- Email: [email protected]
Effect of nitration on protein tyrosine phosphatase and protein phosphatase activity in neuronal cell membranes of newborn piglets
Neuroscience Letters 386 (2005) 78–81
Effect of nitration on protein tyrosine phosphatase and protein phosphatase activity in neuronal cell membranes of newborn piglets Carlos J. Lopez a,b,∗ , Imran Qayyum a,b , Om P. Mishra a,b , Maria Delivoria-Papadopoulos a,b a
Department of Pediatrics, Drexel University College of Medicine, Philadelphia, PA, USA b St. Christopher’s Hospital for Children, 3300 Henry Avenue, Philadelphia, PA, USA Received 18 April 2005; received in revised form 28 April 2005; accepted 30 April 2005
Abstract Protein tyrosine phosphatase predominantly determines the status of protein tyrosine kinase-dependent phosphorylation of specific proteins and controls the survival and death of neurons. Previous studies have shown that protein tyrosine phosphatase activity is decreased during hypoxia in cortical membranes of the newborn piglet. We have also shown that nitric oxide (NO) free radicals are generated during hypoxia, and may result in modification of protein tyrosine phosphatase via peroxynitrite-mediated modification. The present study tests the hypothesis that the hypoxia-induced decrease in protein tyrosine phosphatase activity is NO-mediated. To test this hypothesis, in vitro experiments were conducted by measuring protein tyrosine phosphatase activity in the presence of an NO donor, sodium nitroprusside (SNP), or peroxynitrite. Since 3-nitrotyrosine is produced as a consequence of peroxynitrite reactions, we have also examined the effect of 3-nitrotyrosine on protein phophatase activity. Cerebral cortical P2 membranes were prepared from seven normoxic newborn piglets and each sample was divided into three aliquots: a control group, a SNP group (exposed to 200 M SNP), and a peroxynitrite group (exposed to 100 M peroxynitrite). Protein tyrosine phosphatase activity was determined spectrophotometrically in the presence or absence of 2 M bpV(phen), a highly selective inhibitor of protein tyrosine phosphatase. The protein tyrosine phosphatase activity was 198 ± 25 nmol/mg protein/h in the normoxic group, 177 ± 30 nmol/mg protein/h in the SNP group (p = NS versus normoxic) and 77 ± 20 nmol/mg protein/h in the peroxynitrite group (p < 0.001 versus normoxic). The results show that peroxynitrite but not SNP exposure results in decreased protein tyrosine phosphatase activity in vitro. Furthermore 3-nitrotyrosine (100 m), a product of peroxynitrite, decreased the enzyme activity from 926 ± 102 to 200 ± 77 (p < 0.001). We conclude that protein tyrosine phosphatase regulation is mediated by peroxynitrite. We propose that hypoxia-induced NO production leading to peroxynitrite formation is a potential mechanism of protein tyrosine phosphatase inactivation in vivo. The NO-induced decrease in protein tyrosine phosphatase and protein phosphatase activity, leading to Bcl-2 protein phosphorylation and loss of its antiapoptotic activity may be a NO-mediated mechanism of programmed cell death in the hypoxic brain. © 2005 Published by Elsevier Ireland Ltd. Keywords: Nitric oxide: protein tyrosine phosphatase; Peroxynitrite; Brain
Phosphorylation of proteins on tyrosine residues is one of the most important regulatory cell signaling mechanisms, controlling cell proliferation, differentiation, and migration [6]. Cellular phosphotyrosine levels are regulated by the antagonistic activities of two classes of enzymes, the protein tyrosine ∗ Corresponding author at: Pediatrics/Neonatal Research, 7th Floor Heritage Building, Room 701, Drexel University College of Medicine, 3300 Henry Avenue, Philadelphia, PA 19129, USA. Tel.: +1 215 842 4960; fax: +1 215 843 3505. E-mail address: [email protected] (C.J. Lopez).
0304-3940/$ – see front matter © 2005 Published by Elsevier Ireland Ltd. doi:10.1016/j.neulet.2005.04.089
kinases (PTKs) and the protein tyrosine phosphatases (PTPs). Although much work has focused on activation of protein tyrosine kinases, the ultimate physiological and pathological effects of phosphotyrosine-mediated signaling are closely related to the duration of the phosphorylated state, which is highly regulated by specific tyrosine phosphatases [17]. PTPs are regulated through multiple mechanisms. Reversible oxidation of the active site cysteine residue has recently been identified as a general mechanism for regulation of PTPs [13]. PTP activity is also regulated through subcellular translocations such as the calpain-mediated release of PTP-1B from
C.J. Lopez et al. / Neuroscience Letters 386 (2005) 78–81
the endoplasmatic reticulum, and the SH2-domain-mediated recruitment of SHP-1 and SHP-2 to tyrosine-phosphorylated proteins [9]. Nitric oxide and nitrogen oxides formed by the slow reaction of nitric oxide with oxygen have been suggested to modulate thiol-dependent enzymes through the formation of S-nitrosothiols and subsequent oxidation to give disulfides. The highly reactive thiolate group in tyrosine phosphatases could also be a potential target for transnitrosation by Snitrosothiols [15]. The chemical reactivity of nitric oxide is greatly increased by its diffusion-limited reaction with superoxide (O2 − ) to form peroxynitrite (ONOO− ). Peroxynitrite nitrates tyrosine residues in proteins to form 3-nitrotyrosine, which can potentially interfere with subsequent phosphorylation of the affected tyrosine [11]. In addition, peroxynitrite readily oxidizes both free and protein-bound sulfhydryls and induces oxidative inactivation of other sulfhydryl-containing enzymes such as alcohol dehydrogenase, creatine kinase and succinate dehydrogenase. Consequently, peroxynitrite could potentially inactivate tyrosine phosphatases by either oxidizing the thiolate or nitrating the tyrosine residue in the active site [14]. The formation of 3-nitrotyrosine is considered a marker of in vivo production of peroxynitrite but should more correctly be considered a marker of NO related oxidants, since different mechanisms may lead to tyrosine nitration [7]. One of the oxidative reactions induced by peroxynitrite is the modification of tyrosine residues in proteins to yield 3-nitrotyrosine, and it has been suggested that this modification impairs phosphotyrosine signaling. However, several in vitro studies have shown that peroxynitrite may promote rather than inhibit tyrosine phosphorylation [7]. Previous studies from our laboratory have shown that PTP activity decreases during hypoxia potentially contributing to phosphorylation of a number of proteins including anti-apoptotic protein Bcl-2 [1,2]. We have also shown that hypoxia results in nitration of N-methyl-d-aspartate (NMDA) receptor subunits and altered receptor function [19]. The present study tests the hypothesis that in vitro exposure to sodium nitroprusside (SNP), will decrease protein tyrosine phosphatase enzyme activity in cortical cell membranes of newborn piglets and that the decrease in protein tyrosine phosphatase activity is NO-mediated. To test this hypothesis, in vitro experiments were conducted by measuring protein tyrosine phosphatase activity in the presence of an NO donor, sodium nitroprusside (SNP), or peroxynitrite. In addition, since 3-nitrotyrosine is produced in vivo, a marker of peroxynitrite formation in tissues, we have determined the effect of 3-nitrotyrosine on protein phosphatase activity, a mechanism that regulates protein phosphorylation/dephosphorylation. Studies were performed on 2- to 4-day-old Yorkshire piglets as per standard protocol as described by Ashraf et al. [1]. The study was approved by the Institutional Animal Care and Use Committee of the Drexel University College of Medicine. Cerebral cortical P2 membranes [5], which have high levels of PTP activity [1] were prepared from seven nor-
79
moxic newborn piglets and protein was determined by the method of Lowry et al. [8] and each sample was divided into three aliquots: a control group, a SNP group (exposed to 200 M SNP), and a peroxynitrite group (exposed to 100 M peroxynitrite). Protein tyrosine phosphatase activity was determined in a medium containing 100 mM HEPES (pH 7.0), 1 mM EDTA, 0.05% Tween 20 and 1.25 mM pnitrophenylphosphate (pNPP) at 37 ◦ C for 20 min in the presence or absence of 2 M bpV(phen), a highly selective inhibitor of protein tyrosine phosphatase. The enzyme activity was determined spectrophotometrically at 405 nm and the bpV(phen)-sensitive activity was expressed as nmol/mg protein/h. Negative controls were also performed. In addition, effect of 3-nitrotyrosine (100 M) on protein phosphatase activity was determined in the above assay system with 1 mM sodium orthovandate. Protein nitration was confirmed by immunoblotting [1] with anti-nitrotyrosine antibody. The data are presented as mean ± standard deviation and analyzed by one-way analysis of variance. A p-value <0.05 was considered significant. The protein tyrosine phosphatase activity (Fig. 1) was 198 ± 25 nmol/mg protein/h in the normoxic group, 177 ± 30 nmol/mg protein/h in the SNP group (p = NS versus normoxic) and 77 ± 20 nmol/mg protein/h in the peroxynitrite group (p < 0.001 versus normoxic). In the presence of 3nitrotyrosine protein phosphatase activity (Fig. 2) decreased from 926 ± 102 to 200 ± 77 (p < 0.001). PTPs can regulate the phosphorylation status of apoptotic proteins. The phosphorylation state also determines the antiapoptotic as well as pro-apoptotic potential of these proteins. These proteins belong to the Bcl-2 multigene family and regulate cell death and survival. The Bcl-2 family consists of both cell death promoter (Bax) and cell death repressor (Bcl-2) proteins. Overexpression of Bax is known to promote programmed cell death and overexpression of Bcl-2 leads to cell survival [18]. We have shown that hypoxia results in increased tyrosine phosphorylation and serine phosphorylation of Bcl-2. Unphosphorylated Bcl-2 is anti-apoptotic, but
Fig. 1. The activity of protein tyrosine phosphatase in the control, sodium nitropruside (SNP) treated and peroxynitrite treated cortical cell membranes of normoxic newborn piglet. The data are expressed as mean ± S.D.
80
C.J. Lopez et al. / Neuroscience Letters 386 (2005) 78–81
Fig. 2. The activity of protein phosphatase in the control and 3-nitrotyrosine treated cortical cell membranes of normoxic newborn piglets. The data are expressed as mean ± S.D.
phosphorylated Bcl-2 is pro-apoptotic [2,18]. In a separate study, we have observed that cerebral hypoxia resulted in increased tyrosine phosphorylation of a number of neuronal proteins whereas a number of proteins were dephosphorylated and some of the proteins were not altered in terms of phosphorylation/dephosphorylation [4]. These results indicate that the tyrosine phosphorylation/dephosphorylation equilibrium during hypoxia may further be determined by the protein substrate itself and its specificity to phosphatases. There is a possibility that the translocation of PTP from membrane into cytosol occurs during hypoxia and this translocation of PTP is NO-mediated. Peroxynitrite readily nitrates tyrosine residues in proteins, and peptides containing nitrotyrosine are resistant to phosphorylation in vitro [9], suggesting that peroxynitrite could alter phosphotyrosine-mediated signaling. Peroxynitrite has been shown to affect signal transduction in cultured cells and to induce apoptosis [3,16]. However, tyrosine phosphorylation of proteins is typically increased rather than inhibited when cells are treated with peroxynitrite [12]. Peroxynitrite shows in vitro characteristics of a signaling molecule able to upregulate tyrosine-dependent and -independent phosphorylations. This effect could be related to the inhibition of phosphatases and/or to the activation of kinases. Whatever the activation mechanism, it is interesting to stress that direct or indirect effects of peroxynitrite are focused toward the upregulation of tyrosine-dependent signaling. Tyrosine nitration is a protein modification characteristic of peroxynitrite. Results of the present study indicate that peroxynitrite and 3-nitrotyrosine inhibit protein phosphatases indicating that tyrosine nitration is a potential mechanism of regulation of protein phosphorylation/dephosphorylation. Since 3-nitrotyrosine, is produced during hypoxia, the present in vitro experiments demonstrate that 3-nitrotyrosine, a product of peroxynitrite-dependent nitration by itself can lead to inhibition of protein tyrosine phosphatase in vivo. The data show that peroxynitrite but not SNP exposure results in decreased protein tyrosine phosphatase activity in vitro, indicating that the mechanism of PTP inactivation is peroxynitrite-mediated.
In previous studies, we have observed that hypoxia resulted in increased expression of PTP and other proteins such as the pro-apoptotic proteins Bax and Bad [20]. Expression of anti-apoptotic proteins Bcl-2 and Bcl-xl was not increased. These studies also demonstrated that the hypoxiainduced increased expression of the pro-apoptotic proteins can be blocked by the administration of NOS inhibitors [20]. In a series of studies we have observed that cerebral hypoxia resulted in increased neuronal nuclear Ca2+ -influx, increased calcium calmodulin-dependent protein kinase IV (CaM kinase IV) activity, increased phosphorylation of cyclic AMP response element binding (CREB) protein and increased expression of pro-apoptotic protein Bax [10,21]. Each of these steps were also blocked by the administration of NOS inhibitors during hypoxia. Therefore, hypoxia-induced increased expression and decreased activity of PTPs are due to a mechanism that is mediated by NO generated during hypoxia. In summary, we have shown that exposure to peroxynitrite decreased protein tyrosine phosphatase enzyme activity in cortical cell membranes of newborn piglets. However, the exposure to SNP did not decrease the enzyme activity. We conclude that protein tyrosine phosphatase inactivation is mediated by peroxynitrite, a product of NO. We speculate that hypoxia-induced NO production leading to peroxynitrite formation is a potential mechanism of protein tyrosine phosphatase inhibition. The NO-induced decrease in protein tyrosine phosphatase and protein phosphatase activity leading to Bcl-2 protein phosphorylation and loss of its antiapoptotic activity is a proposed NO-mediated mechanism of programmed cell death in the hypoxic brain.
Acknowledgements The authors thank Mrs. Joanna Kubin and Mrs. Anli Zhu for their technical assistance. This research was supported by grants from the National Institutes of Health, NIH HD-20337 and NIH HD-38079.
References [1] Q.M. Ashraf, S.H. Haider, C.D. Katsetos, M. DelivoriaPapadopoulos, O. Mishra, Nitric oxide-mediated alterations of protein tyrosine phosphatase activity and expression during hypoxia in the cerebral cortex of newborn piglets, Neurosci. Lett. 362 (2004) 108–112. [2] Q.M. Ashraf, S.A. Zanelli, O.P. Mishra, M. Delivoria-Papadopoulos, Phosphorylation of Bcl-2 and Bax proteins during hypoxia in newborn piglets, Neurochem. Res. 26 (2001) 1–9. [3] C. Brito, M. Naviliat, A.C. Tiscornia, F. Vuillier, G. Gualco, G. Dighiero, R. Radi, A.M. Cayota, Peroxynitrite inhibits T lymphocyte activation and proliferation by promoting impairment of tyrosine phosphorylation and peroxynitrite-driven apoptotic death, J. Immunol. 162 (1999) 3356–3366. [4] G. Buonocore, S. Liberatori, L. Bini, O.P. Mishra, M. DelivoriaPapadopoulos, V. Pallini, R. Bracci, Hypoxic response of
C.J. Lopez et al. / Neuroscience Letters 386 (2005) 78–81
[5]
[6] [7]
[8]
[9]
[10]
[11]
[12]
[13]
synaptosomal proteins in term guinea pig fetuses, J. Neurochem. 73 (1999) 2139–2148. S.I. Harik, G.H. Doull, A.P. Dick, Specific ouabain binding to brain microvessels and choroid plexus, J. Cereb. Blood Flow Metab. 5 (1985) 156–160. J. den Hertog, A. Groen, T. vander Wijk, Redox regulation of proteintyrosine phosphatases, Arch. Biochem. Biophys. 434 (2005) 11–15. H. Ischiropoulos, Biological tyrosine nitration: a pathophysiological function of nitric oxide and reactive oxygen species, Arch. Biochem. Biophys. 356 (1998) 1–11. O. Lowry, N.J. Rosenbrough, A.L. Farr, R.J. Randall, Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193 (1951) 265–275. B.L. Martin, D. Wu, S. Jakes, D.J. Graves, Chemical influences on the specificity of tyrosine phosphorylation, J. Biol. Chem. 265 (1990) 7108–7111. O.P. Mishra, Q.M. Ashraf, M. Delivoria-Papadopoulos, Phosphorylation of cAMP response element binding (CREB) protein during hypoxia in cerebral cortex of newborn piglets and the effect of nitric oxide synthase inhibition, Neuroscience 115 (2002) 985–991. O.P. Mishra, D. Maulik, Q.M. Ashraf, M. Delivoria-Papadopoulos, Nitration of N-methyl-d-aspartate receptor subunits following in vitro dephosphorylation of cerebral cortical membranes of newborn piglets, Neurosci. Lett. 317 (2002) 115–118. T.H. Mondoro, B.C. Shafer, J.G. Vostal, Peroxynitrite-induced tyrosine nitration and phosphorylation in human platelets, Free Radic. Biol. Med. 22 (1997) 1055–1063. A.D.B. Pannifer, A.J. Flint, N.K. Tonks, D. Barford, Visualization of the cysteinyl-phosphate intermediate of a protein-tyrosine
[14]
[15]
[16]
[17] [18]
[19]
[20]
[21]
81
phosphatase by X-ray crystallography, J. Biol. Chem. 273 (1998) 10454–10462. R. Radi, J.S. Beckman, K.M. Bush, B.A. Freeman, Peroxynitrite oxidation of sulfhydryls. The cytotoxic potential of superoxide and nitric oxide, J. Biol. Chem. 266 (1991) 4244–4250. R.J. Singh, N. Hogg, J. Joseph, B. Kalyanaraman, Mechanism of nitric oxide release from S-nitrosothiols, J. Biol. Chem. 271 (1996) 18596–18603. N. Spear, A.G. Estevez, L. Barbeito, J.S. Beckman, G.V. Johnson, Nerve growth factor protects PC12 cells against peroxynitriteinduced apoptosis via a mechanism dependent on phosphatidylinositol 3-kinase, J. Neurochem. 69 (1997) 53–59. K.M. Walton, J.E. Dixon, Protein tyrosine phosphatases, Annu. Rev. Biochem. 62 (1993) 101–120. H. Wei, W. Wei, D.E. Bredesen, D.C. Perry, Bcl-2 protects against apoptosis in neuronal cell line caused by thapsigargin-induced depletion of intracellular calcium stores, J. Neurochem. 70 (1998) 2305–2314. S.A. Zanelli, Q.M. Ashraf, O.P. Mishra, Nitration is a mechanism of regulation of the NMDA receptor function during hypoxia, Neuroscience 112 (2002) 869–877. A.B. Zubrow, M. Delivoria-Papadopoulos, Q.M. Ashraf, J.R. Ballesteros, K.I. Fritz, O.P. Mishra, Nitric oxide-mediated expression of Bax protein and DNA fragmentation during hypoxia in neuronal nuclei from newborn piglets, Brain Res. 954 (2002) 60–70. A.B. Zubrow, M. Delivoria-Papadopoulos, Q.M. Ashraf, K.I. Fritz, O.P. Mishra, Nitric oxide-mediated Ca2+ /calmodulin-dependent protein kinase IV activity during hypoxia in neuronal nuclei from newborn piglets, Neurosci. Lett. 335 (2002) 5–8.
Effect of nitration on protein tyrosine phosphatase and protein phosphatase activity in neuronal cell membranes of newborn piglets Carlos J. Lopez a,b,∗ , Imran Qayyum a,b , Om P. Mishra a,b , Maria Delivoria-Papadopoulos a,b a
Department of Pediatrics, Drexel University College of Medicine, Philadelphia, PA, USA b St. Christopher’s Hospital for Children, 3300 Henry Avenue, Philadelphia, PA, USA Received 18 April 2005; received in revised form 28 April 2005; accepted 30 April 2005
Abstract Protein tyrosine phosphatase predominantly determines the status of protein tyrosine kinase-dependent phosphorylation of specific proteins and controls the survival and death of neurons. Previous studies have shown that protein tyrosine phosphatase activity is decreased during hypoxia in cortical membranes of the newborn piglet. We have also shown that nitric oxide (NO) free radicals are generated during hypoxia, and may result in modification of protein tyrosine phosphatase via peroxynitrite-mediated modification. The present study tests the hypothesis that the hypoxia-induced decrease in protein tyrosine phosphatase activity is NO-mediated. To test this hypothesis, in vitro experiments were conducted by measuring protein tyrosine phosphatase activity in the presence of an NO donor, sodium nitroprusside (SNP), or peroxynitrite. Since 3-nitrotyrosine is produced as a consequence of peroxynitrite reactions, we have also examined the effect of 3-nitrotyrosine on protein phophatase activity. Cerebral cortical P2 membranes were prepared from seven normoxic newborn piglets and each sample was divided into three aliquots: a control group, a SNP group (exposed to 200 M SNP), and a peroxynitrite group (exposed to 100 M peroxynitrite). Protein tyrosine phosphatase activity was determined spectrophotometrically in the presence or absence of 2 M bpV(phen), a highly selective inhibitor of protein tyrosine phosphatase. The protein tyrosine phosphatase activity was 198 ± 25 nmol/mg protein/h in the normoxic group, 177 ± 30 nmol/mg protein/h in the SNP group (p = NS versus normoxic) and 77 ± 20 nmol/mg protein/h in the peroxynitrite group (p < 0.001 versus normoxic). The results show that peroxynitrite but not SNP exposure results in decreased protein tyrosine phosphatase activity in vitro. Furthermore 3-nitrotyrosine (100 m), a product of peroxynitrite, decreased the enzyme activity from 926 ± 102 to 200 ± 77 (p < 0.001). We conclude that protein tyrosine phosphatase regulation is mediated by peroxynitrite. We propose that hypoxia-induced NO production leading to peroxynitrite formation is a potential mechanism of protein tyrosine phosphatase inactivation in vivo. The NO-induced decrease in protein tyrosine phosphatase and protein phosphatase activity, leading to Bcl-2 protein phosphorylation and loss of its antiapoptotic activity may be a NO-mediated mechanism of programmed cell death in the hypoxic brain. © 2005 Published by Elsevier Ireland Ltd. Keywords: Nitric oxide: protein tyrosine phosphatase; Peroxynitrite; Brain
Phosphorylation of proteins on tyrosine residues is one of the most important regulatory cell signaling mechanisms, controlling cell proliferation, differentiation, and migration [6]. Cellular phosphotyrosine levels are regulated by the antagonistic activities of two classes of enzymes, the protein tyrosine ∗ Corresponding author at: Pediatrics/Neonatal Research, 7th Floor Heritage Building, Room 701, Drexel University College of Medicine, 3300 Henry Avenue, Philadelphia, PA 19129, USA. Tel.: +1 215 842 4960; fax: +1 215 843 3505. E-mail address: [email protected] (C.J. Lopez).
0304-3940/$ – see front matter © 2005 Published by Elsevier Ireland Ltd. doi:10.1016/j.neulet.2005.04.089
kinases (PTKs) and the protein tyrosine phosphatases (PTPs). Although much work has focused on activation of protein tyrosine kinases, the ultimate physiological and pathological effects of phosphotyrosine-mediated signaling are closely related to the duration of the phosphorylated state, which is highly regulated by specific tyrosine phosphatases [17]. PTPs are regulated through multiple mechanisms. Reversible oxidation of the active site cysteine residue has recently been identified as a general mechanism for regulation of PTPs [13]. PTP activity is also regulated through subcellular translocations such as the calpain-mediated release of PTP-1B from
C.J. Lopez et al. / Neuroscience Letters 386 (2005) 78–81
the endoplasmatic reticulum, and the SH2-domain-mediated recruitment of SHP-1 and SHP-2 to tyrosine-phosphorylated proteins [9]. Nitric oxide and nitrogen oxides formed by the slow reaction of nitric oxide with oxygen have been suggested to modulate thiol-dependent enzymes through the formation of S-nitrosothiols and subsequent oxidation to give disulfides. The highly reactive thiolate group in tyrosine phosphatases could also be a potential target for transnitrosation by Snitrosothiols [15]. The chemical reactivity of nitric oxide is greatly increased by its diffusion-limited reaction with superoxide (O2 − ) to form peroxynitrite (ONOO− ). Peroxynitrite nitrates tyrosine residues in proteins to form 3-nitrotyrosine, which can potentially interfere with subsequent phosphorylation of the affected tyrosine [11]. In addition, peroxynitrite readily oxidizes both free and protein-bound sulfhydryls and induces oxidative inactivation of other sulfhydryl-containing enzymes such as alcohol dehydrogenase, creatine kinase and succinate dehydrogenase. Consequently, peroxynitrite could potentially inactivate tyrosine phosphatases by either oxidizing the thiolate or nitrating the tyrosine residue in the active site [14]. The formation of 3-nitrotyrosine is considered a marker of in vivo production of peroxynitrite but should more correctly be considered a marker of NO related oxidants, since different mechanisms may lead to tyrosine nitration [7]. One of the oxidative reactions induced by peroxynitrite is the modification of tyrosine residues in proteins to yield 3-nitrotyrosine, and it has been suggested that this modification impairs phosphotyrosine signaling. However, several in vitro studies have shown that peroxynitrite may promote rather than inhibit tyrosine phosphorylation [7]. Previous studies from our laboratory have shown that PTP activity decreases during hypoxia potentially contributing to phosphorylation of a number of proteins including anti-apoptotic protein Bcl-2 [1,2]. We have also shown that hypoxia results in nitration of N-methyl-d-aspartate (NMDA) receptor subunits and altered receptor function [19]. The present study tests the hypothesis that in vitro exposure to sodium nitroprusside (SNP), will decrease protein tyrosine phosphatase enzyme activity in cortical cell membranes of newborn piglets and that the decrease in protein tyrosine phosphatase activity is NO-mediated. To test this hypothesis, in vitro experiments were conducted by measuring protein tyrosine phosphatase activity in the presence of an NO donor, sodium nitroprusside (SNP), or peroxynitrite. In addition, since 3-nitrotyrosine is produced in vivo, a marker of peroxynitrite formation in tissues, we have determined the effect of 3-nitrotyrosine on protein phosphatase activity, a mechanism that regulates protein phosphorylation/dephosphorylation. Studies were performed on 2- to 4-day-old Yorkshire piglets as per standard protocol as described by Ashraf et al. [1]. The study was approved by the Institutional Animal Care and Use Committee of the Drexel University College of Medicine. Cerebral cortical P2 membranes [5], which have high levels of PTP activity [1] were prepared from seven nor-
79
moxic newborn piglets and protein was determined by the method of Lowry et al. [8] and each sample was divided into three aliquots: a control group, a SNP group (exposed to 200 M SNP), and a peroxynitrite group (exposed to 100 M peroxynitrite). Protein tyrosine phosphatase activity was determined in a medium containing 100 mM HEPES (pH 7.0), 1 mM EDTA, 0.05% Tween 20 and 1.25 mM pnitrophenylphosphate (pNPP) at 37 ◦ C for 20 min in the presence or absence of 2 M bpV(phen), a highly selective inhibitor of protein tyrosine phosphatase. The enzyme activity was determined spectrophotometrically at 405 nm and the bpV(phen)-sensitive activity was expressed as nmol/mg protein/h. Negative controls were also performed. In addition, effect of 3-nitrotyrosine (100 M) on protein phosphatase activity was determined in the above assay system with 1 mM sodium orthovandate. Protein nitration was confirmed by immunoblotting [1] with anti-nitrotyrosine antibody. The data are presented as mean ± standard deviation and analyzed by one-way analysis of variance. A p-value <0.05 was considered significant. The protein tyrosine phosphatase activity (Fig. 1) was 198 ± 25 nmol/mg protein/h in the normoxic group, 177 ± 30 nmol/mg protein/h in the SNP group (p = NS versus normoxic) and 77 ± 20 nmol/mg protein/h in the peroxynitrite group (p < 0.001 versus normoxic). In the presence of 3nitrotyrosine protein phosphatase activity (Fig. 2) decreased from 926 ± 102 to 200 ± 77 (p < 0.001). PTPs can regulate the phosphorylation status of apoptotic proteins. The phosphorylation state also determines the antiapoptotic as well as pro-apoptotic potential of these proteins. These proteins belong to the Bcl-2 multigene family and regulate cell death and survival. The Bcl-2 family consists of both cell death promoter (Bax) and cell death repressor (Bcl-2) proteins. Overexpression of Bax is known to promote programmed cell death and overexpression of Bcl-2 leads to cell survival [18]. We have shown that hypoxia results in increased tyrosine phosphorylation and serine phosphorylation of Bcl-2. Unphosphorylated Bcl-2 is anti-apoptotic, but
Fig. 1. The activity of protein tyrosine phosphatase in the control, sodium nitropruside (SNP) treated and peroxynitrite treated cortical cell membranes of normoxic newborn piglet. The data are expressed as mean ± S.D.
80
C.J. Lopez et al. / Neuroscience Letters 386 (2005) 78–81
Fig. 2. The activity of protein phosphatase in the control and 3-nitrotyrosine treated cortical cell membranes of normoxic newborn piglets. The data are expressed as mean ± S.D.
phosphorylated Bcl-2 is pro-apoptotic [2,18]. In a separate study, we have observed that cerebral hypoxia resulted in increased tyrosine phosphorylation of a number of neuronal proteins whereas a number of proteins were dephosphorylated and some of the proteins were not altered in terms of phosphorylation/dephosphorylation [4]. These results indicate that the tyrosine phosphorylation/dephosphorylation equilibrium during hypoxia may further be determined by the protein substrate itself and its specificity to phosphatases. There is a possibility that the translocation of PTP from membrane into cytosol occurs during hypoxia and this translocation of PTP is NO-mediated. Peroxynitrite readily nitrates tyrosine residues in proteins, and peptides containing nitrotyrosine are resistant to phosphorylation in vitro [9], suggesting that peroxynitrite could alter phosphotyrosine-mediated signaling. Peroxynitrite has been shown to affect signal transduction in cultured cells and to induce apoptosis [3,16]. However, tyrosine phosphorylation of proteins is typically increased rather than inhibited when cells are treated with peroxynitrite [12]. Peroxynitrite shows in vitro characteristics of a signaling molecule able to upregulate tyrosine-dependent and -independent phosphorylations. This effect could be related to the inhibition of phosphatases and/or to the activation of kinases. Whatever the activation mechanism, it is interesting to stress that direct or indirect effects of peroxynitrite are focused toward the upregulation of tyrosine-dependent signaling. Tyrosine nitration is a protein modification characteristic of peroxynitrite. Results of the present study indicate that peroxynitrite and 3-nitrotyrosine inhibit protein phosphatases indicating that tyrosine nitration is a potential mechanism of regulation of protein phosphorylation/dephosphorylation. Since 3-nitrotyrosine, is produced during hypoxia, the present in vitro experiments demonstrate that 3-nitrotyrosine, a product of peroxynitrite-dependent nitration by itself can lead to inhibition of protein tyrosine phosphatase in vivo. The data show that peroxynitrite but not SNP exposure results in decreased protein tyrosine phosphatase activity in vitro, indicating that the mechanism of PTP inactivation is peroxynitrite-mediated.
In previous studies, we have observed that hypoxia resulted in increased expression of PTP and other proteins such as the pro-apoptotic proteins Bax and Bad [20]. Expression of anti-apoptotic proteins Bcl-2 and Bcl-xl was not increased. These studies also demonstrated that the hypoxiainduced increased expression of the pro-apoptotic proteins can be blocked by the administration of NOS inhibitors [20]. In a series of studies we have observed that cerebral hypoxia resulted in increased neuronal nuclear Ca2+ -influx, increased calcium calmodulin-dependent protein kinase IV (CaM kinase IV) activity, increased phosphorylation of cyclic AMP response element binding (CREB) protein and increased expression of pro-apoptotic protein Bax [10,21]. Each of these steps were also blocked by the administration of NOS inhibitors during hypoxia. Therefore, hypoxia-induced increased expression and decreased activity of PTPs are due to a mechanism that is mediated by NO generated during hypoxia. In summary, we have shown that exposure to peroxynitrite decreased protein tyrosine phosphatase enzyme activity in cortical cell membranes of newborn piglets. However, the exposure to SNP did not decrease the enzyme activity. We conclude that protein tyrosine phosphatase inactivation is mediated by peroxynitrite, a product of NO. We speculate that hypoxia-induced NO production leading to peroxynitrite formation is a potential mechanism of protein tyrosine phosphatase inhibition. The NO-induced decrease in protein tyrosine phosphatase and protein phosphatase activity leading to Bcl-2 protein phosphorylation and loss of its antiapoptotic activity is a proposed NO-mediated mechanism of programmed cell death in the hypoxic brain.
Acknowledgements The authors thank Mrs. Joanna Kubin and Mrs. Anli Zhu for their technical assistance. This research was supported by grants from the National Institutes of Health, NIH HD-20337 and NIH HD-38079.
References [1] Q.M. Ashraf, S.H. Haider, C.D. Katsetos, M. DelivoriaPapadopoulos, O. Mishra, Nitric oxide-mediated alterations of protein tyrosine phosphatase activity and expression during hypoxia in the cerebral cortex of newborn piglets, Neurosci. Lett. 362 (2004) 108–112. [2] Q.M. Ashraf, S.A. Zanelli, O.P. Mishra, M. Delivoria-Papadopoulos, Phosphorylation of Bcl-2 and Bax proteins during hypoxia in newborn piglets, Neurochem. Res. 26 (2001) 1–9. [3] C. Brito, M. Naviliat, A.C. Tiscornia, F. Vuillier, G. Gualco, G. Dighiero, R. Radi, A.M. Cayota, Peroxynitrite inhibits T lymphocyte activation and proliferation by promoting impairment of tyrosine phosphorylation and peroxynitrite-driven apoptotic death, J. Immunol. 162 (1999) 3356–3366. [4] G. Buonocore, S. Liberatori, L. Bini, O.P. Mishra, M. DelivoriaPapadopoulos, V. Pallini, R. Bracci, Hypoxic response of
C.J. Lopez et al. / Neuroscience Letters 386 (2005) 78–81
[5]
[6] [7]
[8]
[9]
[10]
[11]
[12]
[13]
synaptosomal proteins in term guinea pig fetuses, J. Neurochem. 73 (1999) 2139–2148. S.I. Harik, G.H. Doull, A.P. Dick, Specific ouabain binding to brain microvessels and choroid plexus, J. Cereb. Blood Flow Metab. 5 (1985) 156–160. J. den Hertog, A. Groen, T. vander Wijk, Redox regulation of proteintyrosine phosphatases, Arch. Biochem. Biophys. 434 (2005) 11–15. H. Ischiropoulos, Biological tyrosine nitration: a pathophysiological function of nitric oxide and reactive oxygen species, Arch. Biochem. Biophys. 356 (1998) 1–11. O. Lowry, N.J. Rosenbrough, A.L. Farr, R.J. Randall, Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193 (1951) 265–275. B.L. Martin, D. Wu, S. Jakes, D.J. Graves, Chemical influences on the specificity of tyrosine phosphorylation, J. Biol. Chem. 265 (1990) 7108–7111. O.P. Mishra, Q.M. Ashraf, M. Delivoria-Papadopoulos, Phosphorylation of cAMP response element binding (CREB) protein during hypoxia in cerebral cortex of newborn piglets and the effect of nitric oxide synthase inhibition, Neuroscience 115 (2002) 985–991. O.P. Mishra, D. Maulik, Q.M. Ashraf, M. Delivoria-Papadopoulos, Nitration of N-methyl-d-aspartate receptor subunits following in vitro dephosphorylation of cerebral cortical membranes of newborn piglets, Neurosci. Lett. 317 (2002) 115–118. T.H. Mondoro, B.C. Shafer, J.G. Vostal, Peroxynitrite-induced tyrosine nitration and phosphorylation in human platelets, Free Radic. Biol. Med. 22 (1997) 1055–1063. A.D.B. Pannifer, A.J. Flint, N.K. Tonks, D. Barford, Visualization of the cysteinyl-phosphate intermediate of a protein-tyrosine
[14]
[15]
[16]
[17] [18]
[19]
[20]
[21]
81
phosphatase by X-ray crystallography, J. Biol. Chem. 273 (1998) 10454–10462. R. Radi, J.S. Beckman, K.M. Bush, B.A. Freeman, Peroxynitrite oxidation of sulfhydryls. The cytotoxic potential of superoxide and nitric oxide, J. Biol. Chem. 266 (1991) 4244–4250. R.J. Singh, N. Hogg, J. Joseph, B. Kalyanaraman, Mechanism of nitric oxide release from S-nitrosothiols, J. Biol. Chem. 271 (1996) 18596–18603. N. Spear, A.G. Estevez, L. Barbeito, J.S. Beckman, G.V. Johnson, Nerve growth factor protects PC12 cells against peroxynitriteinduced apoptosis via a mechanism dependent on phosphatidylinositol 3-kinase, J. Neurochem. 69 (1997) 53–59. K.M. Walton, J.E. Dixon, Protein tyrosine phosphatases, Annu. Rev. Biochem. 62 (1993) 101–120. H. Wei, W. Wei, D.E. Bredesen, D.C. Perry, Bcl-2 protects against apoptosis in neuronal cell line caused by thapsigargin-induced depletion of intracellular calcium stores, J. Neurochem. 70 (1998) 2305–2314. S.A. Zanelli, Q.M. Ashraf, O.P. Mishra, Nitration is a mechanism of regulation of the NMDA receptor function during hypoxia, Neuroscience 112 (2002) 869–877. A.B. Zubrow, M. Delivoria-Papadopoulos, Q.M. Ashraf, J.R. Ballesteros, K.I. Fritz, O.P. Mishra, Nitric oxide-mediated expression of Bax protein and DNA fragmentation during hypoxia in neuronal nuclei from newborn piglets, Brain Res. 954 (2002) 60–70. A.B. Zubrow, M. Delivoria-Papadopoulos, Q.M. Ashraf, K.I. Fritz, O.P. Mishra, Nitric oxide-mediated Ca2+ /calmodulin-dependent protein kinase IV activity during hypoxia in neuronal nuclei from newborn piglets, Neurosci. Lett. 335 (2002) 5–8.