A chemical model for redox regulation of protein tyrosine phosphatase 1B (PTP1B) activity

Article abstract of DOI:10.1021/ja052599e

Growing evidence indicates that endogenously produced hydrogen peroxide acts as a cellular signaling molecule that (among other things) can regulate the activity of some protein phosphatases. Recent X-ray crystallographic studies revealed an unexpected chemical transformation underlying the redox regulation of protein tyrosine phosphatase 1B, in which oxidative inactivation of the enzyme yields an intrastrand protein cross-link between the catalytic cysteine residue and its neighboring amide nitrogen. This work describes a small organic molecule that serves as an effective model for the redox-sensing assembly of functional groups at the active site of PTP1B. Findings obtained using this model system suggest that the oxidative transformation of PTP1B to its "crosslinked" inactive form can proceed directly via oxidation of the active-site cysteine to a sulfenic acid (RSOH). The remarkably facile nature of this protein cross-link-forming reaction, along with the widespread cellular occurrence of protein sulfenic acids generated via oxidation of cysteine residues, suggests that the type of oxidative protein cross-link formation first seen in the context of PTP1B represents a potentially general mechanism for redox "switching" of protein function. Thus, the chemistry characterized here could have broad relevance to both redox-regulated signal transduction and the toxic effects of oxidative stress. Copyright

Full text of DOI:10.1021/ja052599e

Published on Web 07/19/2005
A Chemical Model for Redox Regulation of Protein Tyrosine Phosphatase 1B
(PTP1B) Activity
Santhosh Sivaramakrishnan, Kripa Keerthi, and Kent S. Gates*
Departments of Chemistry and Biochemistry, UniVersity of Missouri, Columbia, Missouri 65211
Received April 21, 2005; E-mail: gatesk@missouri.edu
Scheme 1
The functional properties of many proteins involved in cellular
signal transduction are modulated by the addition and removal of
phosphoryl groups at selected serine, threonine, and tyrosine resi-
dues.1 The phosphorylation status of target amino acid residues in
relevant proteins is controlled by the coordinated action of protein
kinases2 that catalyze the addition of phosphoryl groups and protein
phosphatases3 that catalyze the removal of these groups. The cellular
activity of various kinases and phosphatases must be tightly regu-
lated1-3 for normal cell function, and it is important to seek an
understanding of the molecular processes governing the activity of
these enzymes.
Growing evidence indicates that endogenously produced hydro-
gen peroxide acts as a cellular signaling molecule that (among other
things) can regulate the activity of some protein phosphatases.4 For
example, exposure of cells to agents such as insulin, growth factors,
and cytokines elicits a burst of hydrogen peroxide that inactivates
selected phosphatases and leads to an increase in phosphorylation
levels of relevant substrate proteins.5 Hydrogen peroxide-mediated
inactivation of phosphatases is typically thought to involve oxidation
of a catalytic thiol residue as shown in Scheme 1.5d,6 This process
Scheme 2
is reversible, and when levels of hydrogen peroxide decline, reac-
tions with the cellular thiol glutathione convert the inactivated pro-
tein back to its catalytically active form (this process may be cata-
lyzed by disulfide reductases such as thioredoxin or glutaredoxin).5e,6f
Protein tyrosine phosphatase 1B (PTP1B) plays a central role in
insulin signaling.7 A key feature of this signal transduction pathway
involves the transient inactivation of PTP1B by an insulin-stimulated
burst of hydrogen peroxide.5a Recent X-ray crystallographic studies
revealed an unexpected chemical transformation underlying the
redox regulation of PTP1B in which oxidative inactivation of the
enzyme yields an intrastrand protein cross-link between the catalytic
cysteine residue and a neighboring amide nitrogen (Scheme 2).8,9
Several possible mechanisms were offered8,9 to explain the forma-
tion of this protein-derived 3-isothiazolidinone heterocycle, with
the most direct route involving peroxide-mediated oxidation of the
active-site thiol residue to a sulfenic acid, followed by attack of
the neighboring amide nitrogen (Scheme 2). This transformation
is unprecedented in the literature of both organic chemistry and
protein chemistry and is striking because nitrogen atoms of amide
groups are generally considered poor nucleophiles.10
Scheme 3
Here, we set out to develop a small organic model system11 that
could be used to characterize the unusual chemical reactions
involved in the redox regulation of PTP1B. A central goal of this
initial study was to determine whether the sulfenic acid intermediate
is, in fact, competent to generate the 3-isothiazolidinone heterocycle
as shown in Scheme 2. For this purpose, we prepared compounds
1a and 1b in three steps from thiosalicylic acid. Importantly, we
determined that the thiol form of this model system (e.g. 5a, Scheme
3) has a pKa of 5.7, similar to that measured for the active-site
thiol in PTP1B (pKa ) 5.6).12 Sulfenic acids typically are too
unstable to be isolated;13 thus, we employed â-sulfinyl propionic
acid ester groups as a means14,15 for in situ generation of the desired
sulfenic acid intermediate under physiologically relevant conditions.
Finally, the ortho-substituted benzene scaffold of 1 provides a good
model for the enforced proximity of the amide and cysteine thiol
groups at the active site of PTP1B.8,9
We find that incubation of 1b in aqueous buffered solution (250
mM sodium phosphate, pH 7.5, containing 30% MeCN at 37 °C)
affords a 92% yield of the 1,2-benzisothiazolin-3(2H)-one 3b
(Scheme 3). The reaction is rather robust and does not depend
strongly upon the nature of the solvent or the substituent on nitrogen.
For example, the phenyl-substituted amide 1a gave an 88% yield
of 3a in the sodium phosphate buffer system described above, and
1b provides a 60% yield of 3b when the reaction is conducted in
9
10830
J. AM. CHEM. SOC. 2005, 127, 10830-10831
10.1021/ja052599e CCC: $30.25 © 2005 American Chemical Society

C O M M U N I C A T I O N S
Scheme 4
Supporting Information Available: Experimental procedures and
spectral data for all compounds. This material is available free of charge
via the Internet at http://pubs.acs.org.
References
(1) (a) Hunter, T. Cell 2000, 100, 113-127. (b) Ahn, N. Chem. ReV. 2001,
101, 2207. (c) Lawrence, D. S. Acc. Chem. Res. 2003, 36, 353-354.
(2) Manning, G.; Whyte, D. B.; Martinez, R.; Hunter, T.; Sudarsanam, S.
Science 2002, 298, 1912-1934.
(3) (a) Neel, B. G.; Tonks, N. K. Curr. Opin. Cell Biol. 1997, 9, 193-204.
(b) Zhang, Z.-Y. Acc. Chem. Res. 2003, 36, 385-392. (c) Jackson, M.
D.; Denu, J. M. Chem. ReV. 2001, 101, 2313-2340.
(4) (a) Finkel, T. Curr. Opin. Cell Biol. 2003, 15, 247-254. (b) Rhee, S. G.;
Chang, T.-S.; Bae, Y. S.; Lee, S.-R.; Kang, S. W. J. Am. Soc. Nephrol.
2003, 14, S211-S215. (c) Alder, V.; Yin, Z.; Tew, K. D.; Ronai, Z.
Oncogene 1999, 18, 6104-6111. (d) Mikkelsen, R. B.; Wardman, P.
Oncogene 2003, 22, 5734-5754.
(5) (a) Mahedev, K.; Zilbering, A.; Zhu, L.; Goldstein, B. J. J. Biol. Chem.
2001, 276, 21938-21942. (b) Meng, T.-C.; Fukada, T.; Tonks, N. K.
Mol. Cell 2002, 9, 387-399. (c) Bae, Y. S.; Sung, J.-Y.; Kim, O.-S.;
Kim, Y. J.; Hur, K. C.; Kazlauskas, A.; Rhee, S. G. J. Biol. Chem. 2000,
275, 10527-10531. (d) Chiarugi, P.; Fiaschi, T.; Taddei, M. L.; Talini,
D.; Giannoni, E.; Raugei, G.; Ramponi, G. J. Biol. Chem. 2001, 276,
33478-33487. (e) Downes, C. P.; Walker, S.; McConnachie, G.; Lindsay,
Y.; Batty, I. H.; Leslie, N. R. Biochem. Soc. Trans. 2004, 32, 338-342.
(e) Lee, S. R.; Kwon, K. S.; Kim, S. R.; Rhee, S. G. J. Biol. Chem. 1998,
273, 15366-15372.
dichloromethane. Consistent with our expectation14,15 that the
â-sulfinyl propionic acid ester group would decompose to produce
the desired sulfenic acids under these reaction conditions, we find
that the analogue 6, in which cyclization to the benzisothiazolinone
ring system is blocked by dialkyl substitution on the amide nitrogen,
yields the characteristic13 product (7) resulting from alkylation of
the sulfenic acid intermediate when incubated in the presence of
excess methyl iodide (Scheme 4).16 In the absence of methyl iodide,
this compound affords the expected13 products arising from sulfenic
acid dimerization (see Supporting Information). In direct analogy
to the oxidative inactivation of PTP1B shown in Scheme 2, we
find that treatment of the thiol 5a with H2O2 produces a good yield
of 3a (see Supporting Information).
(6) (a) Denu, J. M.; Tanner, K. G. Biochemistry 1998, 37, 5633-5642. (b)
Barrett, W. C.; DeGnore, J. P.; Keng, Y.-F.; Zhang, Z.-Y.; Yim, M. B.;
Chock, P. B. J. Biol. Chem. 1999, 274, 34543-34546. In some
phosphatases, the sulfenic acid intermediate undergoes further reaction
with a “back door” thiol at the active site; see: (c) Sohn, J.; Rudolph, J.
Biochemistry 2003, 42, 10060-10070. (d) Lee, S.-R.; Yang, K.-S.; Kwon,
J.; Lee, C.; Jeong, W.; Rhee, S. G. J. Biol. Chem. 2002, 277, 20336-
20342.
(7) (a) Johnson, T. O.; Ermolieff, J.; Jirousek, M. R. Nat. ReV. Drug DiscoVery
2002, 1, 696-709. (b) Hooft van Huijsduijnen, R.; Sauer, W. H. B.;
Bombrun, A.; Swinnen, D. J. Med. Chem. 2004, 47, 4142-4146.
(8) Salmeen, A.; Anderson, J. N.; Myers, M. P.; Meng, T.-C.; Hinks, J. A.;
Tonks, N. K.; Barford, D. Nature 2003, 423, 769-773.
(9) van Montfort, R. L. M.; Congreeve, M.; Tisi, D.; Carr, R.; Jhoti, H. Nature
2003, 423, 773-777.
(10) Gajda, T.; Zwierzak, A. Synthesis 1981, 1005-1008.
(11) Appropriately designed small organic molecules provide a powerful tool
for exploring the fundamental chemical reactivity of structurally complex
biopolymers and secondary metabolites. (a) Gates, K. S. Chem. Res.
Toxicol. 2000, 13, 953-956. (b) Greenberg, M. M. Chem. Res. Toxicol.
1998, 11, 1235-1248. (c) Myers, A. G.; Dragovich, P. S. J. Am. Chem.
Soc. 1989, 111, 9130-9132. (d) Robins, M. J.; Ewing, G. J. J. Am. Chem.
Soc. 1999, 121, 5823-5824. (e) D’Souza, V. T.; Bender, M. L. Acc. Chem.
Res. 1987, 20, 146-152. (f) Breslow, R. Acc. Chem. Res. 1995, 28, 146-
153.
Taken together, the results indicate first that the compounds 1
decompose to yield the sulfenic acid intermediates 2 and second
that the sulfenic acids (2) undergo efficient conversion to the 3-iso-
thiazolidinone products 3.17 In contrast, the mixed disulfide 4b
(Scheme 3, R′ ) CH2CH2OH) does not undergo rapid cyclization
to 3b either in organic solvent (CDCl3) or our standard sodium
phosphate buffer system.18 Overall, our findings indicate that the
oxidative transformation of PTP1B to its inactive 3-isothiazolidinone
form can proceed directly via oxidation of the active-site thiol to a
sulfenic acid intermediate (as shown in Scheme 2). This argues
against the need to invoke alternative mechanisms9 involving further
conversion of the sulfenic acid to a sulfenyl peroxide or a mixed
disulfide.
In addition to modeling the chemistry underlying oxidative
inactivation of PTP1B, the small organic system reported here also
mimics the thiol-mediated reduction of the inactive isothiazolidinone
form of the enzyme back to its catalytically active thiol form.8,9
Specifically, 3b is rapidly and completely converted (<1 min) to
the aromatic thiol 5b (Scheme 3) upon treatment with excess thiol
(10 µM 3b, 10 equiv of 2-mercaptoethanol, in 50 mM pH 7.0
phosphate buffer containing 30% acetonitrile).
In summary, we have developed a small organic molecule that
serves as an effective model for the redox-sensing assembly of
functional groups found at the active site of the enzyme PTP1B.
Importantly, results obtained with this model system show that the
sulfenic acid residue possesses sufficient electrophilicity to drive
the cyclization reaction with a neighboring amide group, thus
generating a 3-isothiazolidinone heterocycle analogous to that
recently characterized at the active site of oxidatively inactivated
PTP1B.8,9 Protein sulfenic acids are common intermediates gener-
ated during the oxidation of cysteine thiol residues in cells.19 This
fact, along with the remarkably facile nature of the sulfenic acid
chemistry reported here, suggests that the reversible formation of
a protein-derived 3-isothiazolidinone residue first seen in the context
of PTP1B represents a potentially general mechanism for redox
“switching” of protein function. Thus, this chemistry could have
broad relevance to both redox-regulated signal transduction and
the toxic effects of oxidative stress.
(12) Lohse, D. L.; Denu, J. M.; Santoro, N.; Dixon, J. E. Biochemistry 1997,
36, 4568-4575.
(13) O’Donnell, J. S.; Schwan, A. L. J. Sulfur Chem. 2004, 25, 183-211.
(14) (a) Cubbage, J. W.; Guo, Y.; McCulla, R. D.; Jenks, W. S. J. Org. Chem.
2001, 66, 8722-8736. (b) Adams, H.; Anderson, J. C.; Bell, R.; Neville
Jones, D.; Peel, M. R.; Tomkinson, N. C. O. J. Chem. Soc., Perkin 1
1998, 3967-3973. (c) Hashmi, M.; Vamvakas, S.; Anders, M. W. Chem.
Res. Toxicol. 1992, 5, 360-365.
(15) In addition, we successfully employed 2-(4-pyridyl)ethyl sulfoxides as
sulfenic acid precursors in this reaction: Katritzky, A. R.; Takahashi, I.;
Marson, C. M. J. Org. Chem. 1986, 51, 4914-4920. See Supporting
Information.
(16) In Scheme 1, we see the sulfenic acid group acting as an electrophile,
while in Scheme 4 it is a nucleophile. It is well documented that sulfenic
acids can act as either electrophiles or nucleophiles. See, for example:
Hogg, D. R.; Robertson, A. Tetrahedron Lett. 1974, 43, 3783-3784 and
Goto, K.; Holler, M.; Okazaki, R. J. Am. Chem. Soc. 1997, 119, 1460-
1461.
(17) The formation of 3 from 1, in principle, could occur via a process involving
dimerization of the sulfenic acid 2 to the corresponding thiosulfinate (RS-
(O)SR), followed by conversion of the thiosulfinate to 3. However, this
possibility is rendered unlikely based upon our observation (see Supporting
Information) that the yield of the cyclization product 3 (Scheme 3) is not
diminished when the reaction is conducted in the presence of methyl iodide
concentrations that completely preVent dimerization of a sulfenic acid
intermediate as shown in Scheme 4.
(18) In contrast, 2,2′-dithiobisbenzamides exist in equilibrium with the corre-
sponding 1,2-benzisothiazolin-3(2H)-ones, see: Domagala, J. M.; Bader,
J. P.; Gogliotti, R. D.; Sanchez, J. P.; Stier, M. A.; Song, Y.; Prasad, J.
V. N. V.; Tummino, P. J.; Scholten, J.; Harvey, P.; Holler, T.; Gracheck,
S.; Hupe, D.; Rice, W. G.; Schultz, R. Bioorg. Med. Chem. 1997, 5, 569-
579.
Acknowledgment. We thank Professors Peter Tipton, Richard
Loeppky, and Christina Wells for critical review of the manuscript
and are grateful to the NIH (CA 83925) for partial support of this
work.
(19) Claiborne, A.; Yeh, J. I.; Mallet, T. C.; Luba, J.; Crane, E. J.; Charrier,
V.; Parsonage, D. Biochemistry 1999, 38, 15407-15416.
JA052599E
9
J. AM. CHEM. SOC. VOL. 127, NO. 31, 2005 10831