaction are significantly higher (mM) than those used in these
(Supplementary Fig. 2). A time dependent study with BiPhYn-1 and
DYn-0 showed that BiPhYn-1 detects the oxidized PTP within 5 min
as opposed to 30 min with the parent compound (Fig. 2b).
To determine whether the RBPs recognize and bind non-
covalently to the phosphatase, we measured their potential to
inhibit phosphatase activity using
a fluorogenic substrate, 4-
methylumberlliferyl phosphate (4-MUP). Aryl compounds can be
prone to an aggregation-based mechanism of inhibition [24]. To test
whether the RBPs inhibit PTP1B through such a mechanism, we
performed activity assays in the presence of increasing concentra-
tions of Triton-X 100 detergent. Notably, no differences were
observed in the inhibition of PTP1B by BiPhYn-1 as the concentration
of detergent was increased (Supplementary Fig. 3), indicating that
this probe does not aggregate and subsequently inhibit PTP1B.
BiPhYn-1 showed modest inhibition towards PTP1B (IC50 ¼ 0.49 mM)
as opposed to the parent compound DYn-0 (IC50 ¼ 4.7 mM) and
NaphYn-1 (IC50 ¼ 2.3 mM) (Supplementary Fig. 4a). Since NaphYn-1
exhibited poor inhibition, we focused all subsequent efforts on
further characterization of BiPhYn-1. First order reaction rates of the
RBPs towardstheRSOH were determined using a RSOHmodel system
and revealed that the binding modules did not enhance the nucleo-
philicity of the dimedone warhead (Supplementary Fig. 4b) [25].
We used glutathione peroxidase 3 (Gpx3) and glyceraldehyde 3-
phosphate dehydrogenase (GAPDH) to evaluate the reactivity of the
RBPs towards detection of RSOH in non-PTP proteins. Gpx3 and
GAPDH were treated with BiPhYn-1 and DYn-0 in the presence of
H2O2 followed by conjugation of the biotin tag and avidin blotting.
Both compounds demonstrated equal labeling of non-PTPs sug-
gesting that BiPhYn-1 maintains the ability to detect RSOH modi-
fications in non-PTPs (Supplementary Fig. 5). The PTP super family
is comprised of classical PTPs and dual specificity phosphatases
(DUSPs), which dephosphorylate phospho-tyrosine and phospho-
serine/threonine/tyrosine residues respectively. Since all PTPs
contain a highly conserved catalytic site, we envisioned BiPhYn-1
would act as a global probe for all PTPs. We therefore tested the
capacity of BiPhYn-1 in detecting a panel of oxidized PTPs in
comparison to DYn-0. BiPhYn-1 was able to detect oxidized YopH,
PTP1B, CDC25B, SHP-1, and VHR more robustly then DYn-
0 (Supplementary Fig. 6). Taken together, these results designate
the BiPhYn-1 RBP as a general tool for detecting oxidized PTPs.
Catalytically inactive PTP1B (C215S) was generated to ensure
RBPs were targeting the catalytic cysteine. A loss of signal was
observed when labeling C215S with BiPhYn-1 in the presence of
H2O2, as opposed to wild type (Supplementary Fig. 7a). Competi-
tion studies with phenyl vinyl sulfone (PVSF) [26], a mechanism
based probe for PTPs, exhibited loss of detection of oxidized PTP1B
with BiPhYn-1 in the presence of increasing concentrations of PVSF
(Supplementary Fig. 7b) within cells. Finally, LC/MS/MS was used to
map the site of modification by BiPhYn-1 (Supplementary Fig. 7c).
To ascertain whether BiPhYn-1 would detect oxidized phos-
phatases in a cellular context, COS1 cells were transfected with
pJ3H-PTP1B to overexpress HA-tagged PTP1B (Supplementary
Fig. 8). BiPhYn-1 was titrated into transfected cells for 1 h at
37 ꢂC, 5% CO2. The cells were washed then lysed followed by
immunoprecipitation of HA-tagged PTP1B using anti-HA agarose
resin overnight. The probe-labeled phosphatase was visualized by
appending a biotin tag via bioorthogonal ligation of the immuno-
complex followed by avidin blot. BiPhYn-1 detects basal levels of
oxidized PTP1B in a dose dependent manner (Fig. 2c).
experiments (mM) (unpublished data). Nonetheless, to further rule
out this possibility we generated the sulfenic acid form of PTP1B,
quenched this reaction with catalase, and then exposed the
oxidized enzyme to the RBPs. Using this alternate workflow, no
differences in PTP1B labeling by RBPs were observed, as expected
Fig. 2. RBPs exhibit enhanced selectivity towards oxidized PTP1B in vitro and in situ.
Several works have reported that PTP1B is transiently inacti-
vated by H2O2 produced by various cell stimuli and that phospha-
tase inactivation is important for induction of optimal tyrosine
phosphorylation response [27e29]. One signaling pathway for
which H2O2 production occurs is in response to the hormone in-
sulin [30]. To verify that BiPhYn-1 can detect increased oxidized
Labeling of recombinant PTP1B (20
mM) with 10 equivalences H2O2 (200 mM) in the
presence of 50 M or increasing concentrations of RBP in (a) dose- and (b) time-
m
dependent fashion displays RBPs selectivity towards oxidized PTP1B. (c) COS1 cells
that had been transfected with pJ3H-PTP1B were treated with BiPhYn-1 and exhibit a
dose-dependent detection of basal oxidized PTP1B. (d) 250 mM BiPhYn-1, but not DYn-
0, detects increased levels of oxidized PTP1B initiated by insulin (100 nM) induced
H2O2 production.

F.J. Garcia, K.S. Carroll / European Journal of Medicinal Chemistry 88 (2014) 28e33
31
PTP1B upon ligand induced ROS formation, pJ3H-PTP1B transfected
COS1 cells were treated with 100 nM insulin followed by the
addition of BiPhYn-1. BiPhYn-1 was able to detect an increase in the
levels of oxidized PTP1B upon insulin stimulation (Fig. 2d). This
data suggests that the RBPs are robust tools to directly monitor
levels of oxidized PTPs within a cell.
PTP1B has been shown to function as a negative regulator in the
insulin signaling pathway. PTP1B influences the duration and
amplitude of the insulin signaling response by catalyzing the
removal of phosphoryl groups on tyrosine residues of the insulin
scaffold associated with the RBPs, it is speculated that the probes
may behave promiscuously and give rise to off-target effects via the
inhibition of opposing but structurally related phosphatases in the
PTP super family, ultimately leading to the reduction in the overall
levels of phosphorylation at high RBP concentrations [33].
Insulin increases glucose uptake in cells by stimulating the
translocation of the glucose transporter (GLUT4) from intracellular
sites to the cell surface [34]. We next sought to determine whether
the increased levels of phosphorylation of IRb translated into an
increase in glucose uptake. A fluorescent derivative of glucose, 2-
NBD Glucose (2-NBDG), was used to ascertain whether the RBPs
could trap the oxidized PTP1B to enhance glucose uptake in cells.
COS1 cells were treated with increasing concentrations of the RBPs
followed by simultaneous treatment with insulin and 2-NBDG. A
dose-dependent increase in 2-NBDG uptake was observed in cells
treated with BiPhYn-1 as opposed to cells treated with DYn-
0 (Fig. 3b,c). Taken together, this data conveys that the RBPs can
be used as a direct approach to facilitate cellular investigation of
PTP redox regulation in cell signaling.
receptor (IRb, pYpY 1162/1163) and insulin receptor substrates
protein (IRS-1) [31,32]. Since BiPhYn-1 was able to detect increased
levels of oxidized PTP1B in cells, the next step was to determine if
the RBPs could modulate the extent of tyrosine phosphorylation.
We hypothesized that the BiPhYn-1 RBP would react with the
oxidized inactivated PTP in cells to enable prolonged signaling and
downstream physiological effects for further investigations
(Fig. 3a). To this end, CHO cells that express the human insulin
receptor (CHO/hIRc) were incubated with a range of concentrations
of BiPhYn-1 followed by treatment with or without insulin. BiPhYn-
1 was able to increase the phosphorylation levels of IR
maximal effect of BiPhYn-1 on the levels of phosphorylation in
CHO/hIRc cells peaked at 250 M (Fig. 3b). At higher BiPhYn-1
concentrations the phosphorylation levels of the insulin receptor
decreased potentially due to nonspecific effects. Due to the generic
b. The
3. Conclusion
m
In summary, we report the use of redox-based probes to directly
detect and monitor oxidized phosphatases in a cellular setting. These
RBPs can facilitate the detection of ROS-inactivated PTPs associated
Fig. 3. (a) Reversible oxidative inactivation of protein tyrosine phosphatases (PTPs) is triggered by hydrogen peroxide (H2O2) reacting with the catalytic cysteine to generate a
sulfenic acid (RSOH). Phosphatase activity is restored upon reaction with thiols. We hypothesize that trapping oxidized PTP1B with BiPhYn-1 can enhance tyrosyl phosphorylation
of the insulin receptor
b. (b) CHO/hIRc cells stimulated with 10 nM insulin in the presence of increasing concentrations of the RBP. (c, d) Enhanced glucose uptake observed in the
presence of increasing concentrations of BiPhYn-1, but not DYn-0, when stimulating cells with 250 nM insulin in the presence of 50
mM 2-NBD glucose (2-NBDG).

32
F.J. Garcia, K.S. Carroll / European Journal of Medicinal Chemistry 88 (2014) 28e33
with specific cell signaling pathways and help to outline the physi-
ological roles of redox regulation. Our experiments supports and
communicates the importance and ease of incorporating chemo-
selective small molecule approaches to assist in the study of aberrant
phosphatase activity brought about by increased ROS that is associ-
ated with pathophysiological states such as cancer. Additionally,
PTP1B has been implicated as an important target for the treatment
of type 2 diabetes and obesity. The development of cell permeable
and bioavailable small molecule PTP inhibitors has been constrained
by the highly conserved and highly polar phospho-tyrosine binding
pocket [35]. As a result, many reported PTP inhibitors conform to
highly charged anionic phosphatase mimetics that cannot cross the
cell membrane [36]. Previously, aryl diketoacids were identified as
pTyr surrogates that targeted and stabilized the “open” inactive
conformation of PTP1B, thereby inhibiting the enzyme [37]. Recently
it was demonstrated that stabilization of oxidized PTP1B, using
single-chain variable fragment antibodies that target the unique
conformation of oxidized PTP1B, enhanced phosphorylation and
sustained insulin signaling [18]. We illustrate that our redox-based
probes serve as a proof-of-concept for the development of a new
class of small molecule inhibitors that target the oxidized inactive
phosphatase via nucleophilic trapping of the oxidized catalytic
cysteine. Our results revealed an increase in levels of phosphoryla-
tion of the insulin receptor as well as an increase in glucose uptake in
the presence of the RPBs. This demonstrates that trapping of the
oxidized phosphatase by way of small molecules bearing a nucleo-
philic site may be a practical means to inhibit further catalytic ac-
tivity. The design of more selective and reactive “nucleophile-based
inhibitors” may be used as a new approach for the treatment of
diabetes and other disease states, which have been associated with
aberrant phosphatase activity and amplified ROS production.
by film. Equal loading of recombinant PTP1B was assessed by
treating the membrane with a solution of 1:1 MeOH:R-250 Coo-
massie blue for 10 min, then allowing membrane to dry.
4.2. Cell culture
COS1 cells (ATCC) were maintained in a humidified atmosphere
of 5% CO2 at 37 ꢂC and cultured in DMEM media (Invitrogen) sup-
plemented with 10% FBS (Invitrogen), 1% penicillinestreptomycin
(Invitrogen), 1% GlutaMax (Invitrogen), and 1% non-essential amino
acids (Invitrogen). CHO cells overexpressing the human insulin
receptor (CHO/hIRc) were a kind gift from Dr. Michael L. Tremblay.
CHO/hIRc cells were maintained in a humidified atmosphere of 5%
CO2 at 37 ꢂC and cultured in Ham's F-12 media (Corning) supple-
mented with 10% FBS, 1% penicillin-streptomycin, and 1% non-
essential amino acids. For insulin treatment, cells were serum
starved for 16 h prior to experimentation.
4.3. Labeling of oxidized PTP1B in COS1 cells
COS1 cells were plated on 100 mm dishes and transfected at 90%
confluency according to manufactures instructions with pJ3H-
PTP1B (Addgene plasmid 8601) for 48 h. COS1 cells were then
washed with PBS, lifted with 0.25% trypsineEDTA, harvested by
centrifugation at 1500 g for 2 min, and then resuspended in serum-
free DMEM at a density of 3e4 ꢃ106 cells/mL. The resuspended cells
were treated with DMSO or the indicated concentration of the
redox-based probe for 1 h at 37 ꢂC in a 5% CO2 humidified atmo-
sphere with periodic gentle agitation. Following treatment, cells
were collected and washed with PBS (3ꢃ). For insulin treatment,
cells were cultured in a similar fashion as above with the exception
of being serum starved for 16 h prior to the experiment. After serum
deprivation, cells were treated with 100 nM insulin (Calbiochem) for
4. Materials and methods
2 min followed by the addition of the 250 m
M RBPs for 1 h at 37 ꢂC in
More comprehensive experimental procedures, supplemental
figures, and compound characterization can be found in the
Supporting information.
a 5% CO2 humidified atmosphere with periodic gentle agitation.
4.4. Generation of cell lysate
4.1. Recombinant protein labeling with RBPs
COS1 and CHO/hIRc cells were harvested in a NP-40 lysis buffer
[50 mM TriseHCl pH 8.0,137 mM NaCl,10% glycerol,1% NP-40, 50 mM
PTP1B and C215S PTP1B were buffer exchanged using a Nap-5
column (GE Healthcare Illustra) pre-equilibrated with 50 mM
HEPES, 100 mM NaCl, 1 mM EDTA, pH 7.0. Simultaneous labeling of
NaF, 10 mM
b
-glycerolphosphate, 1 mM sodium vanadate, 1 ꢃ EDTA-
free protease cocktail inhibitors (Roche), and 200 U/mL catalase
(Sigma)]. After 20 min incubation on ice with frequent mixing, cell
debris wasremoved bycentrifugation at 14,000 rpm at4ꢂC for 15 min.
Protein concentrations were determined by BCA assay (Pierce).
PTP1B or C215S PTP1B was performed by taking 20
mM phospha-
tase and treating it with 10 equivalences (200 M) of hydrogen
m
peroxide and indicated concentrations of sulfenic acid probes
(DYn-0, NaPhYn-1, BiPhYn-1, or dimedone) for 1 h at room tem-
perature while rocking. Excess RBP and EDTA were removed by
4.5. Immunoprecipitation
passing the samples through
a
P30 column (Bio-Rad) pre-
HA-PTP1B was immunoprecipitated from 500
mg of cell lysate
equilibrated with 50 mM HEPES, 100 mM NaCl, 0.1% SDS, pH 7.4.
Time-dependent analyses were performed by taking an aliquot of
the reaction mixture at various time points and passing through a
pre-equilibrated P30 column to quench the reaction. RBP modified
with 20 L anti-HA agarose (Pierce) as specified by manufacturer.
m
The following day, the resin was pelleted with a 10 s burst at
12,000 ꢃ g and the supernatant was saved. The resin was washed
three times with TBST and twice with 50 mM HEPES, 100 mM NaCl,
proteins were then treated with 100
mM AzBiotin-PEG4 (Invi-
pH 7.4. The resin was then treated with 20
mL of a pre-mixed click
trogen), a pre-mixed BTTP:CuSO4 solution (200
mM BTTP:100
mM
chemistry mix (100 M AzBiotin-PEG4, 500
m
mM BTTP, 250 mM
CuSO4), and 2.5 mM sodium ascorbate for 1 h at room temperature
while rocking. The click chemistry reaction was quenched with the
addition of 1 mM EDTA. Protein samples were resolved by SDS-
PAGE using Mini-Protean TGX 4e15% Tris-Glycine gels (BioRad)
and transferred to a polyvinylidene difluoride (PVDF) membrane
(BioRad). The PVDF membrane was blocked with 3e5% BSA in TBST
for 1 h at room temperature, washed with TBST (3ꢃ), and immu-
noblotting was performed with HRP-streptavidin (GE Healthcare,
1:80,000). The PVDF membrane was washed with TBST (3ꢃ) and
developed with ECL Plus chemiluminescence (Pierce) and imaged
CuSO4, and 2.5 mM sodium ascorbate in 50 mM HEPES, 100 mM
NaCl, pH 7.4). The click reaction was allowed to mix for 1 h then
terminated by the addition of 10
-Me and boiling for 10 min.
mL Laemmli sample buffer without
b
4.6. Detection of phosphorylated IRb in CHO/hIRc cells
CHO/hIRc cells were plated onto 6-well pates and allowed to
become adherent in complete media overnight at 37 ꢂC in a hu-
midified atmosphere of 5% CO2. The cells were serum starved for

F.J. Garcia, K.S. Carroll / European Journal of Medicinal Chemistry 88 (2014) 28e33
33
[10] R. Karisch, B.G. Neel, Methods to monitor classical protein-tyrosine phos-
phatase oxidation, Febs. J. 280 (2013) 459e475.
16 h prior to the experiment. CHO/hIRc cells were pre-treated with
DMSO, 1 mM vanadate, and DYn-0 or BiPhYn-1 at various concen-
trations for 1 h at 37 ꢂC, 5% CO2. Media containing DMSO, vanadate,
or RBPs was removed and cells were washed with PBS (3ꢃ) then
stimulated with 10 nM insulin for 5 min. After stimulation, the
media was removed and the cells were washed with ice cold PBS
(3ꢃ). The cells were then lysed with the aid of a rubber policeman.
[11] T.C. Meng, T. Fukada, N.K. Tonks, Reversible oxidation and inactivation of
protein tyrosine phosphatases in vivo, Mol. Cell. 9 (2002) 387e399.
[12] B. Boivin, S. Zhang, J.L. Arbiser, Z.Y. Zhang, N.K. Tonks, A modified cysteinyl-
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[13] C. Persson, T. Sjoblom, A. Groen, K. Kappert, U. Engstrom, U. Hellman,
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25 mg of clarified cell lysatewas resolved by SDS-PAGE, transferred to
PVDF membrane, and probed with appropriate antibodies.
[14] R. Karisch, M. Fernandez, P. Taylor, C. Virtanen, J.R. St-Germain, L.L. Jin,
I.S. Harris, J. Mori, T.W. Mak, Y.A. Senis, A. Ostman, M.F. Moran, B.G. Neel,
Global proteomic assessment of the classical protein-tyrosine phosphatome
and “Redoxome”, Cell 146 (2011) 826e840.
4.7. 2-NBD glucose uptake assay
COS1 cells were plated in triplicate at a density of 3 ꢃ 104 cells/
well in black clear bottom 96-well microplates (Corning) and
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This work was supported by the Camile Henry Dreyfus Teacher
Scholar Award (K.S.C) and the American Heart Association Scientist
Development Award (0835419N, K.S.C.). We thank K.S. Gates (U.
Missouri) for the PTP1B constructs, M.L. Tremblay (McGill Univer-
sity) for the CHO/hIRc cells, J. Yang (Vanderbilt) and D. Liebler
(Vanderbilt) for mass spectrometry support. Finally, we would like
to acknowledge T.H. Truong (Scripps) and M. LoConte (Scripps) for
reviewing the manuscript.
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Appendix A. Supplementary data
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