EDITORS’ PICK cro

Bicarbonate is essential for protein-tyrosine phosphatase 1B

(PTP1B) oxidation and cellular signaling through EGF-
triggered phosphorylation cascades
Received for publication, April 23, 2019, and in revised form, June 6, 2019 Published, Papers in Press, June 13, 2019, DOI 10.1074/jbc.RA119.009001
X Markus Dagnell‡1, Qing Cheng‡, Syed Husain Mustafa Rizvi§, Paul E. Pace¶, Benoit Boivin§,
X Christine C. Winterbourn¶2, and X Elias S. J. Arnér‡3
From the ‡Division of Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-171 77,
Stockholm, Sweden, the ¶Centre for Free Radical Research, Department of Pathology and Biomedical Science, University of Otago,
Christchurch 8011, New Zealand, and the §Department of Nanobioscience, SUNY Polytechnic Institute, Albany, New York 12203
Edited by Ruma Banerjee

Protein-tyrosine phosphatases (PTPs) counteract protein Receptor-tyrosine kinase (RTK)4 activation leads to trans-
tyrosine phosphorylation and cooperate with receptor-tyro- mission of downstream phosphorylation cascades upon growth
sine kinases in the regulation of cell signaling. PTPs need to factor stimulation, and has major importance in physiology and
undergo oxidative inhibition for activation of cellular cas- proliferative diseases such as cancer. Protein-tyrosine phospha-
cades of protein-tyrosine kinase phosphorylation following tases (PTPs), including PTP1B, counteract protein tyrosine
growth factor stimulation. It has remained enigmatic how phosphorylation and thereby act together with RTKs to regulate
such oxidation can occur in the presence of potent cellular cell signaling (1). PTP activity depends upon a conserved ac-
reducing systems. Here, using in vitro biochemical assays tive-site Cys residue (1–4), which renders these enzymes suscep-
with purified, recombinant protein, along with experiments tible to oxidative inactivation. This has physiological importance,
in the adenocarcinoma cell line A431, we discovered that because RTK activation triggers transient H2O2 production
bicarbonate, which reacts with H2O2 to form the more reac- from NADPH oxidases (NOXs) (5), which in turn leads to
tive peroxymonocarbonate, potently facilitates H2O2-medi- reversible oxidation and inhibition of PTPs. Inactivation of
ated PTP1B inactivation in the presence of thioredoxin PTPs is believed to be absolutely required for RTK signaling (4,
reductase 1 (TrxR1), thioredoxin 1 (Trx1), and peroxiredoxin 6). NOXs are membrane-localized (typically on the plasma,
2 (Prx2) together with NADPH. The cellular experiments endosomal, or endoplasmic reticulum membrane) and use
revealed that intracellular bicarbonate proportionally dic- cytoplasmic NADPH to produce superoxide and H2O2 on the
tates total protein phosphotyrosine levels obtained after opposing membrane surface. The H2O2 must then enter the
stimulation with epidermal growth factor (EGF) and that cell, facilitated through aquaporins (7, 8), to exert its signaling
bicarbonate levels directly correlate with the extent of PTP1B actions. During stimulation of platelet-derived growth factor
oxidation. In fact, EGF-induced cellular oxidation of PTP1B and epidermal growth factor (EGF) receptor pathways, the cat-
was completely dependent on the presence of bicarbonate. alytic Cys residue of PTP1B becomes reversibly oxidized and
These results provide a plausible mechanism for PTP inacti- thus inhibited (4, 9). The initial oxidation product is a sulfenic
vation during cell signaling and explain long-standing obser- acid (-SOH) (10, 11), which can condense to an internal sulfe-
vations that growth factor responses and protein phosphory- nylamide (12–14) or undergo glutathionylation (15, 16).
lation cascades are intimately linked to the cellular acid– base Reversibly oxidized PTP1B can be reactivated by the thiore-
balance. doxin (Trx) system (thioredoxin reductase 1 (TrxR1) and
NADPH with or without Trx1 or thioredoxin-related protein of
14 kDa (TRP14) (10, 17–19), thus enabling the Trx system to
modulate cellular RTK signaling (17).
This work was supported by the Karolinska Institutet, the Knut and Alice Wal- It is much debated exactly how oxidation of PTP1B occurs in
lenberg Foundations, the Swedish Cancer Society, the Karolinska Institutet a cellular environment, especially considering that members of
Research Foundations (to E. S. J. A.), Swedish Research Council Grant
(to E. S. J. A. and M. D.) 537-2014-360, Swedish Society of Medicine Grant
the peroxiredoxin (Prx) family of thiol proteins, which are
SLS-786841, Åke Wiberg Stiftelse Grants M17-0101 and M18-0141 (to expressed at high levels in cells and also recycled predominantly
M. D.), National Institutes of Health Grant HL138605, American Heart Asso- by the Trx/TrxR/NADPH system, are many orders of magni-
ciation Grant 17GRNT33700265 (to B. B.), and the New Zealand Marsden tude more reactive and therefore likely to react with nearly all
Fund (to C. C. W.). The authors declare that they have no conflicts of interest
with the contents of this article. The content is solely the responsibility of intracellular H2O2 (20 –22). Some cytosolic thiol-containing
the authors and does not necessarily represent the official views of proteins can be oxidized via Prx-mediated relays (23, 24), but
the National Institutes of Health.
This article was selected as one of our Editors’ Picks.
This article contains Figs. S1–S4.
1 4
To whom correspondence may be addressed. E-mail: [email protected]. The abbreviations used are: RTK, receptor-tyrosine kinase; PTP, protein-ty-
2
To whom correspondence may be addressed. E-mail: Christine.Winterbourn@ rosine phosphatase; EGF, epidermal growth factor; Trx, thioredoxin; Prx,
otago.ac.nz. peroxiredoxin; NBC, sodium and bicarbonate exchanger protein; NOX,
3
To whom correspondence may be addressed. E-mail: [email protected]. NADPH oxidase.
This is an open access article under the CC BY license.

12330 J. Biol. Chem. (2019) 294(33) 12330 –12338

© 2019 Dagnell et al. Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc.
 EDITORS’ PICK: Bicarbonate in PTP1B oxidation/signaling by EGF

this mechanism has not been demonstrated for PTP1B (25).

With PTP1B being a therapeutic target with key roles in pathol-
ogies such as type 2 diabetes, obesity, and cancer (26, 27), as well
as an archetypical example of oxidative inactivation in redox
signaling, it is important that the mechanism of this process
becomes understood.
We recently characterized the effect of H2O2 on PTP1B
activity and found that a reconstituted Prx2/Trx1/TrxR1/
NADPH system prevents PTP1B inactivation through both the
removal of H2O2 and recycling of the oxidized PTP1B (25). It
thus remains unclear how PTP1B can be inactivated by H2O2 in
the presence of a functional Prx/Trx system. One possibility is
that oxidation could be mediated by the more reactive per-
oxymonocarbonate (HCO⫺ 4 ), which is spontaneously formed
in a reversible reaction between H2O2 and CO2/bicarbonate
(HCO⫺ 3 ) (28, 29).

H 2 O 2 ⫹ CO 2 /HCO ⫺ ⫺ ⫹
3 ª HCO 4 ⫹ H /H 2 O

Reaction 1

The X-ray structure of peroxymonocarbonate shows a true

hydroperoxide (30), and it has been structurally and kinetically
characterized using 13C NMR (28). Bicarbonate at physiological
concentrations accelerates the reaction of H2O2 with thiols,
thioethers, amines, and other small molecules such as GSH and
albumin via formation of peroxymonocarbonate (29, 31, 32).
However, of particular note is the finding by Gates and co-
workers (33) that isolated PTP1B is especially reactive and
reacts 7000 times faster with HCO⫺ 4 than with H2O2 itself. Figure 1. Bicarbonate potentiates H2O2-dependent inactivation of
Although the impact of this mechanism is modulated by the PTP1B. A, recombinant PTP1B (600 nM) was treated with increasing concen-
equilibrium shown above, it was suggested as a possible route trations of H2O2, 0 ␮M buffer control (F), 3.4 ␮M (E), 6.3 ␮M (⽧), 12.5 ␮M () 25
␮M (Œ), 50 ␮M (f) (20 mM HEPES, 100 mM NaCl buffer, pH 7.4, containing 0.1
for PTP1B oxidation during cell signaling (20), but this proposal mM diethylenetriaminepentaacetic acid, 0.05% BSA, 1 mM sodium azide) and
has not been tested experimentally. Importantly, detection then assayed for PTP activity at the indicated times. PTP1B activity is given in
min⫺1 (mol of product/mol of enzyme/min). Data points represent means ⫾
of the peroxymonocarbonate species in vitro requires up to S.D. (error bars) (n ⫽ 3). B, PTP1B was treated as in A but in the presence of 25
0.1–1 M concentrations, making it virtually impossible to detect mM bicarbonate (final concentration after adding H2O2/bicarbonate and
directly in cellular systems. It is a highly reactive, short-lived assay buffer). Data points represent means ⫾ S.D. (n ⫽ 4). A representative run
is presented in Fig. S1.
chemical species that is in equilibrium with H2O2, which gives
the same reaction products as hydrogen peroxide. Thus, there
are no specific probes or reagents that would distinguish per- ceutical disruption of several NBCs (SLCA4, SLC4A7, and
oxymonocarbonate from H2O2. However, recent studies have SLC4A9) suppresses tumor growth of breast cancer spheroids
identified links between regulation of bicarbonate levels, cell and murine xenografts (43, 44), and chemical inhibition of
growth, and cancer progression (34). Signaling through the bicarbonate influx using S0859 decreases spheroid growth of
EGF receptor, which is an important event therapeutically tar- HCT116 cells (45). Considering these observations, we asked
geted for cancer treatment (35–37), has been directly linked to whether bicarbonate can directly facilitate PTP1B oxidation in
bicarbonate-regulating enzymes. Notably, carbonic anhydrase the presence of a peroxiredoxin-recycling system and in a cel-
IX, a glycoprotein with an extracellular domain that catalyzes lular setting. Surprisingly, our results reveal that bicarbonate is
hydration of CO2 (CO2 ⫹ H2O 3 HCO⫺ ⫹
3 ⫹H ), becomes acti-
an obligate component of cellular PTP1B oxidation and EGF
vated upon EGF ligand stimulation (38). Carbonic anhydrase IX responsiveness.
also localizes at the leading edge of focal adhesions, and its
Results
overexpression increases the rate of adhesion and spreading
(39). Electroneutral sodium and bicarbonate cotransporter Bicarbonate potently facilitates H2O2-dependent inactivation
proteins (NBCs), such as NBCn1, facilitate chloride-HCO⫺ 3 ex-
of PTP1B
change with a Na⫹/HCO⫺ 3 stoichiometry of 2:1. These mem- Using a direct activity assay with pure recombinant PTP1B,
brane-bound proteins are important regulators of intracellular we first confirmed the previously described facilitation of
bicarbonate levels and help to control cellular pH (40). In MCF7 H2O2-mediated inactivation of PTP1B by bicarbonate (33).
breast cancer cells, NBCn1 expression is highly up-regulated Compared with the inefficient dose- and time-dependent direct
upon HER2 expression (41) and is correlated with tumor pro- inactivation of PTP1B by H2O2 (Fig. 1A), inclusion of a physio-
gression and metastasis (42). Furthermore, genetic or pharma- logical concentration of 25 mM bicarbonate potently increased

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EDITORS’ PICK: Bicarbonate in PTP1B oxidation/signaling by EGF

Figure 2. Modulation of bicarbonate- and H2O2-dependent inactivation of PTP1B in the presence of a Prx2/TrxR1/Trx1 redox system. A, scheme
showing relevant reactions in the experimental PTP1B/Prx2/thioredoxin system. Sox of PTP1B could be either the sulfenic acid or sulfenylamide species, with
the former also reducible by TrxR/NADPH in the absence of Trx. B, PTP1B (600 nM) was treated with 50 ␮M H2O2 together with 25 mM bicarbonate in the presence
of Trx1 (5 ␮M), Prx2 (10 ␮M), NADPH (300 ␮M), and increasing TrxR1 concentrations of 0 nM (f), 2 nM (Œ), 10 nM (), 50 nM (⽧), 250 nM (E), and 1 ␮M (䡺) and with
1 ␮M TrxR1 using buffer-treated control without H2O2 (F). PTP activity was measured after the indicated times. Data points represent means ⫾ S.D. (error bars)
(n ⫽ 3). C, analyses were performed as in B with TrxR1 (1 ␮M) and increasing Trx1 concentration, 0 ␮M (⽧), 0.5 ␮M (), 1 ␮M (Œ), 2 ␮M (f), and 5 ␮M (F) (n ⫽ 1).
D, analyses performed as in A with TrxR1 (15 nM) and increasing concentrations of bicarbonate as indicated, 0 mM (f), 5 mM (Œ), 10 mM (), 15 mM (⽧), 25 mM
(E), 35 mM (䡺), and 45 mM (‚) (n ⫽ 1).

inactivation (Fig. 1B). Notably, bicarbonate alone had no effect 2D). These results show that there is an interplay between
(Fig. 1B, control). We next examined whether inclusion of bicarbonate, PTP1B, and the Trx/Prx redox cycling systems
bicarbonate facilitated H2O2-mediated inactivation of PTP1B and that there are conditions whereby bicarbonate, presumably
in the presence of a functional Trx system. through the spontaneous production of peroxymonocarbonate
(33), promotes transient inactivation of PTP1B by H2O2. To
H2O2 and bicarbonate in combination can overcome rule out possible inhibitory effects of bicarbonate and H2O2 on
protection of PTP1B activity by the Trx/Prx system the Trx system, NADPH consumption was measured using
As reported previously, in the absence of bicarbonate, Prx2/ insulin as a substrate (46). The results showed no difference in
Trx1/TrxR1/NADPH (at 10, 2, 0.5, and 200 ␮M, respectively) consumption by bicarbonate alone or in combination with
fully protects PTP1B against inactivation by 100 ␮M H2O2 (25). H2O2 as compared with buffer control (Fig. S2).
This is due to a combination of H2O2 removal by the peroxidase
activity of Prx2 and reduction of the sulfenic acid and sulfenyl- Inhibition of bicarbonate cotransporters in A431 cells
amide forms of PTP1B by TrxR1 and Trx1, as illustrated in Fig. decreases EGF-induced phosphorylation cascades
2A. In the presence of bicarbonate (25 mM), where HCO⫺ 4 can We next addressed whether the mechanism identified with
cause faster PTP1B inactivation, Prx2/Trx1/TrxR1/NADPH at the purified enzymes applies to cellular systems, by examining
the same concentrations still gave almost complete protection the effect of bicarbonate on protein phosphorylation cascades
(Fig. 2B, 1 ␮M TrxR1). However, by lowering the TrxR1 concen- triggered by growth factor stimulation. We used human A431
tration (Fig. 2B) or Trx1 concentration (Fig. 2C), it was possible epidermal squamous carcinoma cells, as these cells are known
to obtain conditions where PTP1B was inactivated for the dura- to inactivate PTP1B by oxidation in response to treatment with
tion of the experiment or inactivated at early time points, fol- EGF (10, 47). Extracellularly produced bicarbonate is trans-
lowed by a time-dependent regain in activity during the assay. ported into cells through membrane-bound cotransporter pro-
The latter pattern is reminiscent of the transient PTP1B inac- teins, NBCs, that control intracellular bicarbonate levels and
tivation expected to occur in cells during an oxidative burst thereby cellular pH (40). We thus stimulated A431 cells with
after growth factor stimulation. A higher concentration of pH- EGF ligand for 2, 4, and 6 min with or without 1-h pretreatment
adjusted bicarbonate (45 mM, pH 7.4) was also able to fully with S0859, a well-characterized inhibitor of NBCs (48). As
overcome the complete protection of PTP1B against 50 ␮M shown in Fig. 3 (A and B), EGF ligand induced a rapid increase
H2O2 provided by the cycling Prx/Trx system, as seen by a rapid in total protein tyrosine phosphorylation, primarily associated
and sustained loss of PTP1B activity (Fig. 2D). At intermediate with the ⬃198 kDa band representing the EGF receptor (Fig.
concentrations of bicarbonate, maintaining the same pH, H2O2 3A). This phosphorylation was notably inhibited when bicar-
caused transient PTP1B inactivation followed by recovery (Fig. bonate influx was blocked with S0859. The EGFR phosphory-

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 EDITORS’ PICK: Bicarbonate in PTP1B oxidation/signaling by EGF

ered (50 mM) medium, with the addition of 0, 40, or 60 mM

bicarbonate, adjusted to pH 7.4, and equilibrated in air with
varying percentages of corresponding CO2 levels (0, 5, and 10%,
respectively). The A431 cells were stimulated with EGF ligand
after 1 day of these culture conditions and harvested 2, 4, and 6
min after the EGF addition. Analyses of total protein phospho-
tyrosine revealed increases in phosphorylation at the higher
bicarbonate concentration, most pronounced for a band (not
identified) at about 28 kDa seen at longer exposures (Fig. 5).
Thus, increased cellular bicarbonate levels augment EGFR-
linked protein phosphorylation in A431 cells independently of
pH-derived effects.

Oxidation of PTP1B in A431 cells during EGF stimulation

depends on the presence of bicarbonate
The observed bicarbonate-dependent increases seen in EGF-
triggered phosphorylation are consistent with oxidative inacti-
vation of PTPs. However, this is an indirect readout, and other
explanations are possible. We therefore looked for direct evi-
Figure 3. Pharmacological inhibition of bicarbonate cotransporters in dence of oxidative PTP1B inactivation using a cysteine-labeling
A431 cells decreases EGF ligand-induced phosphotyrosine formation. assay that detects reversible thiol oxidation (50, 51). The basis
Overnight starved A431 cells in regular bicarbonate-containing DMEM 0.1%
FCS) were pretreated with the NBC inhibitor S0859 (100 ␮M) or DMSO control
of the assay is to use a biotin tag that labels only reversibly
for 1 h prior to EGF stimulation for 2, 4, and 6 min. Cell extracts were separated oxidized thiol proteins and isolates the labeled proteins with
by SDS-PAGE and Western blotted (IB) for total phosphotyrosine and EGF streptavidin. Immunoblotting against the protein of interest
receptor (A) and specific phosphorylation of the EGF receptor Tyr-992 (pY992)
residue (C). Bar diagrams show densitometry ratios for total signal over all will give a positive response only if it has been oxidized.
phosphotyrosine bands (B) or the specific pTyr-992 signal (D), relative to the When this procedure was applied to A431 cells maintained
immunoblot staining for the EGF receptor. Every ratio is compared with in regular bicarbonate-containing DMEM, we detected a
that in unstimulated control, which was set to 1. Results are mean ⫾ S.D.
(error bars) for three independent experiments with filled circles showing time-dependent increase in oxidized PTP1B peaking at 2
individual results. Statistically significant differences are indicated (*, p ⬍ min after the EGF addition (Fig. 6A). This is in agreement
0.05). The membranes in the figure stained with Ponceau for total protein
loading are shown in Fig. S3. with earlier reports (10). Most strikingly, pretreatment with
the NBC inhibitor S0859 completely abrogated the EGF-de-
lation site Tyr-992 (pY992), previously shown to be regulated pendent PTP1B oxidation at the 2-min time point (Fig. 6A).
by PTP1B (49), was also significantly inhibited at all time points Furthermore, PTP1B oxidation was not seen when the cells
by S0859 (Fig. 3, C and D). were treated with EGF in HEPES-buffered bicarbonate-free
DMEM. Finally, the addition of pH-equilibrated bicarbonate
Lactic acid pretreatment of A431 cells decreases EGF-induced to the bicarbonate-free cells before EGF stimulation brought
phosphorylation oxidation of PTP1B back to the level seen in bicarbonate-
We next investigated whether protein tyrosine phosphoryla- containing DMEM (Fig. 6A). Thus, the presence of bicarbon-
tion patterns were affected by physiologically relevant lactic ate in cells is a crucial and apparently obligate component for
acid concentrations, which decrease cellular bicarbonate as EGF-dependent phosphorylation cascades that require oxi-
well as pH at events of metabolic acidosis. Lactic acid (up to 40 dation and inactivation of PTP1B.
mM) was added to the growth medium of A431 cells 2 min prior
to the addition of EGF ligand. Measurements made after 5-min Discussion
stimulation showed that lactic acid concentrations above 10 Reversible oxidative inhibition of PTPs is a key regulatory
mM resulted in a progressive decrease in total protein tyrosine event during growth factor signaling (52). The molecular mech-
phosphorylation (Fig. 4A). Time course measurements showed anisms leading to PTP oxidation have remained unclear,
that the addition of 15 mM lactic acid slowed the onset of EGF- although it is well-known that RTK activation involves cellular
dependent phosphorylation, whereas 30 mM gave very strong production of H2O2 derived from NOX enzymes (5). Several
inhibition at all time points (Fig. 4B). models for PTP inactivation have been proposed, such as the
“floodgate” hypothesis in which inactivation of Prxs enables
Bicarbonate depletion at constant pH decreases EGF-induced oxidation of less-reactive proteins (53) or redox relay–me-
protein tyrosine phosphorylation in A431 cells diated oxidation via Prxs (23, 24). Our results add another
As the results in Figs. 3 and 4 could be attributed to effects of dimension and reveal that a concerted action of H2O2 together
bicarbonate or to general pH effects, we next assessed whether with bicarbonate is required for inactivation of PTP1B and the
lowering cellular levels of bicarbonate without changing the pH initiation of protein phosphorylation cascades.
would still affect protein tyrosine phosphorylation patterns. The results with the recombinant proteins clearly showed
A431 cells were cultured in regular DMEM and subsequently that H2O2 triggered inactivation of PTP1B in the presence of a
starved overnight in low-serum bicarbonate-free HEPES-buff- fully active and cycling Prx system only when bicarbonate was

J. Biol. Chem. (2019) 294(33) 12330 –12338 12333

EDITORS’ PICK: Bicarbonate in PTP1B oxidation/signaling by EGF

Figure 4. Treatment of A431 cells with lactic acid prior to EGF ligand stimulation results in less phosphorylation. A, concentration dependence.
Serum-starved A431 cells in regular DMEM containing bicarbonate were pretreated for 2 min with 0, 5, 10, 20, and 40 mM lactic acid and subsequently
stimulated with EGF (100 ng/ml) for 5 min. Lysates were analyzed for total phosphotyrosine. B, time course. A431 cells were pretreated for 2 min with 0, 15, and
30 mM lactic acid and subsequently stimulated with EGF (100 ng/ml) for 2, 4, and 6 min. Lysates were analyzed for total phosphotyrosine. The right-hand panels
in A and B show densitometry analyses as in Fig. 3 with filled circles showing individual results (n ⫽ 3; mean ⫾ S.D. (error bars); *, p ⬍ 0.05). The membranes in
this figure stained with Ponceau for total protein loading are shown in Fig. S4.

present. We used all reactants at physiologically relevant con- vated as the H2O2 is consumed and the reducing mechanisms
centrations and were able to vary conditions within these to see dominate.
transient inactivation and reactivation, reminiscent of a signal- To test the relevance of this mechanism of PTP1B inactiva-
ing step. We propose a mechanism to explain these results in tion during EGF-triggered signaling context, we modulated the
which H2O2 can either be scavenged by Prx2 or react with cellular bicarbonate levels using three different approaches.
bicarbonate to generate peroxymonocarbonate, which is Lowering bicarbonate uptake with the NBC inhibitor S0859
responsible for PTP1B inactivation (Fig. 2A). Increasing the resulted in less EGFR-dependent protein tyrosine phosphory-
bicarbonate favors the latter, whereas regeneration of reduced lation in A431 cells. Likewise, the addition of lactic acid, which
Prx2 by the thioredoxin system enhances H2O2 removal and is lowers bicarbonate levels by a shift in acid– base equilibrium,
protective. It is possible that peroxymonocarbonate could also also decreased phosphotyrosine levels following EGF ligand
react with Prx2 in this system. However, we have recently found stimulation in a dose-dependent manner. These observations
that oxidation of the reduced protein is unaffected by the pres- are compatible with the notion that lower intracellular levels of
ence of bicarbonate.5 Although hyperoxidation was enhanced, bicarbonate give less inhibition of cellular PTP activity, but
this would cause only minor inactivation of the Prx2 under the other effects of acidification could not be excluded. However,
conditions of our current study. Nevertheless, in other situa- the experiments with cells grown in pH-controlled HEPES-
tions, this could amplify the effect on PTP1B. Complementing buffered DMEM revealed signaling effects of bicarbonate not
these oxidative reactions are the reductive steps involving related to changes in pH.
reduction of Prx2 disulfide to maintain its scavenging activity as Our results collectively suggest a molecular mechanism by
well as recycling of PTP1B, either through a reaction of the which physiological changes in bicarbonate levels affect
sulfenic acid intermediate directly with TrxR1 (25) or recycling
growth factor signaling responses. This conclusion is sub-
of the sulfenylamide by TrxR1 together with Trx1 or TRP14
stantiated by the finding that the cysteinyl-labeling assay
(17). The balance between these steps determines the extent to
detected EGF-dependent oxidation of PTP1B only in the
which PTP1B is initially inactivated and subsequently reacti-
presence of bicarbonate. Thus, bicarbonate seems to be an
obligate factor for EGF-dependent PTP1B oxidation. We
5
A. V. Peskin and C. C. Winterbourn, unpublished observations. hence propose a model for redox regulation of PTP1B during

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 EDITORS’ PICK: Bicarbonate in PTP1B oxidation/signaling by EGF

Figure 5. Bicarbonate increases total phosphorylation in A431 cells after

EGF ligand stimulation. A, A431 cells were grown and incubated overnight
in low-serum HEPES-buffered (50 mM) DMEM at pH 7.4 with 0, 40, and 60 mM
bicarbonate added and equilibrated with 0, 5, and 10% CO2, respectively, as
indicated. The cells were then stimulated with EGF and analyzed for total
phosphotyrosine. B, densitometry for three independent experiments, of
short- and long-exposed membranes, for high-molecular weight (HMW) phos- Figure 6. A, bicarbonate is required for EGF-dependent reversible PTP1B oxi-
phorylated proteins, and ⬃28 kDa band, respectively (dashed rectangles in A) dation in A431 cells. A431 cells were incubated overnight either in low-serum
quantified in relation to Ponceau staining with 2-, 4-, and 6-min time points regular DMEM-containing bicarbonate or in HEPES-buffered DMEM (50 mM,
combined (n ⫽ 3; mean ⫾ S.D. (error bars); *, p ⬍ 0.05). Symbols indicate pH 7.4). Pretreatment of the cells in bicarbonate-containing DMEM with the
individual results. IB, immunoblotting. NBC inhibitor S0859 (50 ␮M) was for 1 h prior to stimulation. Bicarbonate (60
mM) was added to cells in HEPES-buffered DMEM and subsequently stimu-
lated. At the indicated times after EGF stimulation, cells were subjected to the
growth factor signaling, in which the growth factor initiates cysteinyl-labeling assay using biotinylated iodoacetyl-PEG2-biotin for analy-
sis of reversible PTP1B oxidation. Biotinylated proteins were purified on
transient production of H2O2, which must act jointly with streptavidin-Sepharose beads, resolved by SDS-PAGE, and visualized using
bicarbonate to achieve PTP1B inactivation and initiate phos- antibodies against PTP1B. PTP1B control levels were determined from total
cell lysate by SDS-PAGE and blotting (IB) against PTP1B. Representative West-
phorylation cascades. For the reasons given in the Introduc- ern blotting of three independent experiments is shown. B, model for regu-
tion, we believe that, although it is not possible to validate lation of PTP1B activity during growth factor signaling. EGFR activation
experimentally, this mechanism most likely occurs through induces a transient burst of H2O2, which reacts with bicarbonate to give
PTP1B oxidation via peroxymonocarbonate (red) and activation of phosphor-
the formation of peroxymonocarbonate (Fig. 6B). ylation pathways (green arrow). Prxs compete for the H2O2, and the Trx system
Our investigation so far has focused on PTP1B, but it is pos- decreases the availability of H2O2 by supporting the Prx cycle and also acts by
reactivating oxidized PTP1B. See “Discussion” for further details.
sible that the redox state of other thiol proteins could be regu-
lated by bicarbonate. Gates and co-workers (33) showed that
SHP1 is also highly reactive with peroxymonocarbonate, but Experimental procedures
this was not a general property of all of the thiol proteins they
Expression of recombinant proteins
studied. Thus, there appears to be selectivity, and our findings
give credence to the possibility that peroxymonocarbonate has The catalytic domain (residues 1–322) of PTP1B was sub-
a wider, yet unexplored, role in H2O2-mediated redox signaling. cloned into pD441-H6 vector using PCR with primers 5⬘-
They also provide a plausible explanation for how some thiol GTAGGTCTCGGTGGTATGGAGATGGAAAAGGAG-
proteins that react slowly with H2O2 in isolation (when bicar- TTC-3⬘ and 5⬘-GTAGGTCTCTTATTACCCATTGTGTG-
bonate is typically not present) appear to be oxidant-sensitive GCTCCAGG-3⬘ and Eco31I and DpnI restriction sites. The
during cell signaling. PTP1B protein was expressed and purified under reducing
Another important consideration is that intracellular bicar- conditions according to previously described procedures,
bonate-regulating enzymes such as CA II, IX, XII, and NBCs including subsequent tag removal (25, 55). Prx2 protein was
have been shown to contribute do disease pathology, such as expressed and purified with a His tag, which was cleaved off
promotion of tumor development, through a more alkaline as described previously (56). Recombinant human Trx1 and
intracellular milieu (34, 38, 39, 54). Although most focus has rat TrxR1 WT were expressed and purified as described pre-
been on pH effects of alkalinization of the intracellular milieu, viously (55). PTP1B was subjected to buffer exchange to
we here propose an alternative and yet unexplored mechanism remove reductant before all experiments, using Zeba Spin
whereby redox changes associated with growth factor re- Desalting Columns (Thermo Scientific catalog no. 87766).
sponses, such as the inhibition of PTP1B, are dependent specif- Protein concentrations were determined using Bradford
ically upon bicarbonate. reagents.

J. Biol. Chem. (2019) 294(33) 12330 –12338 12335

EDITORS’ PICK: Bicarbonate in PTP1B oxidation/signaling by EGF

Treatment of PTP1B with H2O2 and bicarbonate SDS-PAGE and analysis of protein tyrosine phosphorylation
Reduced PTP1B (600 nM) was pre-incubated for 20 min in 20 Treated cells were washed with ice-cold PBS (pH 7.4) and
mM HEPES, 100 mM NaCl buffer, pH 7.4, containing 0.1 mM lysed with lysis buffer (0.5% Triton X-100, 0.5% sodium deoxy-
EDTA, 0.05% BSA, 1 mM sodium azide, with the indicated con- cholate salt/deoxycholic acid, 150 mM NaCl, 20 mM Tris (pH
centrations of Trx1, TrxR1, NADPH (N7505, Sigma-Aldrich), 7.5), 10 mM EDTA, and 30 mM sodium pyrophosphate (pH 7.5),
and Prx2. Sodium azide was used to inhibit any trace amounts supplemented with 200 ␮M sodium orthovanadate and a prote-
of catalase. Each reaction mixture was exposed to H2O2 with ase inhibitor mixture (Roche Applied Science)). Protein con-
and without bicarbonate for the indicated times. Bicarbon- centration of lysates was determined using the Bradford assay.
ate and H2O2 were premixed prior to treatment. Subsequent Equal amounts of lysate protein were resolved by SDS-PAGE,
to treatment, measurement of PTP activity was performed. transferred to polyvinylidene difluoride membranes (Milli-
pore), blocked with 5% milk in TBS, and immunoblotted for
PTP activity assay total phosphotyrosine 4G10 (Merck, catalog no. 05-321)
PTP activity was assessed using 15 mM chromogenic sub- (1:1000) and EGFR pTyr-992 (Cell Signaling). The total amount
strate 4-nitrophenyl phosphate (P4744, Sigma-Aldrich) as of loading was verified using Ponceau staining of blotted mem-
described previously (57). Rates of absorbance increase were branes or EGFR intensities (AF231, R&D Systems). Protein
measured at 410 nm at 22 °C using an Infinite M200 Pro plate phosphotyrosine intensities were quantified using ImageJ and
reader (Tecan) and calibrated using a 4-nitrophenol standard shown as a ratio of loading control, as described.
curve.
Detection of reversible PTP oxidation in cells
Treatment of PTP1B with H2O2 The cysteinyl-labeling assay of reversibly oxidized PTP1B
Buffer-exchanged reduced PTP1B was exposed to H2O2 and was performed as described previously (50, 51). The lysis buffer
different components of the Trx system at the indicated time (50 mM sodium acetate, pH 5.5, 150 mM NaCl, 10% glycerol, 1%
points followed by the addition of substrate and measurement Surfact-Amp Nonidet P-40, 5 ␮g/ml aprotinin, 5 ␮g/ml leupep-
of activity. We initially observed that incubation of PTP1B tin, 50 units/ml superoxide dismutase, 50 units/ml catalase, 10
alone resulted in some time-dependent inactivation that was mM iodoacetic acid) was degassed and placed on ice into a
partially prevented in the presence of BSA, so BSA (0.05%) was hypoxic glove-box station equilibrated with 100% argon. Cells
added to the buffer. The activity after each H2O2 treatment cultured in 100-mm dishes were treated and then transferred
was related to the activity of untreated PTP1B incubated for from a 37 °C/5% CO2 environment into a hypoxic glove-box
the same time. A 0 –24% loss of activity was seen in control station, medium was carefully removed, and cells were rapidly
conditions at 30 min. lysed with 800 ␮l of ice-cold lysis buffer. Lysates were trans-
ferred to amber-colored microcentrifuge tubes and shaken for
Insulin-coupled TrxR assay 1 h at room temperature to alkylate reduced thiols. Protein
concentrations were determined, and 1 mg of lysate was applied
NADPH consumption was measured through incubation of
to a spin column to remove excess iodoacetic acid. Tris(2-car-
the Trx system (1 ␮M TrxR1, 20 ␮M Trx1, and 300 ␮M NADPH)
boxyethyl)phosphine (1 mM) was then added to reduce revers-
together with 0.16 mM insulin in 20 mM HEPES, 100 mM NaCl
ibly oxidized protein thiols. Following this step, 5 mM EZ-link
buffer, pH 7.4, containing 0.1 mM EDTA, 0.05% BSA with and
iodoacetyl-PEG2-biotin probe (Pierce) was added to the lysate
without 25 mM bicarbonate and 100 ␮M H2O2.
to label the reactivated thiols. Labeled proteins were then
Cell culture conditions and treatments pulled down by streptavidin-Sepharose beads, which were
washed with lysis buffer (pH 5.5), resuspended in 20 ␮l of 4⫻
Adenocarcinoma cell line A431 cells (ATCC) were typically, Laemmli sample buffer, and heated at 90 °C for 90 s. Samples
unless stated otherwise, cultured in DMEM (44 mM sodium were resolved by SDS-PAGE and blotted with anti-PTP1B.
bicarbonate, 5% CO2) ⫹ 10% (v/v) fetal bovine serum, 2 mM Immunoblots of total cell lysates were run as controls.
L-glutamine, penicillin, and streptomycin. Cells were grown
until 90% confluence and then starved for 24 h in 0.1% fetal Statistical analyses
bovine serum. Pretreatment of starved cells prior to ligand
Analyses of data were performed using GraphPad Prism with
stimulation was performed with the indicated concentrations
two-way analysis of variance followed by Bonferroni post hoc
of either lactic acid (Sigma-Aldrich, catalog no. L6402), S0859
tests for multiple comparisons.
(Sigma-Aldrich, catalog no. SML0638), or sodium bicarbonate
(Merck, catalog no. 9018415). Ligand stimulations were per-
Author contributions—M. D. performed a majority of the experi-
formed with 100 ng/ml EGF ligand (R&D Systems, catalog no.
ments and analysis of data and contributed to writing the paper.
236-EG-200). For cell culture conditions with varying concen- Q. C. expressed and purified TrxR1 and Trx1. S. H. M. R. performed
trations of bicarbonate, bicarbonate-free DMEM (D5648) the CLA assay. P. E. P. expressed Prx2. B. B. performed the CLA
(0.1% (v/v) FBS, 2 mM L-glutamine, penicillin, and streptomy- assay and provided intellectual input. C. C. W. designed experi-
cin) was supplemented with 50 mM HEPES and 0, 40, and 60 mM ments, analyzed data, provided essential intellectual input, and con-
bicarbonate, set to pH 7.4, and pre-equilibrated in 0, 5, and 10% tributed to writing the paper. E. S. J. A. was responsible for the pro-
CO2, respectively, for 3 days prior to the addition to cells, as ject overall and contributed to design of experiments, analyzed data,
stated above. provided intellectual input, and wrote the paper.

12336 J. Biol. Chem. (2019) 294(33) 12330 –12338

 EDITORS’ PICK: Bicarbonate in PTP1B oxidation/signaling by EGF

18. Schwertassek, U., Haque, A., Krishnan, N., Greiner, R., Weingarten, L.,
Acknowledgment—We thank Dr. Mark Hampton for helpful Dick, T. P., and Tonks, N. K. (2014) Reactivation of oxidized PTP1B and
PTEN by thioredoxin 1. FEBS J. 281, 3545–3558 CrossRef Medline
discussions.
19. Parsons, Z. D., and Gates, K. S. (2013) Thiol-dependent recovery of cata-
lytic activity from oxidized protein tyrosine phosphatases. Biochemistry
52, 6412– 6423 CrossRef Medline
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