BIOCHEMICAL
Vol. 188, No. 2, 1992 October
AND BIOPHYSICAL
30, 1992
RESEARCH COMMUNICATIONS Pages 77X-779
SELECTIVE INHIBITION OF PROTEIN TYROSINE PHOSPHATASE ACTIVITIES BY Hz02 AND VANADATE IN .V/TRO Dalit
Department
Received
September
Hecht and Yehiel Zick#
of Chemical Immunology, the Weizmann Institute of Science, Rehovot 76100, Israel 9,
1992
Summary: Acute (lo-30 min) treatment of intact rat hepatoma (Fao) cells with H202, inhibits in vivo protein tyrosine phosphatase activity. Vanadate markedly potentiates this effect although it has only trivial effects of its own. Here we show that Hz02 inhibits a protein tyrosine phosphatase activity, but not a p-nitro phenyl phosphate hydrolysing activity, in cytosolic extracts of these cells. This effect is completely reversed by 10 mM dithiothreitol. Other oxidants have similar inhibitory effects. Vanadate inhibits the protein tyrosine phosphatase activity in vitro, and its effects are additive with those of H202. These findings suggest that Hz02 and vanadate interact with the protein tyrosine phosphatases at two independent sites. They also suggest that in intact cells HsOs has a direct inhibitory effect on protein tyrosine phosphatase activity and an indirect effect 0 1992Academic Press,Inc. of facilitating the entry of vanadate.
Acute (lo-30 potentiates tyrosine
min) treatment
protein
tyrosine
phosphatases
(3),
markedly augments
phosphorylation
(PTPs)
inhibited under these conditions
of intact rat hepatoma
(Fao) cells with H202,
in a reversible
manner
localized in the cytosolic fraction of these cells, are (2).
Sodium orthovanadate,
a known inhibitor of PTPs
the effect of H202, although vanadate
trivial inhibitory effects on PTP activity under these conditions
on its own exerts only (2).
A common feature shared by PTPs is their absolute dependence compounds
for activity
(4).
Comparison
of protein sequences
family indicates that two cysteine residues [numbers totally conserved.
(I). Protein
on sulfhydryl
between members of the
121 and 215 in PTPlB
(5)],
are
This suggests that either one or both of these residues plays an
# To whom correspondence should be addressed. The abbreviations used are; PTP, protein tyrosine phosphatase; PAO, phenylarsine oxide; Hepes, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; NBT, nitroblue tetrazolium; PNPP, p-nitro phenyl phosphate. 0006-291X/92 773
$4.00
Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
188,
No.
essential
1992
BIOCHEMICAL
AND
role in the catalytic mechanism.
equivalent) enzyme
2,
both in receptor-like (6,7).
Additional
cysteine-phosphate presumably
BIOPHYSICAL
RESEARCH
Site-directed
mutagenesis
PTPs and in non-receptor
evidence
suggests
intermediate
takes place at Cys2ls
where
that the
COMMUNICATIONS
of Cys2ls (or its
PTPs resulted in an inactive
PTP catalysis
covalent
thiol
proceeds
via a
phosphate
bond
In view of the above we examined the possibility
(8).
that H202 inhibits the PTP activity in intact cells through
a direct oxidation.
We also
studied the combined effects of Hz02 and vanadate on PTP activity in vitro.
MATERIALS
AND
METHODS
Cell cultures- Monolayer cultures of rat hepatoma (Fao) cells were grown in 100 mm Nunk tissue culture dishes at 37OC in a humidified atmosphere composed of 95% air and 5% Con, in RPM1 1640 medium supplemented with 10% fetal calf serum. Extraction of PTP activity from Fao Cells- Cytosolic and particulate extracts of Fao cells were prepared as described in (9). Briefly, the cells were washed with ice-cold phosphate buffer saline and frozen on liquid nitrogen. Solubilization was performed in 600 ul buffer composed of 25 mM imidazole-HCI, 1mM EDTA, 1mM EGTA, 10% glycerol, 0.5 mM digitonin, pH 7.2, and the extracts were centrifuged for 10 min at 4OC at 400xg. The supernatants were collected and re-centrifuged for 15 min at 4OC at 12000xg. The supernatants were defined as cytosolic extracts, and the pellets were suspended in the same buffer and defined as particulate extracts. The two fractions served as sources for PTP activity, immediately after extraction. Assay of PTP activity- The assay was carried out as described in (2). Briefly, 40 ul of extract were mixed with 40 ul reaction mixture containing 10 mg/ml [32P]poly-(Glu,Tyr) 4:1, 2.5 mM ATP and 50 mM Hepes pH 7.4. Reactions were carried out for 8 min at 3OOC and terminated by applying 60 ul aliquots onto Whatman 3 MM filter papers. The papers were extensively washed in 10% (v/v) trichloroacetic acid, rinsed in ethanol, dried and counted by liquid scintillation. The extent of reduction of the trichloroacetic acid-precipitable [aaP]poly(Glu;Tyr) 4:l was taken as a measure of PTP activity. Assay of p-nifrophenyl
phosphate
(PNPP) hydrolyzing
activity30
ul of extract, 150 ul of
50 mM Hepes pH 7.4 and 20 ul of 0.1 M PNPP were incubated at 22OC for 20 min. The reaction was terminated with 50 ul of 2 M NaOH. The concentration of the hydrolysis product, p-nitrophenol, was estimated from the absorbance at 410 nm. materials- [y-s2P]-ATP (3000 Ci/mmol) was from Amersham. Sodium orthovanadate was from BDH. Poole, England. Phenylarsine oxide was from Aldrich. H202, Digitonin, and KMn04 were from Merck. All other materials were from Sigma.
RESULTS To determine
whether
Hz02 directly interacts with intracellular
extracts of Fao cells were preincubated the
PTPs, cytosolic
with Hz02 at 4OC for 30 min. As seen in Fig 1,
PTP activity was inhibited in vitro by H202 in a dose dependent 774
manner.
1mM
Vol.
188,
No.
2,
1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Cytosolic extracts (0.15 mg protein/ml) of Fao cells were incubated for 30 min at 4% with H202 at the indicated concentrations. At the end of the incubation PTP activity was assayed using [ssP] labeled poly-(Glu,Tyr) 4:l as substrate, as described under “Materials and Methods”. Results are the mean+/-SE. values of four independent experiments. Insert: Cytosolic extracts (0.15 mg protein/ml) of Fao cells were incubated for the indicated times at 4% with 0.5mM H202. At the end of incubation PTP activity was assayed using [32P] labeled poly-(Glu,Tyr) 4:l as substrate, as described under “Materials and Methods”. Fia.2. Reversal of H909 effects by DlT. Cytosolic extracts (0.7 mg protein/ml) of Fao cells were incubated with (0) or without (0) 1mM Hz02 for 15 min at 4OC. DTT at the indicated final concentrations was added for additional 15 min. At the end of incubation PTP activity was assayed using [32P] labeled poly-(Glu,Tyr) 4:l as substrate, as described under “Materials and Methods”. The data is of a representative experiments (out of four) done in duplicate.
Hz02 induced 80-90%
inhibition,
with half maximal effect being obtained
This inhibitory effect was time dependent incubation
and maximal inhibition required up to 10 min
at 4OC (Fig 1, insert). Consistent
activity could be completely
inhibited
at 30 uM.
with previous studies (IO), the in vitro PTP
in the presence
of 1mM vanadate
with half
maximal inhibition being obtained at 10 uM (not shown). The HnO@timulated reversed compatible
protein tyrosine
once dithiothreitol with a mechanism
reversible manner.
phosphorylation
(DTT) is added to the medium where
in intact cells is readily (I). These findings
Ha02 inhibits an intracellular
are
PTP activity in a
Indeed, as seen in Fig 2, the inhibition of PTP activity by Hz02 in
vitro was reversed by DTT in a dose dependent achieved at 10 mM DTT.
manner and complete
reversion
was
Vol.
188,
No.
2,
1992
BIOCHEMICAL
The aforementioned
AND
BIOPHYSICAL
RESEARCH
effects of H202 on cultured cells are markedly potentiated
in the presence of vanadate
(2).
To study the combined action of these agents in vitro,
cytosolic extracts were incubated with, or without, a sub-optimal in the presence of increasing concentrations vanadate
COMMUNICATIONS
of vanadate.
concentration
of Hz02
As seen in Fig 3, Hz02 and
inhibited the in vitro PTPs activity in an additive manner as evidenced by the
fact that the binding inhibition curves in the absence or in the presence of Hs02 almost paralleled each other. Alkaline phosphatases, (ii,
utilize p-nitrophenyl
12),
pre-incubation were
combination findings
03
0
(PNPP) as a substrate.
of the PNPP hydrolysing insensitive
to inhibition
of H202 and vanadate
suggest
.’
phosphate
phosphatases
As shown in Fig. 4
of either soluble or particulate Fao extracts with 1mM vanadate
in 60 % inhibition activities
which also function as protein tyrosine
10s
activity. In contrast, by up to 10mM
10-s
104
[vanadate]
(M)
PNPP hydrolysing
H202.
The effect of a
was identical to that of vanadate
that the PNPP hydrolysing
10 -3
activity is associated
0 4
H202
vanadate
resulted
alone. These with enzyme(s)
A
B
C
D
-
+
-
+
-
-
+
+
Fia.3. Additive inhibitorv effects of H909 and Vanadate on PTP activify, Cytosolic extracts (0.15 mg protein/ml) of Fao cell were incubated with the indicated concentrations of vanadate, in the presence (0) or in the absence (0) of Hz02 (final concentration 10-3 M), for 30 min at 4%. At the end of the incubation PTP activity was assayed using [ssP] labeled poly-(Glu,Tyr) 4:l as substrate, as described under “Materials and Methods”. The data are of representative experiments ( out of five ) done in duplicate. Fio.4. Effects of H9Q9 and vanadate on PNPP hvdrolvsina act ivi tv. Cytosolic ( 0.3 mg protein/ml) or particulate ( 0.08 mg protein/ml) extracts of Fao cells, were incubated for 30 min at 4OC with: A- buffer, B- Hz02 (lmM, final concentration), Cvanadate (lmM, final concentration) or D- H202 and vanadate (each at 1mM final
concentration). At the end of the incubation, PNPP hydrolysing activity was assayed as described under experiments.
“Materials
and
Methods”.
776
Results
are the mean+/-SE.
of four
Vol. 188, No. 2, 1992
BIOCHEMICALAND BIOPHYSICALRESEARCHCOMMUNICATIONS . . Table 1: Effects of various oxidants on PTP actrvrty OXIDANT
PTP Activity
(1mM)
(% of Maximum)
NONE
100
f
0
Hz02
40.7
5
1.3
PA0
12.2
5
8.0
16.0
+
12.3
37.7
2
10.9
10.9
+
3.4
KMnO
4
SPERMIDINE NBT
Cytosolic extracts (0.35 mg protein/ml) of Fao cells were incubated with the indicated oxidants for 30 min at 4OC. At the end of the incubation PTP activity was assayed using [a2P] labeled poly-(Glu,Tyr) 4:l as substrate, as described under ‘Materials and Methods”. Results are the mean+/-SE. values of five experiments.
having somewhat assumption activities. fractions
different characteristics
is supported
then those possessing the PTP activity. This
by the difference
in the subcellular
distribution
While the specific activity of PTPs isolated from cytosolic of Fao ceils was roughly the same, about
of the two
or particulate
5 fold higher PNPP hydrolysing
activity was associated with the particulate fraction (not shown). The ability
of other oxidants
to mimic the inhibitory
effects
of Hz02
examined.
As seen in table 1, phenylarsine
spermidine
and KMn04, (all present at 1 mM), inhibited the PTP activity with a potency
even
greater
than
H202.
These
oxide (PAO), nitroblue tetrazolium
was
effects
were
partially
reversed
(NBT),
by DTT or
2,3-dimercaptopropanol (DMP), (not shown). By contrast, the PNPP hydrolysing activity was not inhibited by any of these oxidants (not shown).
DISCUSSION In the present study we have demonstrated from rat hepatoma
that the activity of PTPs extracted
(Fao) cells is reversibly inhibited by Hz02 in vitro . Two lines of
evidence support the assumption that oxidation of sulfhydryl groups could provide a molecular basis for this inhibitory effect. First, we have shown that the inhibition of PTP activity by HZ02 is reversed by the reducing agent DTT (Fig. 2) which is consistent with the ability of DTT to reverse phosphorylation
the stimulatory
effects of Hz02 on protein
tyrosine
in intact cells (1). Second, the inhibitory effect is not specific for H202,
and can be mimicked
by a series of agents that are capable of oxidizing 777
sulfhydryl
Vol.
188,
No.
groups,
2,
1992
BIOCHEMICAL
including
AND
PAO, NBT, spermidine
BIOPHYSICAL
and KMnO+
RESEARCH
COMMUNICATIONS
Our results are in agreement
with other studies where PA0 was shown to inhibit PTP activity in vivo as well as in vitro in rat adipocytes
and to inhibit the activity of the PTP CD45
(13),
PTPs share an homologous PTPlB)
core sequence,
Many of the
(14).
including a cysteine residue (Cys 215 in
This residue was shown to be crucial for the activity of these enzymes
(4).
based on site directed mutagenesis
Hence, oxidation of this sulfhydryl group
(7,15,16).
by H202 and other oxidants, could inhibit the catalytic activity of the PTPs. Although
either
Hz02
effects when added together
or vanadate at suboptimal
These findings suggest that Ha02 sites. Moreover
it appears
whereas the vanadate as those expressing
could fully inhibit the PTPs activity, their concentrations,
and vanadate
were additive
(Fig. 3).
inhibit the PTPs at two independent
that the site sensitive to oxidation
is unique to the PTPs
binding site is also shared by other kinds of phosphatases PNPP hydrolysing
activity. The different subcellular
such
distribution
of
the two activities supports this notion. In a previous studies a combination
of H202 and vanadate
now demonstrate The difference explained
we demonstrated
(2)
that when intact cells are treated with
a synergistic inhibition of PTPs is observed.
that the direct effects of these agents on PTP activity are additive. between
the in vivo and in vitro effects
if we assume that Hz02 enhances
the diffusion
of HaOs/vanadate of oxidized
cells. It appears that in certain cell types, including hepatocytes the rate of accumulation H202, vanadate
We
of intracellular
vanadate
is oxidized to pervanadate
Once inside the cell, vanadate
(18)
(17)
can be
vanadate
into
and Fao cells
(2),
is relatively slow. In the presence of
which could facilitate its entry into cells.
could inhibit PTP activity independently
by HsOs. Thus in intact cells, and in the presence of vanadate,
of the inhibition
Hz02 seems to have a
dual effect on PTP activity, a direct inhibitory effect and an indirect effect of facilitating the entry of oxidized vanadate. known insulinomimetic which exceeds
This could explain why Ha02 and vanadate,
agents,
together
induce
that seen with insulin, vanadate,
explain the marked potentiation
which are
insulin’s bioeffects
with a potency
or H202 alone (is,
20)~
of protein tyrosine
phosphorylation
and
(2, 21, 22)
could in cells
treated with H202 and vanadate.
ACKNOWLEDGMENTS We thank Drs. Ronit Sagi-Eisenberg for helpful discussions and a critical review of the manuscript. We thank Ruth Dror for excellent technical assistance. This work was supported by grants from the Israel Cancer Research Fund, the Israel Cancer Association and the Juvenile Diabetes Foundation International. Y.Z. is the incumbent of the Philip Harris and Gerald Ronson Career Development Chair in Diabetes Research. 778
Vol.
188, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
REFERENCES
1. 2. 3. 4. 5.
Heffetz, D. and Zick, Y. (1989) J Biol Chem 264, 10126-10132. Heffetz, D., Bushkin, I., Dror, R. and Zick, Y. (1990) J Biol Chem 265, 2896-2902. Lau, K. H., Farley, J. R. and Baylink, D. J. (1989) Biochem J 257, 23-36. Fischer, E. H., Charbonneau, H., and Tonks, N. K. (1991) Science 235, 401-406. Charbonneau, H., Tonks, N. K., Kumar, S., Diltz, C. D., Harrylock, M., Cool, D. E., Krebs, E.G., Fischer, E. H. and Walsh, K. A. (1989) Proc. Nat/. Acad. Sci. USA 86, 5252-5256. 6. Streuli, M., Krueger, N. X., Tsai, A.Y.M., and Saito, H. (1989) Proc. Nat/. Acad. Sci. USA 86,
8698-8702.
7. Streuli, M., Krueger, N. X., Thai, T., Tang, M. and Saito, H. (1990) EMBO J. 9, 2399- 2407. 8. Guan, K., and Dixson, J.E. (1991) J. Biol. Chem. 266, 17026-I 7030. 9. Pelech, S. L., Meier, K. E. and Krebs E.G. (1986) Biochemistry 25, 8348-8353. 10. Swarup, G., Cohen, S. and Garbers, D. L. (1982) Biochem Biophys Res Commun 107, 1104-I 109. 11. Shriner, C. L. and Brautigan, D. L. (1984) J Biol Chem 259, 11383-I 1390. 12. Swarup, G., Cohen, S. and Garbers, D. L. (1981) J. Biol. Chem. 256, 8197-8201. 13. Liao, K., Hoffman, Ft. D., and Lane, D. M. (1991) J. biol. Chem. 266, 6544-6553. 14. Garcia-Morales, P., Minami, Y., Luong, E., Klausner, Ft. D., and Samelson, L. E. (1990) Proc. Nat/. Acad. Sci. USA 67, 9255-9259. 15. Guan, K., and Dixson, J.E. (1990) Science 249, 553-556. 16. Guan, K., Broyles, S.S., and Dixson, J.E. (1991) Nature 350, 359. 17. Seglen, P. O., and Gordon P.B. (1981) J. biol. Chem. 256, 7699-7701. 18. Howarth, 0. W., and Hunt J.R. (1979) J.C.S. Dalton 1388-1391. 19. Kadota, S., Fantus, I. G., Deragon, G., Gutda, H. J. and Posner, 8. I. (1987) J Biol Chem
262,
8252-8256.
20. Kadota, S., Fantus, I. G., Deragon, G., Gutda, H. J., Hersh, B. and Posner, B. I. (1987) Biochem biophys res Commun 147, 259-266. 21. Zick, Y. and Sagi, E. R. (1990) Biochemistry 29, 10240-10245. 22. Bushkin, I., Roth, J., Heffetz, D. and Zick, Y. (1991) J Biol Chem 266, 1189011895.
779
Vol. 188, No. 2, 1992 October
AND BIOPHYSICAL
30, 1992
RESEARCH COMMUNICATIONS Pages 77X-779
SELECTIVE INHIBITION OF PROTEIN TYROSINE PHOSPHATASE ACTIVITIES BY Hz02 AND VANADATE IN .V/TRO Dalit
Department
Received
September
Hecht and Yehiel Zick#
of Chemical Immunology, the Weizmann Institute of Science, Rehovot 76100, Israel 9,
1992
Summary: Acute (lo-30 min) treatment of intact rat hepatoma (Fao) cells with H202, inhibits in vivo protein tyrosine phosphatase activity. Vanadate markedly potentiates this effect although it has only trivial effects of its own. Here we show that Hz02 inhibits a protein tyrosine phosphatase activity, but not a p-nitro phenyl phosphate hydrolysing activity, in cytosolic extracts of these cells. This effect is completely reversed by 10 mM dithiothreitol. Other oxidants have similar inhibitory effects. Vanadate inhibits the protein tyrosine phosphatase activity in vitro, and its effects are additive with those of H202. These findings suggest that Hz02 and vanadate interact with the protein tyrosine phosphatases at two independent sites. They also suggest that in intact cells HsOs has a direct inhibitory effect on protein tyrosine phosphatase activity and an indirect effect 0 1992Academic Press,Inc. of facilitating the entry of vanadate.
Acute (lo-30 potentiates tyrosine
min) treatment
protein
tyrosine
phosphatases
(3),
markedly augments
phosphorylation
(PTPs)
inhibited under these conditions
of intact rat hepatoma
(Fao) cells with H202,
in a reversible
manner
localized in the cytosolic fraction of these cells, are (2).
Sodium orthovanadate,
a known inhibitor of PTPs
the effect of H202, although vanadate
trivial inhibitory effects on PTP activity under these conditions
on its own exerts only (2).
A common feature shared by PTPs is their absolute dependence compounds
for activity
(4).
Comparison
of protein sequences
family indicates that two cysteine residues [numbers totally conserved.
(I). Protein
on sulfhydryl
between members of the
121 and 215 in PTPlB
(5)],
are
This suggests that either one or both of these residues plays an
# To whom correspondence should be addressed. The abbreviations used are; PTP, protein tyrosine phosphatase; PAO, phenylarsine oxide; Hepes, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; NBT, nitroblue tetrazolium; PNPP, p-nitro phenyl phosphate. 0006-291X/92 773
$4.00
Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
188,
No.
essential
1992
BIOCHEMICAL
AND
role in the catalytic mechanism.
equivalent) enzyme
2,
both in receptor-like (6,7).
Additional
cysteine-phosphate presumably
BIOPHYSICAL
RESEARCH
Site-directed
mutagenesis
PTPs and in non-receptor
evidence
suggests
intermediate
takes place at Cys2ls
where
that the
COMMUNICATIONS
of Cys2ls (or its
PTPs resulted in an inactive
PTP catalysis
covalent
thiol
proceeds
via a
phosphate
bond
In view of the above we examined the possibility
(8).
that H202 inhibits the PTP activity in intact cells through
a direct oxidation.
We also
studied the combined effects of Hz02 and vanadate on PTP activity in vitro.
MATERIALS
AND
METHODS
Cell cultures- Monolayer cultures of rat hepatoma (Fao) cells were grown in 100 mm Nunk tissue culture dishes at 37OC in a humidified atmosphere composed of 95% air and 5% Con, in RPM1 1640 medium supplemented with 10% fetal calf serum. Extraction of PTP activity from Fao Cells- Cytosolic and particulate extracts of Fao cells were prepared as described in (9). Briefly, the cells were washed with ice-cold phosphate buffer saline and frozen on liquid nitrogen. Solubilization was performed in 600 ul buffer composed of 25 mM imidazole-HCI, 1mM EDTA, 1mM EGTA, 10% glycerol, 0.5 mM digitonin, pH 7.2, and the extracts were centrifuged for 10 min at 4OC at 400xg. The supernatants were collected and re-centrifuged for 15 min at 4OC at 12000xg. The supernatants were defined as cytosolic extracts, and the pellets were suspended in the same buffer and defined as particulate extracts. The two fractions served as sources for PTP activity, immediately after extraction. Assay of PTP activity- The assay was carried out as described in (2). Briefly, 40 ul of extract were mixed with 40 ul reaction mixture containing 10 mg/ml [32P]poly-(Glu,Tyr) 4:1, 2.5 mM ATP and 50 mM Hepes pH 7.4. Reactions were carried out for 8 min at 3OOC and terminated by applying 60 ul aliquots onto Whatman 3 MM filter papers. The papers were extensively washed in 10% (v/v) trichloroacetic acid, rinsed in ethanol, dried and counted by liquid scintillation. The extent of reduction of the trichloroacetic acid-precipitable [aaP]poly(Glu;Tyr) 4:l was taken as a measure of PTP activity. Assay of p-nifrophenyl
phosphate
(PNPP) hydrolyzing
activity30
ul of extract, 150 ul of
50 mM Hepes pH 7.4 and 20 ul of 0.1 M PNPP were incubated at 22OC for 20 min. The reaction was terminated with 50 ul of 2 M NaOH. The concentration of the hydrolysis product, p-nitrophenol, was estimated from the absorbance at 410 nm. materials- [y-s2P]-ATP (3000 Ci/mmol) was from Amersham. Sodium orthovanadate was from BDH. Poole, England. Phenylarsine oxide was from Aldrich. H202, Digitonin, and KMn04 were from Merck. All other materials were from Sigma.
RESULTS To determine
whether
Hz02 directly interacts with intracellular
extracts of Fao cells were preincubated the
PTPs, cytosolic
with Hz02 at 4OC for 30 min. As seen in Fig 1,
PTP activity was inhibited in vitro by H202 in a dose dependent 774
manner.
1mM
Vol.
188,
No.
2,
1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Cytosolic extracts (0.15 mg protein/ml) of Fao cells were incubated for 30 min at 4% with H202 at the indicated concentrations. At the end of the incubation PTP activity was assayed using [ssP] labeled poly-(Glu,Tyr) 4:l as substrate, as described under “Materials and Methods”. Results are the mean+/-SE. values of four independent experiments. Insert: Cytosolic extracts (0.15 mg protein/ml) of Fao cells were incubated for the indicated times at 4% with 0.5mM H202. At the end of incubation PTP activity was assayed using [32P] labeled poly-(Glu,Tyr) 4:l as substrate, as described under “Materials and Methods”. Fia.2. Reversal of H909 effects by DlT. Cytosolic extracts (0.7 mg protein/ml) of Fao cells were incubated with (0) or without (0) 1mM Hz02 for 15 min at 4OC. DTT at the indicated final concentrations was added for additional 15 min. At the end of incubation PTP activity was assayed using [32P] labeled poly-(Glu,Tyr) 4:l as substrate, as described under “Materials and Methods”. The data is of a representative experiments (out of four) done in duplicate.
Hz02 induced 80-90%
inhibition,
with half maximal effect being obtained
This inhibitory effect was time dependent incubation
and maximal inhibition required up to 10 min
at 4OC (Fig 1, insert). Consistent
activity could be completely
inhibited
at 30 uM.
with previous studies (IO), the in vitro PTP
in the presence
of 1mM vanadate
with half
maximal inhibition being obtained at 10 uM (not shown). The HnO@timulated reversed compatible
protein tyrosine
once dithiothreitol with a mechanism
reversible manner.
phosphorylation
(DTT) is added to the medium where
in intact cells is readily (I). These findings
Ha02 inhibits an intracellular
are
PTP activity in a
Indeed, as seen in Fig 2, the inhibition of PTP activity by Hz02 in
vitro was reversed by DTT in a dose dependent achieved at 10 mM DTT.
manner and complete
reversion
was
Vol.
188,
No.
2,
1992
BIOCHEMICAL
The aforementioned
AND
BIOPHYSICAL
RESEARCH
effects of H202 on cultured cells are markedly potentiated
in the presence of vanadate
(2).
To study the combined action of these agents in vitro,
cytosolic extracts were incubated with, or without, a sub-optimal in the presence of increasing concentrations vanadate
COMMUNICATIONS
of vanadate.
concentration
of Hz02
As seen in Fig 3, Hz02 and
inhibited the in vitro PTPs activity in an additive manner as evidenced by the
fact that the binding inhibition curves in the absence or in the presence of Hs02 almost paralleled each other. Alkaline phosphatases, (ii,
utilize p-nitrophenyl
12),
pre-incubation were
combination findings
03
0
(PNPP) as a substrate.
of the PNPP hydrolysing insensitive
to inhibition
of H202 and vanadate
suggest
.’
phosphate
phosphatases
As shown in Fig. 4
of either soluble or particulate Fao extracts with 1mM vanadate
in 60 % inhibition activities
which also function as protein tyrosine
10s
activity. In contrast, by up to 10mM
10-s
104
[vanadate]
(M)
PNPP hydrolysing
H202.
The effect of a
was identical to that of vanadate
that the PNPP hydrolysing
10 -3
activity is associated
0 4
H202
vanadate
resulted
alone. These with enzyme(s)
A
B
C
D
-
+
-
+
-
-
+
+
Fia.3. Additive inhibitorv effects of H909 and Vanadate on PTP activify, Cytosolic extracts (0.15 mg protein/ml) of Fao cell were incubated with the indicated concentrations of vanadate, in the presence (0) or in the absence (0) of Hz02 (final concentration 10-3 M), for 30 min at 4%. At the end of the incubation PTP activity was assayed using [ssP] labeled poly-(Glu,Tyr) 4:l as substrate, as described under “Materials and Methods”. The data are of representative experiments ( out of five ) done in duplicate. Fio.4. Effects of H9Q9 and vanadate on PNPP hvdrolvsina act ivi tv. Cytosolic ( 0.3 mg protein/ml) or particulate ( 0.08 mg protein/ml) extracts of Fao cells, were incubated for 30 min at 4OC with: A- buffer, B- Hz02 (lmM, final concentration), Cvanadate (lmM, final concentration) or D- H202 and vanadate (each at 1mM final
concentration). At the end of the incubation, PNPP hydrolysing activity was assayed as described under experiments.
“Materials
and
Methods”.
776
Results
are the mean+/-SE.
of four
Vol. 188, No. 2, 1992
BIOCHEMICALAND BIOPHYSICALRESEARCHCOMMUNICATIONS . . Table 1: Effects of various oxidants on PTP actrvrty OXIDANT
PTP Activity
(1mM)
(% of Maximum)
NONE
100
f
0
Hz02
40.7
5
1.3
PA0
12.2
5
8.0
16.0
+
12.3
37.7
2
10.9
10.9
+
3.4
KMnO
4
SPERMIDINE NBT
Cytosolic extracts (0.35 mg protein/ml) of Fao cells were incubated with the indicated oxidants for 30 min at 4OC. At the end of the incubation PTP activity was assayed using [a2P] labeled poly-(Glu,Tyr) 4:l as substrate, as described under ‘Materials and Methods”. Results are the mean+/-SE. values of five experiments.
having somewhat assumption activities. fractions
different characteristics
is supported
then those possessing the PTP activity. This
by the difference
in the subcellular
distribution
While the specific activity of PTPs isolated from cytosolic of Fao ceils was roughly the same, about
of the two
or particulate
5 fold higher PNPP hydrolysing
activity was associated with the particulate fraction (not shown). The ability
of other oxidants
to mimic the inhibitory
effects
of Hz02
examined.
As seen in table 1, phenylarsine
spermidine
and KMn04, (all present at 1 mM), inhibited the PTP activity with a potency
even
greater
than
H202.
These
oxide (PAO), nitroblue tetrazolium
was
effects
were
partially
reversed
(NBT),
by DTT or
2,3-dimercaptopropanol (DMP), (not shown). By contrast, the PNPP hydrolysing activity was not inhibited by any of these oxidants (not shown).
DISCUSSION In the present study we have demonstrated from rat hepatoma
that the activity of PTPs extracted
(Fao) cells is reversibly inhibited by Hz02 in vitro . Two lines of
evidence support the assumption that oxidation of sulfhydryl groups could provide a molecular basis for this inhibitory effect. First, we have shown that the inhibition of PTP activity by HZ02 is reversed by the reducing agent DTT (Fig. 2) which is consistent with the ability of DTT to reverse phosphorylation
the stimulatory
effects of Hz02 on protein
tyrosine
in intact cells (1). Second, the inhibitory effect is not specific for H202,
and can be mimicked
by a series of agents that are capable of oxidizing 777
sulfhydryl
Vol.
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No.
groups,
2,
1992
BIOCHEMICAL
including
AND
PAO, NBT, spermidine
BIOPHYSICAL
and KMnO+
RESEARCH
COMMUNICATIONS
Our results are in agreement
with other studies where PA0 was shown to inhibit PTP activity in vivo as well as in vitro in rat adipocytes
and to inhibit the activity of the PTP CD45
(13),
PTPs share an homologous PTPlB)
core sequence,
Many of the
(14).
including a cysteine residue (Cys 215 in
This residue was shown to be crucial for the activity of these enzymes
(4).
based on site directed mutagenesis
Hence, oxidation of this sulfhydryl group
(7,15,16).
by H202 and other oxidants, could inhibit the catalytic activity of the PTPs. Although
either
Hz02
effects when added together
or vanadate at suboptimal
These findings suggest that Ha02 sites. Moreover
it appears
whereas the vanadate as those expressing
could fully inhibit the PTPs activity, their concentrations,
and vanadate
were additive
(Fig. 3).
inhibit the PTPs at two independent
that the site sensitive to oxidation
is unique to the PTPs
binding site is also shared by other kinds of phosphatases PNPP hydrolysing
activity. The different subcellular
such
distribution
of
the two activities supports this notion. In a previous studies a combination
of H202 and vanadate
now demonstrate The difference explained
we demonstrated
(2)
that when intact cells are treated with
a synergistic inhibition of PTPs is observed.
that the direct effects of these agents on PTP activity are additive. between
the in vivo and in vitro effects
if we assume that Hz02 enhances
the diffusion
of HaOs/vanadate of oxidized
cells. It appears that in certain cell types, including hepatocytes the rate of accumulation H202, vanadate
We
of intracellular
vanadate
is oxidized to pervanadate
Once inside the cell, vanadate
(18)
(17)
can be
vanadate
into
and Fao cells
(2),
is relatively slow. In the presence of
which could facilitate its entry into cells.
could inhibit PTP activity independently
by HsOs. Thus in intact cells, and in the presence of vanadate,
of the inhibition
Hz02 seems to have a
dual effect on PTP activity, a direct inhibitory effect and an indirect effect of facilitating the entry of oxidized vanadate. known insulinomimetic which exceeds
This could explain why Ha02 and vanadate,
agents,
together
induce
that seen with insulin, vanadate,
explain the marked potentiation
which are
insulin’s bioeffects
with a potency
or H202 alone (is,
20)~
of protein tyrosine
phosphorylation
and
(2, 21, 22)
could in cells
treated with H202 and vanadate.
ACKNOWLEDGMENTS We thank Drs. Ronit Sagi-Eisenberg for helpful discussions and a critical review of the manuscript. We thank Ruth Dror for excellent technical assistance. This work was supported by grants from the Israel Cancer Research Fund, the Israel Cancer Association and the Juvenile Diabetes Foundation International. Y.Z. is the incumbent of the Philip Harris and Gerald Ronson Career Development Chair in Diabetes Research. 778
Vol.
188, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
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