From the Dorrance H. Hamilton Research Laboratories, Division of Endocrinology and Metabolic Diseases, Department of Medicine, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107
Received for publication, March 2, 2001, and in revised form, March 30, 2001
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ABSTRACT |
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The insulin signaling pathway is activated by
tyrosine phosphorylation of the insulin receptor and key post-receptor
substrate proteins and balanced by the action of specific
protein-tyrosine phosphatases (PTPases). PTPase activity, in turn,
is highly regulated in vivo by oxidation/reduction
reactions involving the cysteine thiol moiety required for catalysis.
Here we show that insulin stimulation generates a burst of
intracellular H2O2 in insulin-sensitive hepatoma and adipose cells that is associated with reversible oxidative
inhibition of up to 62% of overall cellular PTPase activity, as
measured by a novel method using strictly anaerobic conditions. The
specific activity of immunoprecipitated PTP1B, a PTPase homolog implicated in the regulation of insulin signaling, was also strongly inhibited by up to 88% following insulin stimulation. Catalase pretreatment abolished the insulin-stimulated production of
H2O2 as well as the inhibition of cellular
PTPases, including PTP1B, and was associated with reduced
insulin-stimulated tyrosine phosphorylation of its receptor and high
Mr insulin receptor substrate (IRS) proteins. These data provide compelling new evidence for a redox signal that
enhances the early insulin-stimulated cascade of tyrosine phosphorylation by oxidative inactivation of PTP1B and possibly other
tyrosine phosphatases.
Protein-tyrosine phosphatases (PTPases)1 play a key
role in the regulation of reversible
tyrosine phosphorylation in the insulin action pathway. Insulin
signaling is initiated by the phosphorylation of specific tyrosyl
residues of the cell surface insulin receptor, which activates its
exogenous kinase activity and promotes the phosphorylation of IRS
proteins on specific tyrosine residues (1). These activation steps are
balanced, in turn, by specific cellular PTPases that dephosphorylate
and inactivate the receptor kinase and reverse the adapter function of
the receptor substrate proteins (2). The cellular role of PTPases is
apparent from the observation that highly purified insulin receptors
and IRS proteins retain their tyrosine phosphorylation and activation state in vitro (3, 4), while in intact or permeabilized cells, receptor activation and substrate tyrosine phosphorylation are
rapidly reversed (5-7).
Since PTPases are high turnover number enzymes, physiological
suppression of PTPase catalytic activity has been postulated to be a
key feature of their regulation within the cellular environment to
allow tyrosine phosphorylation to proceed in a balanced manner (8).
PTPases have in common a conserved ~230-amino acid domain that
contains the cysteine residue that catalyzes the hydrolysis of protein
phosphotyrosine residues by the formation of a cysteinyl-phosphate intermediate (9, 10). Several laboratories have recently provided
evidence that reactive oxygen species, including
H2O2, can oxidize and inactivate PTPases
in vivo (11, 12). Since only the reduced form of the
catalytic site is enzymatically active, stepwise and progressively
irreversible oxidative inhibition is emerging as an important means by
which PTPase activity can be suppressed in specific signal transduction
pathways (13, 14).
In the present work, we show that insulin stimulation of hepatoma and
adipose-like cells causes the rapid formation of intracellular H2O2, which is associated with significantly
decreased overall PTPase activity as well as a reduction in the
specific activity of PTP1B, a PTPase that has been strongly implicated
in the regulation of the insulin signaling pathway. Inhibition of the
insulin-stimulated production of H2O2 by
catalase treatment blocks the PTPase inactivation and reduces
insulin-stimulated receptor autophosphorylation and tyrosine
phosphorylation of IRS proteins. These findings reveal a novel
regulatory mechanism integral to the early steps in insulin signaling
that contribute to the steady-state balance and propagation of the
insulin action cascade.
Cell Culture--
Murine 3T3-L1 preadipocytes were
differentiated with insulin, dexamethasone, and isobutylmethylxanthine
as described previously (15). Cells were serum-starved overnight
in medium containing 0.5% (w/v) bovine serum albumin prior to insulin stimulation.
Visualization of Intracellular
H2O2--
Intracellular
H2O2 production was detected by fluorescence of
5,6-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-H2DCF-DA; catalog number C-6827, Molecular
Probes) on confocal microscopy (Bio-Rad) at an excitation wavelength of
488 nm and emission at 515-540 nm. To avoid photo-oxidation, the
fluorescence image was collected by a single rapid scan with identical
parameters for all samples (16).
Measurement of Cellular PTPase Activities--
Endogenous PTPase
activities as isolated from the cellular environment were measured
using a novel anaerobic technique to avoid air oxidation as we
described recently (41). Briefly, an enclosed anaerobic work
station (Forma Scientific number 901024) provided an oxygen-free
environment (gas mixture of
N2:H2:CO2 = 85:10:5) for cell
homogenization, sealing tubes for centrifugations, immunoprecipitations, and PTPase enzyme assays. After the indicated treatments, cells were snap-frozen in liquid N2, introduced
into the anaerobic chamber in a frozen state, and disrupted by scraping into ice-cold, deoxygenated homogenization buffer (150 mM
NaCl, 5 mM EDTA, 5 mM EGTA, in 50 mM Hepes, pH 7.5, containing a protease inhibitor mixture
(Sigma) followed by brief sonication. The whole cell lysate was
prepared by adding 1% (v/v) Triton X-100, mixing on ice for 45 min,
and clearing of the lysate by centrifugation at 15,000 × g for 20 min. Prior to solubilization, the supernatant resulting from centrifugation of the homogenate at 100,000 × g for 45 min at 4 °C was taken as the cytosol. The
solubilized particulate fraction was prepared by adding Triton X-100 to
the pellet from the ultracentrifugation step and incubating and
clearing as above by centrifugation. Protein was measured using the
method of Bradford (17).
PTPase Assay--
PTPase activity was determined in
cell fractions containing 30 µg of protein in a final volume of 100 µl at 30 °C for 30 min in reaction buffer containing 10 mM para-nitrophenyl phosphate (pNPP;
Sigma) and 2 mM EDTA in 20 mM MES at pH 6.0. Where indicated, assay samples were incubated with 1 mM DTT
on ice for 10 min prior to enzyme assay. The reaction was stopped by
the addition of 50 µl of 1 M NaOH, and the absorption was
determined at 410 nm (18). PTPase activity is reported as the optical
density from hydrolysis of pNPP.
Specific Activity of PTP1B--
Under strictly
anaerobic conditions, PTP1B was immunoprecipitated from cell lysates
with a monoclonal antibody directed at a C-terminal epitope that
preserves its enzymatic activity (Oncogene Sciences; Ab-2) followed by
adsorption to Trisacryl protein G (Pierce). PTPase activity was
measured by the hydrolysis of pNPP in the anaerobic chamber
in washed immunoprecipitates as described above. Control samples using
non-immune mouse IgG showed minimal background PTPase activity (<5%
of the activity with Ab-2).
Immunoblot Analysis of Protein Tyrosine
Phosphorylation--
Samples of 3T3-L1 cell lysates containing 75 µg
of protein were subjected to Western blot analysis using a monoclonal
anti-phosphotyrosine antibody (4G10, Upstate Biotechnology) or
polyclonal antibodies to IRS-1 or insulin receptor An oxidant signal in response to insulin was demonstrated in
3T3-L1 adipocytes loaded with CM-H2DCF-DA, a redox
indicator dye that is trapped intracellularly after cleavage by
cellular esterases (Fig. 1). When
oxidized in situ, DCF generates a signal that can be
visualized by fluorescence confocal microscopy (16). Following
stimulation of 3T3-L1 adipocytes with 100 nM insulin, a
strong oxidant signal was detected by DCF fluorescence within 1 min,
peaked at 5 min, and began to dissipate by 10 min (Fig. 1A).
The oxidant generated was shown to be H2O2,
since preincubation of the cells with catalase completely obliterated
the fluorescent signal. A dose-response study using insulin exposure
for 5 min showed that H2O2 production was
detectable between 0.1 and 1 nM and was maximal at 10 nM (Fig. 1B). Similar results were observed in
human HepG2 hepatoma cells, with an insulin-stimulated
H2O2 signal evident at 1 min that increased
through 10 min of incubation and was completely blocked by catalase
preincubation (Fig. 1C).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-subunit proteins
(Transduction Laboratories) as described previously (19).
Labeled proteins were visualized with horseradish peroxidase-conjugated
anti-mouse IgG for 4G10 or with conjugated anti-rabbit IgG for the
polyclonal antibodies, using conditions supplied by the manufacturer
(Pierce). The blots were quantitated using an ImageStation 440 (Eastman Kodak Co.).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Insulin-stimulated production of
H2O2 in 3T3-L1 adipocytes and HepG2 cells.
Confluent murine 3T3-L1 preadipocytes were differentiated with insulin,
dexamethasone, and isobutylmethylxanthine (15), and human HepG2
hepatoma cells at 80% confluence were serum-starved overnight in
medium containing 0.5% (w/v) bovine serum albumin prior to insulin
stimulation. Intracellular H2O2 production was
detected by fluorescence of CM-H2DCF-DA (catalog
number C-6827, Molecular Probes), which generates a fluorescent
signal in situ visualized by confocal microscopy using
fluorescein parameters for excitation (488 nm) and emission (515-540
nm). Time course studies in 3T3-L1 adipocytes (A) and human
HepG2 hepatoma cells (C) were performed by stimulating the
cells with 100 nM recombinant human insulin (Sigma) for the
indicated period of time prior to rinsing the cells with serum-free
medium and incubation in the dark with CM-H2DCF-DA for an
additional l0 min. Cells were washed with serum-free medium prior to
measurement of DCF fluorescence by confocal microscopy. Where
indicated, the cells were preincubated with 0.008% (w/v) catalase
(Sigma, C-100) for 10 min prior to insulin stimulation as described
previously (16). The dose-response study (B) was performed in
3T3-L1 adipocytes by incubating the serum-starved cells with the
indicated concentration of insulin for 5 min, rinsing with serum-free
medium, and then incubating the cells in the dark with DCF-DA for 10 min prior to confocal microscopy.
To determine whether the insulin-stimulated generation of intracellular
H2O2 affected the endogenous activity of
cellular PTPases, we applied a novel approach recently established in
our laboratory that involves sample handling and analysis under
anaerobic conditions (41). This method preserves the activity of
PTPases as isolated from the cultured cells and avoids oxidation and
artifactual enzyme inhibition that occurs on exposure to air. Treatment
of HepG2 cells with 100 nM insulin for 5 min resulted in a
32-52% reduction in overall PTPase activity in the cell homogenate,
the cytosol, and the solubilized particulate fraction
(p < 0.001) (Fig.
2A). Biochemical reduction of
the enzyme samples with DTT prior to PTPase assay had no significant
effect on the control samples prior to insulin treatment, but fully
restored the reduced PTPase activity of the insulin-treated samples,
indicating that they had been reversibly oxidized and inactivated by
insulin exposure. A similar but more striking effect was observed using
3T3-L1 adipocytes, where insulin treatment caused a 62% drop in PTPase
activity in the cell lysate (p < 0.001), which was
restored to within control levels by treatment of the assay samples
with DTT (Fig. 2B).
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To determine the role of insulin-induced H2O2 in the oxidative inhibition of cellular PTPase activity, cells were preincubated with catalase prior to insulin stimulation and PTPase assay. Catalase had no significant effect on the basal level of PTPase activity. However, the presence of catalase blocked the reduction of PTPase activity in the 3T3-L1 adipocyte cell lysate induced by insulin to a level that was not significantly different from the control samples (Fig. 2). This important finding indicated that H2O2 mediated the oxidative inhibition of cellular PTPase activity associated with insulin stimulation.
To explore whether the insulin-stimulated generation of
H2O2 affected the specific activity of PTP1B
itself as isolated from intact cells, we immunoprecipitated PTP1B from
snap-frozen HepG2 cell lysates under anaerobic conditions and assayed
its activity within the anaerobic chamber. Following insulin treatment
for 2 or 5 min, the activity of immunoprecipitated PTP1B was reduced to
46 and 29% of control, respectively (Fig.
3A). In the continued presence
of insulin, this effect was sustained for at least 10 min (not
shown).
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For comparison, direct treatment of HepG2 cells with 0.5 mM H2O2 for 5 min prior to cell lysis caused a 66% reduction of PTP1B enzyme activity (Fig. 3A). Further insight into the biochemical alterations induced in PTP1B following insulin stimulation of HepG2 cells was obtained by treating the immunoprecipitated enzyme with DTT prior to the PTPase assay (Fig. 3B). As in the previous experiment, insulin stimulation reduced the PTP1B enzyme activity to 28% of control. Treatment of the isolated enzyme with DTT restored the activity to 83% of control, indicating that the insulin-induced oxidative inactivation of PTP1B was largely reversible by biochemical reduction. In contrast, treatment of the cells directly with H2O2 reduced the catalytic activity of immunoprecipitated PTP1B to a similar degree (26% of control), but this effect was less reversible in vitro with DTT incubation, to only 61% of the control level (Fig. 3B). Thus, H2O2 itself causes a greater degree of PTP1B catalytic thiol oxidation compared with cell treatment with insulin. Treatment of immunoprecipitated PTP1B from the control HepG2 cells with DTT increased the enzyme activity slightly but significantly by 16% (p = 0.03), suggesting that in situ, only a small fraction of the enzyme is present in an oxidized state that is activable by biochemical reduction in vitro.
In 3T3-L1 adipocytes, insulin treatment also potently reduced the activity of immunoprecipitated PTP1B to 12% of control, which was reversible to 72% of control by preincubation of the immunoprecipitated enzyme with DTT prior to PTPase assay (Fig. 3C). To further clarify the role of H2O2 in the reduced activity of PTP1B from the insulin-treated cells, 3T3-L1 adipocytes were preincubated with catalase, which completely blocks the insulin-stimulated generation of H2O2 in these cells, as shown above in the experiments utilizing DCF-DA fluorescence. Insulin stimulation of the 3T3-L1 adipocytes reduced PTP1B activity in the control cells by 64%, which was completely prevented by preincubating the cells with catalase (Fig. 3D). Catalase similarly prevented the insulin-stimulated reduction in PTP1B activity in HepG2 cells (not shown), indicating that in both of these cell types, the insulin-induced oxidative inhibition of PTP1B activity, mediated by H2O2, was proportionally greater than the decrease in overall PTPase activity elicited by insulin in the cell lysates.
A receptor-mediated effect of insulin on PTP1B activity was also
suggested by treatment of HepG2 cells with staurosporine (20), which
blocks insulin receptor autophosphorylation in the HepG2 cells (not
shown), and reduced the insulin inhibition of PTP1B enzyme activity by
40% (Fig. 4A). To determine
the reversibility of the insulin effect, insulin bound to the HepG2
cells was dissociated with a mild acid wash after various times of
insulin stimulation, and after an additional 10 min, the cellular PTP1B
activity was determined by immunoprecipitation under anaerobic
conditions (Fig. 4B). At each time point, continued exposure
to insulin led to a persistent reduction in PTP1B activity; however,
removal of the bound insulin fully reversed the inhibition of PTP1B
activity following incubations with insulin of up to 30 min.
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Finally, we evaluated the effect of blocking the insulin-induced
production of H2O2 with catalase on
insulin-stimulated tyrosine phosphorylation of the insulin receptor and
IRS proteins in 3T3-L1 adipocytes (Fig.
5). Catalase treatment had no effect on
the basal level of insulin receptor or IRS phosphorylation in the
serum-starved cells. However, catalase reduced the autophosphorylation
of the insulin receptor by 48 and 44% and similarly reduced the
insulin-stimulated tyrosine phosphorylation of IRS proteins by 34 and
43%, respectively, at 1 or 5 min of insulin treatment. The protein
abundance of IRS-1 and the insulin receptor -subunit was unchanged
by incubation of the cells with catalase (Fig. 5A). Taken
together, these data suggest that by preventing the insulin-induced
inhibition of PTP1B, catalase treatment results in a sustained level of
PTP1B activity, which appears to be inhibitory to insulin-stimulated
autophosphorylation of its receptor and restricts the propagation of
the early insulin signal to IRS proteins.
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DISCUSSION |
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Cross-talk involving oxidative inactivation of PTPases and the signaling pathways they regulate has become evident in recent work showing that reactive oxygen species can be generated by stimulation of cells with growth factors and cytokines (12, 21). Specifically, H2O2 has been implicated in the activation of tyrosine phosphorylation cascades in a manner that mimics the effect of ligands such as epidermal growth factor and platelet-derived growth factor; also, H2O2 can serve as an integral constituent of downstream growth factor signal transduction (16, 22-27).
Recent data has also suggested that signal transduction pathways modulated by reactive oxygen species associated with the action of growth factors and cytokines may involve the stepwise oxidation and inactivation of PTPase enzymes (11, 12). The susceptibility of enzymes in the PTPase family to oxidative inactivation resides in the characteristic active site sequence motif, which requires the catalytic cysteine moiety to be in a reduced state (10). The catalytic thiol hydrogen lies in spatial proximity to amino acid side chains adjacent to the enzyme active site that strongly affect its ionization, effectively lowering its pKa to more than 3 units below that found in a typical cysteine and facilitating its derivatization at physiological pH (9, 28). This cysteine residue is susceptible to oxidation to progressively more inert forms. First, this forms the sulfenic (-SOH) form, which is reversible and amenable to reduction by cellular mechanisms or by reducing agents in vitro; sequential steps of oxidation may then lead to sulfinic (-SO2H) and sulfonic (-SO3H) forms and can lead to irreversible PTPase inactivation. This general scheme may constitute a major regulatory mechanism for PTPases within the cellular environment.
PTP1B, in particular, appears to be a cellular target for oxidative inactivation possibly followed by disulfide conjugation with glutathione (glutathiolation), processes that are at least partially balanced by cellular reductases (13, 14, 29). PTP1B was one of the first specific PTPases to be implicated in the negative regulation of insulin receptor autophosphorylation and post-receptor insulin signaling (7, 18, 30-32). The most compelling evidence for a physiological role of PTP1B in insulin action has been the recent demonstration of enhanced insulin sensitivity and potentiation of insulin-stimulated protein-tyrosine phosphorylation in PTP1B knockout mice (33, 34). Changes in the intracellular enzymatic activity of PTP1B may also affect insulin action in adipose tissue from obese subjects (35). PTP1B has been recognized as a target for drug development to enhance insulin signaling through enhancing protein-tyrosine phosphorylation in the insulin action pathway (36), and novel, relatively specific inhibitors of PTP1B have been reported (37).
In the present work, we provide evidence for a close coupling between the insulin-induced generation of cellular H2O2 and the oxidative inactivation of the catalytic activity of PTP1B. In early studies of insulin action, it was recognized that H2O2 could mimic at least some of the actions of insulin (38), and more recent work has demonstrated that H2O2 is elaborated during physiological insulin signal transduction in adipose cells (39). Some years ago, Meyerovitch and colleagues (40) made the observation that stimulation of Fao rat hepatoma cells with nanomolar concentrations of insulin over a time course of several minutes caused a significant reduction in cytosolic PTPase activity measured against an insulin receptor phosphopeptide. Among the novel findings in our present study is the demonstration that insulin stimulation generates a burst of H2O2 in multiple types of insulin-sensitive cells. In addition to the down-regulation of the specific activity of cellular PTP1B by oxidants elicited by insulin, it is possible that this burst of H2O2 also affects downstream insulin signaling, as shown in response to growth factors in other cell types (12, 21, 26). Further studies detailing the implications of this pathway for distal insulin signaling in various cell types are underway.
In summary, we report here the novel observation that in both 3T3-L1
adipocytes and hepatoma cells, the elaboration of reactive oxygen
species (H2O2) by insulin is associated with
reversible oxidative inactivation of overall cellular PTPase activity
and specifically of PTP1B. This effect, in turn, markedly influences insulin-stimulated receptor autophosphorylation and the tyrosine phosphorylation of IRS proteins. Given the growing body of evidence that PTP1B is integrally involved in the negative regulation of insulin
signaling, this reciprocal regulation of the balance of protein-tyrosine phosphorylation may play a critical role in signal transduction in the insulin action pathway.
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ACKNOWLEDGEMENTS |
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We appreciate helpful discussions during the initiation of this work with Dr. Sue Goo Rhee, Chief, Laboratory of Cell Signaling, NHLBI, National Institutes of Health and thank Nathalie Innocent for technical assistance with confocal microscopy.
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FOOTNOTES |
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* This work was supported by Grants DK43396 and DK53388 from the National Institutes of Health, the Kimmel Cancer Center confocal facility, and a Mentor-based postdoctoral fellowship (in support of L. Z. to B. J. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Director, Division of
Endocrinology and Metabolic Diseases, Jefferson Medical College, Rm.
349 Alumni Hall, 1020 Locust St., Philadelphia, PA 19107-6799. Tel.:
215-503-1272; FAX: 215-923-7932; E-mail: Barry.Goldstein@mail.tju.edu.
Published, JBC Papers in Press, April 10, 2001, DOI 10.1074/jbc.C100109200
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ABBREVIATIONS |
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The abbreviations used are: PTPase, protein-tyrosine phosphatase; IRS, insulin receptor substrate; pNPP, para-nitrophenyl phosphate; MES, 2-(N-morpholino)ethanesulfonic acid; DTT, dithiothreitol; CM-H2DCF-DA, 5,6-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate; PTP1B, protein-tyrosine phosphatase 1B.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Withers, D. J.,
and White, M.
(2000)
Endocrinology
141,
1917-1921 |
2. | Goldstein, B. J. (2000) in Diabetes Mellitus: A Fundamental and Clinical Text (LeRoith, D. , Olefsky, J. M. , and Taylor, S. I., eds), 2nd Ed. , pp. 206-217, Lippincott, Philadelphia |
3. |
Hashimoto, N.,
Feener, E. P.,
Zhang, W. R.,
and Goldstein, B. J.
(1992)
J. Biol. Chem.
267,
13811-13814 |
4. | Kozma, L., Baltensperger, K., Klarlund, J., Porras, A., Santos, E., and Czech, M. P. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 4460-4464[Abstract] |
5. | Bernier, M., Liotta, A. S., Kole, H. K., Shock, D. D., and Roth, J. (1994) Biochemistry 33, 4343-4351[Medline] [Order article via Infotrieve] |
6. | Mooney, R. A., Kulas, D. T., Bleyle, L. A., and Novak, J. S. (1997) Biochem. Biophys. Res. Commun. 235, 709-712[CrossRef][Medline] [Order article via Infotrieve] |
7. |
Calera, M. R.,
Vallega, G.,
and Pilch, P. F.
(2000)
J. Biol. Chem.
275,
6308-6312 |
8. | Hunter, T. (2000) Cell 100, 113-127[Medline] [Order article via Infotrieve] |
9. | Denu, J. M., and Dixon, J. E. (1998) Curr. Opin. Chem. Biol. 2, 633-641[CrossRef][Medline] [Order article via Infotrieve] |
10. | Zhang, Z. Y. (1998) Crit. Rev. Biochem. Mol. Biol. 33, 1-52[Abstract] |
11. | Claiborne, A., Yeh, J. I., Mallett, T. C., Luba, J., Crane, E. J., Charrier, V., and Parsonage, D. (1999) Biochemistry 38, 15407-15416[CrossRef][Medline] [Order article via Infotrieve] |
12. | Herrlich, P., and Bohmer, F. D. (2000) Biochem. Pharmacol. 59, 35-41[CrossRef][Medline] [Order article via Infotrieve] |
13. | Denu, J. M., and Tanner, K. G. (1998) Biochemistry 37, 5633-5642[CrossRef][Medline] [Order article via Infotrieve] |
14. |
Lee, S. R.,
Kwon, K. S.,
Kim, S. R.,
and Rhee, S. G.
(1998)
J. Biol. Chem.
273,
15366-15372 |
15. | Gagnon, A., and Sorisky, A. (1998) Obes. Res. 6, 157-163[Abstract] |
16. |
Bae, Y. S.,
Kang, S. W.,
Seo, M. S.,
Baines, I. C.,
Tekle, E.,
Chock, P. B.,
and Rhee, S. G.
(1997)
J. Biol. Chem.
272,
217-221 |
17. | Bradford, M. M. (1976) Anal. Biochem. 72, 248-254[CrossRef][Medline] [Order article via Infotrieve] |
18. |
Goldstein, B. J.,
Bittner-Kowalczyk, A.,
White, M. F.,
and Harbeck, M.
(2000)
J. Biol. Chem.
275,
4283-4289 |
19. |
Ahmad, F.,
Li, P. M.,
Meyerovitch, J.,
and Goldstein, B. J.
(1995)
J. Biol. Chem.
270,
20503-20508 |
20. | Elberg, G., Li, J., Leibovitch, A., and Shechter, Y. (1995) Biochim. Biophys. Acta 1269, 299-306[Medline] [Order article via Infotrieve] |
21. | Finkel, T. (2000) FEBS Lett. 476, 52-54[CrossRef][Medline] [Order article via Infotrieve] |
22. | Sundaresan, M., Yu, Z. X., Ferrans, V. J., Irani, K., and Finkel, T. (1995) Science 270, 296-299[Abstract] |
23. | Rao, G. N. (1996) Oncogene 13, 713-719[Medline] [Order article via Infotrieve] |
24. |
Guyton, K. Z.,
Liu, Y.,
Gorospe, M.,
Xu, Q.,
and Holbrook, N. J.
(1996)
J. Biol. Chem.
271,
4138-4142 |
25. | Suzuki, Y. J., Forman, H. J., and Sevanian, A. (1997) Free Radic. Biol. Med. 22, 269-285[CrossRef][Medline] [Order article via Infotrieve] |
26. |
Lander, H. M.
(1997)
FASEB J.
11,
118-124 |
27. |
Bae, G. U.,
Seo, D. W.,
Kwon, H. K.,
Lee, H. Y.,
Hong, S.,
Lee, Z. W.,
Ha, K. S.,
Lee, H. W.,
and Han, J. W.
(1999)
J. Biol. Chem.
274,
32596-32602 |
28. | Barford, D., Das, A. K., and Egloff, M. P. (1998) Annu. Rev. Biophys. Biomol. Struct. 27, 133-164[CrossRef][Medline] [Order article via Infotrieve] |
29. | Barrett, W. C., DeGnore, J. P., Konig, S., Fales, H. M., Keng, Y. F., Zhang, Z. Y., Yim, M. B., and Chock, P. B. (1999) Biochemistry 38, 6699-6705[CrossRef][Medline] [Order article via Infotrieve] |
30. | Cicirelli, M. F., Tonks, N. K., Diltz, C. D., Weiel, J. E., Fischer, E. H., and Krebs, E. G. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 5514-5518[Abstract] |
31. |
Kenner, K. A.,
Anyanwu, E.,
Olefsky, J. M.,
and Kusari, J.
(1996)
J. Biol. Chem.
271,
19810-19816 |
32. |
Venable, C. L.,
Frevert, E. U.,
Kim, Y. B.,
Fischer, B. M.,
Kamatkar, S.,
Neel, B. G.,
and Kahn, B. B.
(2000)
J. Biol. Chem.
275,
18318-18326 |
33. |
Elchebly, M.,
Payette, P.,
Michaliszyn, E.,
Cromlish, W.,
Collins, S.,
Loy, A. L.,
Normandin, D.,
Cheng, A.,
Himms-Hagen, J.,
Chan, C. C.,
Ramachandran, C.,
Gresser, M. J.,
Tremblay, M. L.,
and Kennedy, B. P.
(1999)
Science
283,
1544-1548 |
34. |
Klaman, L. D.,
Boss, O.,
Peroni, O. D.,
Kim, J. K.,
Martino, J. L.,
Zabolotny, J. M.,
Moghal, N.,
Lubkin, M.,
Kim, Y. B.,
Sharpe, A. H.,
Stricker-Krongrad, A.,
Shulman, G. I.,
Neel, B. G.,
and Kahn, B. B.
(2000)
Mol. Cell. Biol.
20,
5479-5489 |
35. | Cheung, A., Kusari, J., Jansen, D., Bandyopadhyay, D., Kusari, A., and Bryer-Ash, M. (1999) J. Lab. Clin. Med. 134, 115-123[Medline] [Order article via Infotrieve] |
36. | Kennedy, B. P., and Ramachandran, C. (2000) Biochem. Pharmacol. 60, 877-883[CrossRef][Medline] [Order article via Infotrieve] |
37. | Malamas, M. S., Sredy, J., Gunawan, I., Mihan, B., Sawicki, D. R., Seestaller, L., Sullivan, D., and Flam, B. R. (2000) J. Med. Chem. 43, 995-1010[CrossRef][Medline] [Order article via Infotrieve] |
38. |
Czech, M. P.,
Lawrence, J. C. J.,
and Lynn, W. S.
(1974)
J. Biol. Chem.
249,
1001-1006 |
39. |
Krieger-Brauer, H. I.,
Medda, P. K.,
and Kather, H.
(1997)
J. Biol. Chem.
272,
10135-10143 |
40. | Meyerovitch, J., Backer, J. M., Csermely, P., Shoelson, S. E., and Kahn, C. R. (1992) Biochemistry 31, 10338-10344[Medline] [Order article via Infotrieve] |
41. | Zhu, L., Zilbering, A., Wu, X., Joseph, J. I., Jabbour, S., Deeb, W., and Goldstein, B. J. (2001) FASEB J., in press |