Protein tyrosine phosphatase-1B regulates the tyrosine phosphorylation of the adapter Grb2-associated binder 1 (Gab1) in the retina
© Rajala et al.; licensee BioMed Central Ltd. 2013
Received: 2 January 2013
Accepted: 19 March 2013
Published: 22 March 2013
Gab1 (Grb2-associated binder 1) is a key coordinator that belongs to the insulin receptor substrate-1 like family of adaptor molecules and is tyrosine phosphorylated in response to various growth factors, cytokines, and numerous other molecules. Tyrosine phosphorylated Gab1 is able to recruit a number of signaling effectors including PI3K, SHP2 and PLC-γ. In this study, we characterized the localization and regulation of tyrosine phosphorylation of Gab1 in the retina.
Our immuno localization studies suggest that Gab1 is expressed in rod photoreceptor inner segments. We found that hydrogen peroxide activates the tyrosine phosphorylation of Gab1 ex vivo and hydrogen peroxide has been shown to inhibit the protein tyrosine phosphatase PTP1B activity. We found a stable association between the D181A substrate trap mutant of PTP1B and Gab1. Our studies suggest that PTP1B interacts with Gab1 through Tyrosine 83 and this residue may be the major PTP1B target residue on Gab1. We also found that Gab1 undergoes a light-dependent tyrosine phosphorylation and PTP1B regulates the phosphorylation state of Gab1. Consistent with these observations, we found an enhanced Gab1 tyrosine phosphorylation in PTP1B deficient mice and also in retinas treated ex vivo with a PTP1B specific allosteric inhibitor.
Our laboratory has previously reported that retinas deficient of PTP1B are resistant to light damage compared to wild type mice. Since Gab1 is negatively regulated by PTP1B, a part of the retinal neuroprotective effect we have observed previously in PTP1B deficient mice could be contributed by Gab1 as well. In summary, our data suggest that PTP1B regulates the phosphorylation state of retinal Gab1 in vivo.
KeywordsAdapter protein Gab1 PTP1B Phosphorylation Retina Photoreceptors
Gab1 (Grb2-assoicated binder 1) is a member of a small group of scaffolding adapters that includes Drosophila melanogaster Dos (Daughter of Sevenless), the Caneorhabditis elegans homolog Soc1 (S uppressor-O f C lear), and mammalian Gab2 and Gab3[1–8]. These proteins contain an amino-terminal PH domain, several proline-rich sequences, and multiple binding sites for SH2-domain containing proteins. Upon stimulation of appropriate cells with any of a number of receptor tyrosine kinase ligands, including epidermal growth factor (EGF), hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), nerve growth factor (NGF), and insulin or insulin-like growth factor 1 (IGF-1), Gab1 rapidly becomes tyrosine phosphorylated[3, 8–11]. Tyrosine phosphorylated Gab1 binds multiple signal-relay molecules, including the p85 subunit of phosphoinositide 3′-kinase, Shc, and the protein tyrosine phosphatase (PTP) Shp2[3, 8, 12, 13]. In addition to the binding sites for SHP2 and p85, both Gab1 and Gab2 contain numerous YxxP motifs, potential binding sites for the SH2 domain of PLCγ or Crk family proteins. Further, Grb2 binds to Gab proteins via its C-terminal SH3 domain in a phospho-tyrosine independent manner[15, 16].
The physical association between p85 and Gab1 or Gab2 is critical in mediating the PI3K/Akt signaling pathway induced by a variety of stimuli[9, 10, 17–22]. Overexpression of Gab potentiates FGF-induced Akt activity, whereas overexpression of the p85 binding mutant of Gab1 results in decreased Akt activation. The same mutant is also unable to provide anti-apoptotic signal in response to nerve growth factor stimulation. Mutation in the p85-binding sites of Gab2 was found to impair the ability of IL-3 to activate Akt and to induce cell growth. These studies clearly suggest that Gab-p85 interaction plays an important role in activating the PI3K/Akt pathway in mammalian cells. The activation of PI3K leads to the production of PIP3, which in turn can bind to the PH domain of Gab proteins and presumably promote further activation of PI3K, a positive feedback loop which could be formed to amplify the signal through the Gab proteins. The EGF-dependent positive feedback loop is negatively regulated by SHP2 by dephosphorylating Gab1-p85 binding sites, thereby terminating the Gab1-P3K positive loop.
Many retinal degenerative diseases show an early loss of rod cells followed by cone cell loss, and the pathological phenotype for this loss is apoptosis[24–26]. Blocking of photoreceptor apoptosis is one of the possible therapeutic approaches to protect the morphology and function of the retina and prolong the period of useful vision in patients. The mechanisms of protection are still largely unknown but may involve differential intercellular signaling cascade. We and others have shown that PI3K activation is neuroprotective[27, 28]. Hepatocyte growth factor (HGF) is shown to protect light-induced photoreceptor degeneration and retinal ischemia-reperfusion injury and also attenuates the ceramide-induced apoptosis in retina. All these studies clearly suggest that HGF possesses both neuroprotective and anti-oxidant properties[29, 31]; however, the molecular mechanism behind the neuroprotective effect remains unclear. Both HGF and its receptor c-Met are expressed in the retina. Interaction between Gab1 and the cMet receptor tyrosine kinase is responsible for epithelial morphogenesis. Upon interaction with cMet, Gab1 becomes phosphorylated on several tyrosine residues which, in turn, recruit a number of signaling effectors, including PI3K, SHP2, and PLC-γ. Gab1 phosphorylation by cMet results in a sustained signal that mediates most of the downstream signaling pathways[34, 35]. The association between protein tyrosine phosphatase-1B (PTP1B) and c-Met receptor in the modulation of corneal epithelial wound healing has been reported previously. However, absolutely there are no data available on the expression and regulation of tyrosine phosphorylation of Gab1 in the retina. In this study we have examined the localization of Gab1 and how the phosphorylation state of Gab1 is regulated in the retina as the interaction of Gab1 with effector proteins is phosphorylation-dependent. Our studies suggest that Gab1 is predominantly localized to rod inner segments under both dark- and light-adapted conditions; however, the state of Gab1 phosphorylation is light-dependent. Our studies also suggest that protein tyrosine phosphatase, PTP1B, regulates the Gab1 phosphorylation in vivo as we found enhanced phosphorylation of Gab1 in PTP1B deficient mice and retinas treated ex vivo with a PTP1B specific inhibitor. We also found a region between 1–280 amino acids in Gab1 encompassing Y83 is required for PTP1B binding.
Localization of Gab1 in the retina
Light-dependent phosphorylation of Gab1
Hydrogen peroxide activates the Gab1 phosphorylation
Previously H2O2 has been shown to induce the phosphorylation of Gab1 which results in the binding of SHP2. Therefore we have examined the Gab1 phosphorylation on Y627 (binding site of SHP2) residue in response to H2O2 in retinal ex vivo explants. To determine the effect of H2O2 on Gab1 phosphorylation, we incubated mouse retinal ex vivo explants for 10 min in the presence or absence of 600 μM H2O2. Retinal proteins were prepared and subjected to immunoblot analysis with anti-pGab1-Tyr627 antibody and the results indicate an increased phosphorylation of Gab1 was observed in H2O2 treated retinas compared to control retinas (Figure 3C) while the total Gab1 levels are unchanged (Figure 3C). The blot was reprobed with anti-actin antibody (Figure 3C) to ensure an equal amount of protein in each lane. These results suggest that H2O2 activates the Gab1 phosphorylation.
Binding of Gab1 to p85 (N-SH2) domain of PI3K
Possible mechanism of H2O2-induced Gab1 activation
The exact mechanism of H2O2-induced Gab1 activation is not known. However, it has been shown previously that H2O2 inhibits the PTP1B activity[38, 39]. We also tested in this study the H2O2-induced inhibition of PTP1B activity. We stimulated the rat retinas ex vivo with insulin, and the retinal lysates were immunoprecipitated with anti-IRβ antibody. The IR immunoprecipitates were subjected to dephosphorylation assay by PTP1B in the presence and absence of H2O2 followed by immunoblot analysis with anti-PY99 antibody. The results indicate that PTP1B dephosphorylates the IR and the dephosphorylation of IR by the PTP1B was partially prevented in the presence of H2O2 (Figure 4B). The observed activation of Gab1 in this study could be due to the inhibition of PTP1B activity and that Gab1 could be a substrate of PTP1B.
Light-dependent inhibition of retinal PTP1B activity
To determine whether light regulates PTP1B activity, we immunoprecipitated PTP1B from lysates of dark- and light-adapted rat retinas and measured the PTP1B activity. The PTP1B activity was significantly greater in dark-adapted retinas than in the light-adapted retinas (Figure 4D). To determine whether this greater PTP1B activity was due to increased protein expression in the dark-adapted retinas, we subjected the proteins from dark- and light-adapted retinas to immunoblotting with anti-PTP1B antibody (Figure 4C). No significant differences in the expression of PTP1B was found between the dark- and light-adapted mouse retinas, suggesting that light regulates PTP1B activity in vivo.
Identification of Gab1 as a substrate of PTP1B in vitro
PTP1B dephosphorylates Gab1 in vitro
To determine whether PTP1B dephosphorylates Gab1 in vitro, we expressed the Myc-tagged full-length Gab1 in HEK-293 T cells and the proteins were subjected to immunoprecipitation with anti-Myc tag antibody. The immune complexes were incubated in the presence of either wild type PTP1B or catalytically inactive mutant D181A-PTP1B (GST-fusion proteins) for 30 min at 30°C. At the end of incubation, the immunoprecipitates were washed and subjected to immunoblot analysis with anti-PY99 and anti-Myc antibodies. The results indicate that PTP1B completely dephosphorylated Gab1 and the mutant protein failed to dephosphorylate Gab1 (Figure 5C). The Myc tag blot shows the presence of Gab1 in all the immunoprecipitates (Figure 5C). The blot was also reprobed with anti-GST antibody to ensure equal amount of PTP1B fusion protein in all lanes (Figure 5C). This experiment shows that PTP1B can dephosphorylate Gab1 in vitro.
Gab1 phosphorylation is required for PTP1B binding
In the second approach we expressed Myc-tagged Gab1 in HEK-293 T cells and the cells were treated in the presence or absence of pervanadate. The lysates were subjected to GST pull-down assays with either wild type PTP1B or PTP1B-D181A mutant followed by immune blot analysis with anti-Myc and anti-pGab1 antibodies. The results indicate the binding of Gab1 to PTP1B-D181A mutant only from the cells that were treated with pervanadate (Figure 6B). Pull-downs immunoblotted with anti-pGab1 antibody clearly suggest that the binding of Gab1 to PTP1B mutant is phosphorylation-dependent as we failed to recover the Gab1in PTP1B-D181A pull-down in the absence of its phosphorylation (Figure 6B).
Binding site of PTP1B on Gab1
Prediction of tyrosine phosphorylation on tyrosine residues in Gab1
Position of Tyr
To identify the binding site on Gab1, we expressed the Gab1 protein in HEK-293 T cells from 1–280 amino acids which contain only one likely phosphorylated tyrosine residue 83. This truncated protein is able to interact with PTP1B-D181A mutant (Figure 8A). Our results on Y83F mutant did not abolish the binding interaction between Gab1 and PTP1B-D181A mutant; it is likely that the binding is dictated by the cooperative tyrosine phosphorylation and a region between 50–280 amino acids in Gab1. Examinations of region between 50–280 amino acids clearly indicate the presence of PH domain (1–116 amino acids). When we deleted the PH domain from the Gab1, we failed to observe the interaction with the PTP1B-D181A mutant (Figure 8B), even though the deleted PH domain of Gab1 is tyrosine phosphorylated (Figure 8B, bottom panel). These results clearly suggest that the tyrosine phosphorylation and PH domain of Gab1 is required for substrate recognition of PTP1B.
Increased Gab1 phosphorylation in PTP1B knockout mouse retinas and a PTP1B inhibitor-treated retinas
Tyrosine kinase receptors and downstream pathways used in growth factor signaling are shared by oxygen free radical signaling. Different growth factor receptors and cytokines are known to activate the tyrosine phosphorylation of Gab1 which in turn activates different signaling pathways, including PI3K/Akt[3, 9, 45, 46], ERK[13, 33] and JNK[10, 47]. In this study we observed that H2O2 stimulates the tyrosine phosphorylation of retinal Gab1. On the other hand, light stress decreased the binding of PI3K to Gab1 (data not shown) suggesting a loss of Gab1 phosphorylation under light stress. It has been shown previously that H2O2 stimulates the tyrosine phosphorylation of Gab1 in wild type mouse embryonic fibroblasts and the activated Gab1 recruits molecules such as SHP2, PI3K, and Shc. These studies clearly indicate that Gab1 is a component of oxidative stress signaling. Gab1 is also associated with similar proteins following stimulation with EGF, insulin, NGF, or HGF[3, 8–11]. The Gab1/PI3K interaction with subsequent activation of Akt activation has been shown to protect the PC12 cells or sympathetic neurons from apoptosis induced by serum deprivation[9, 46].
The phosphorylation status of Gab1 after H2O2 treatment has been previously explained due to the activation of EGFR. It is interesting to note in this study that Gab1 is expressed in rod inner segments and its state of phosphorylation is light-dependent. In retina, EGFR expression has shown to be during the first two postnatal weeks in Müller glia and declines as the retina matures; in response to light-damage, EGFR expression is upregulated which has shown to be close to neonatal retina. Insulin-induced Gab1 tyrosine phosphorylation and association of Gab1 with Src homology-2 (SH2) domain-containing proteins has been reported. Retinal ex vivo explants treated with insulin did not induce the tyrosine phosphorylation of Gab1. These studies suggest that light-induced tyrosine phosphorylation of Gab is regulated through an unknown mechanism not known at this time.
It has also been suggested that there is also inactivation of phosphatases in oxidative signaling. Hydrogen peroxide can irreversibly inactivate PTP1B in vivo and contribute to EGFR phosphorylation after EGF treatment. Several studies in literature indicate that PTP1B is somewhat promiscuous in its substrate preference in vitro, dephosphorylating a wide variety of protein and peptide substrates with widely varying Km values[50–52]. Substrate-trapping mutants of PTPs have been shown to be ideal reagents for substrate identification. It was demonstrated that such mutants of PTPs can be produced by mutation of Asp to Ala in the conserved WPD loop. The Asp to Ala mutants of PTP1B, TC-PTP, PTPH1, and PTP-PEST helped identify EGFR, p52shc, VCP (p97/CDC48), TYK2 and JAK2, and p130Cas as candidate substrates, respectively[40, 41, 53–55]. We found that Gab1 stably associates with mutant PTP1B in a tyrosine phosphorylation-dependent manner. These observations suggest that Gab1 could be a putative substrate of PTP1B. Consistent with this observation, Gab1 has previously been identified as one of the PTP1B substrates by Bayesian Integration of Proteome.
Mutational analysis of various tyrosine residues in Gab1 indicated that none of the mutants abolished the binding interaction with PTP1B. However, we found a decreased binding of Y83F with PTP1B. This result is of particular interest since one of the only two Gab1 mutations associated with cancer is Y83C[57–59]. Further studies are required to understand the interaction between PTP1B and Gab1-Y83 in tumorigenesis. Our studies also suggest that a region from 1–280 amino acids in Gab1 is required for PTP1B binding.
It is interesting to note that there are no studies available on the role of Gab1 in the retina, however, deletion of Gab1 binding protein Shp2 (src-homology phosphotyrosyl phosphatase 2) has been shown result in retinal degeneration. Experiments described in this manuscript suggest that PTP1B negatively regulates the Gab1 phosphorylation. Clear evidence comes from the light/dark experiments where higher phosphorylation of Gab1 in light-adapted conditions was correlated with significantly decreased levels of PTP1B and in dark-adapted conditions, higher PTP1B levels correlated with decreased levels of Gab1 phosphorylation. Such a negative relationship has been observed previously between PTP1B and Gab1 in which PTP1B-mediated dephosphorylation of Gab1 has been shown to affect its EGF-induced association with the phosphatase SHP2. Increased Gab1 phosphorylation in PTP1B inhibitor-treated retinas and PTP1B knockout mouse retinas further strengthen the evidence that PTP1B regulates the phosphorylation state of Gab1 in vivo. Our laboratory has previously reported that retinas deficient of PTP1B are resistant to light damage compared to wild type mice. We have also reported that intravenous injection of an allosteric inhibitor of PTP1B protects rats against light stress-induced retinal degeneration through the protection of IR phosphorylation. We have also reported enhanced insulin receptor neuroprective signaling in PTP1B deficient mice. Since Gab1 is negatively regulated by PTP1B, a part of the retinal neuroprotective effect we have observed previously in PTP1B deficient mice could be contributed by Gab1 as well. Further studies are required to determine the Gab1-medited neuroprotective survival signaling in the retina.
In this study we have identified a physical and functional interaction between Gab1 and PTP1B. We also found that Gab1 undergoes a light-dependent phosphorylation and PTP1B regulates the phosphorylation state of Gab1. Consistent with these observations, we found an enhanced Gab1 tyrosine phosphorylation in PTP1B deficient mice and PTP1B-inhibitor treated retinas. Collectively, our data suggest that Gab1 is an endogenous physiological protein substrate of PTP1B.
Anti-PTP1B antibody was obtained from Epitomics (Burlingame, CA). Polyclonal anti-PTP1B, anti-Gab1 antibodies and phosphatase assay reagents were obtained from Upstate Biotechnology (Lake Placid, NY). Monoclonal PY-99 and polyclonal IR antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). An anti-pGab1 antibody was obtained from Cell Signaling (Beverly, MA). The actin antibody was obtained from Affinity BioReagents (Golden, CO). Quick change site-directed mutagenesis kit was obtained from Stratagene (La Jolla, CA). All other reagents were of analytical grade and from Sigma. The PTP1B inhibitor (3-(3,5-Dibromo-4-hydroxy-benzoyl)-2-ethyl-benzofuran-6-sulfonicacid-(4-(thiazol-2- ylsulfamyl)-phenyl)-amide) was obtained from Calbiochem (San Diego, CA).
All animal work was done in strict accordance with the NIH Guide for the Care and Use of Laboratory Animals and the Association for Research in Vision and Ophthalmology on the Use of Animals in Vision Research. All protocols were approved by the IACUC at the University of Oklahoma Health Sciences Center and the Dean McGee Eye Institute. In all experiments, rats and mice were killed by asphyxiation with carbon dioxide before the retinas were harvested. A breeding colony of albino Sprague–Dawley (SD) rats is maintained in our vivarium in cyclic light (5 lux; 12 h on/12 h off). Experiments were carried out on both male and female rats (150–200 g). Breeding colonies of PTP1B and Akt2 knockout mice are maintained in our vivarium. The source of global PTP1B and Akt2 knockout mice have been reported earlier.
Plasmid construction and transfection
The mammalian expression construct of Gab1 was kindly provided by Dr. Ute Schaeper (Berlin, Germany). The Myc-tagged form of full-length Gab1 was generated by adding the Myc-epitope at its C-terminus by PCR and the cDNA encoding Myc-tagged Gab1 was cloned into pCDNA3 vector. All constructs that involved PCR were verified by DNA sequencing. Human embryonic kidney cells (HEK-293 T) were grown in 10% FBS and transfected with 10 μg of DNA in 10-cm plates by calcium phosphate method. Retinal PTP1B was obtained by PCR of reverse transcribed mouse retinal RNA using a 5′ and 3′ oligonucleotide designed based on mouse PTP1B cDNA sequence (accession number NP_035331) (sense: GAA TTC ATG GAG ATG GAG AAG GAG TTC GAG; antisense: GTC GAC TCA GTG AAA ACA CAC CCG GTA GC). Site-directed mutagenesis was carried out according to the method described earlier. Gab1-Y83F; sense: TTT GAA AAC AGC TTT ATC TTT GAT ATC AAC; antisense: GTT GAT ATC AAA GAT AAA GCT GTT TTC AAA; Gab1-Y285F; sense GAC GGG GAG CTG TTC ACC TTT AAC ACC CCA; antisense: TGG GGT GTT AAA GGT GAA CAG CTC CCC GTC; Gab1-Y373F; sense ACT GAC AGC AGT TTC TGT ATC CCT CCT CCA; antisense: TGG AGG AGG GAT ACA GAA ACT GCT GTC AGT; Gab1-Y407F; sense TCT CAA GAT TGC TTT GAT ATT CCA CGG ACC; antisense: GGT CCG TGG AAT ATC AAA GCA ATC TTG AGA; Gab1-Y448F; sense: CTG GAT GAG AAC TTC GTT CCC ATG AAC CCC; antisense: GGG GTT CAT GGG AAC GAA GTT CTC ATC CAG; Gab1-Y473F, sense: CAG GAG CCA AAC TTT GTG CCA AATG ACC CCA; antisense: TGG GGT CAT TGG CAC AAA GTT TGG CTC CTG; Gab1-Y590F; sense: AGT GAA GAG AAC TTT GTC CCC ATG AAT CCA; antisense: TGG ATT CAT GGG GAC AAA GTT CTC TTC ACT; Gab1-Y628F; sense AAA CAA GTC GAA TTC CTG GAT TTA GAC; antisense: GTC TAA ATC CAG GAA TTC GAC TTG TTT; Gab1-Y660F; GAG AGG GTG GAT TTC GTT GTG GTG GAC CAA; antisense: TTG GTC CAC CAC AAC GAA ATC CAC CCT CTC; Gab1-R49T; sense GTC CTG GAG TAT TAC ACA AAC GAT CAT GCC GCA; antisense: GGC ATG ATC GTT TGT GTA ATA CTC CAG GAC; Gab1-R49A; sense: GTC CTG GAG TAT TAC GCA AAC GAT CAT GCC; antisense: GGC ATG ATC GTT TGC GTA ATA CTC CAG GAC; Gab1-Y47F: sense GAT GTC CTG GAG TTT TAC AAA AAC GAT CAT; antisense: ATG ATC GTT TTT GTA AAA CTC CAG GAC ATC. The PTP1B binding motif on Gab1 (ΔEYYK) was deleted and the expression construct (49–695 amino acids) was generated using the following primers: sense: GAA TTC ACC ATG GAC ATC TGT GGA TTC AAT CCC ACA G GAA TTC ACC ATG AAC GAT CAT GCC AAG AAG CC and antisense: GGA TCC CTT CAC ATT CTT GGT GGG TGT CTC GG. Truncated versions of Gab1 were also generated using the following primers: Gab1 (1–280 amino acids) sense, GAA TTC C ACC ATG AGC GGC GGC GAA GTG GTT TGC TCG GG and antisense: GGA TCC GGC CTC CGT GCT TGA TGG GGA TTC C. The PCR products were cloned into TOPO sequencing vector (Invitrogen) and the sequences were verified by DNA sequencing. The inserts were excised as EcoRI/BamHI and cloned into C-terminal Myc-tagged pCDNA3 vector. The primers used in the site-directed mutagenesis are as follows: PTP1B-D181A (sense: ACC ACA TGG CCT GCC TTT GGA GTC CCC; antisense: GGG GAC TCC AAA GGC AGG CCA TGT GGT). The PCR products were cloned into TOPO sequencing vector (Invitrogen) and the sequences were verified by DNA sequencing. The WT and mutant cDNA were excised from the sequencing vector as EcoRI/SalI and cloned into GST fusion vector, pGEX-4 T1. Site-directed mutagenesis was carried out according to the method described earlier. The cloning and expression of N-SH2 domain of p85 subunit of PI3K has been reported previously.
Expression of GST-fusion proteins
An overnight culture of E.coli BL21 (DE3) (pGEX-PTP1B and pGEX-PTP1B-D181A) was diluted 1:10 with 100 μg/ml ampicillin, grown for 1 hr at 37°C, and induced for another hour by addition of IPTG to 1 mM. Bacteria were sonicated three times for 20 s each time in lysis buffer containing 10 mM imidazole-HCl (pH7.2), 1 mM EDTA, 100 mM NaCl, 1 mM dithiothreitol, and 1% Triton X-100. Lysates were clarified by centrifugation, and the supernatants were incubated with 500 μl of 50% glutathione-coupled beads (Amersham Pharmacia) for 30 min at 4°C. The GST-PTP1B fusion proteins were washed in lysis buffer and eluted twice with 1 ml of 5 mM reduced glutathione (Sigma) in phosphatase buffer [20 mM Tris (pH 7.4), 5% glycerol, 0.05% Triton X-100, 2.5 mM MgCl2, aprotinin (2 μg/ml) and leupeptin (5 μg/ml)]. Glycerol was added to a final concentration of 33% (vol/vol), and aliquots of enzyme were stored at −20°C.
Substrate trapping in vitro
The GST fusion proteins were expressed in E.coli and purified on glutathione-Sepharose beads according to the manufacturer’s instructions. Pervanadate stock solution (1 mM) was prepared by adding 10 μl of 100 mM vanadate and 50 μl of 100 mM hydrogen peroxide (diluted from 30% stock in 20 mM HEPES, pH 7.3) to 940 μl of H2O. Excess hydrogen peroxide was removed by adding catalase (100 μg/ml; final concentration = 260 units/ml) 5 min after mixing the vanadate and hydrogen peroxide. The pervanadate solutions were used within 5 min to minimize decomposition of the vanadate-hydrogen peroxide complex. Retinal ex vivo explants or mammalian cells were treated with 1 mM pervanadate for 30 min, washed with phosphate-buffered saline, and lysed in substrate-trapping buffer. The lysates were incubated for 2 h at 4°C with either GST or GST-PTP1B-WT or GST-PTP1B-D181A mutant fusion proteins bound on beads, then the beads were washed 4 times with trapping buffer. Bound proteins were resolved by SDS-PAGE and blotted onto nitrocellulose membranes. Blots were then incubated with anti-PY99 or anti-Gab1 antibodies and developed by ECL.
PTP1B Activity assay
The in vitro PTP activity assay was conducted based on a previously published protocol using the peptide RRLIEDAEPYAARG (Upstate Biotechnology). The reaction was carried out in a 60 μL volume of PTP assay buffer [100 mm HEPES (pH 7.6), 2 mm EDTA, 1 mm dithiothreitol, 150 mm NaCl, 0.5 mg/ml bovine serum albumin] at 30°C. At the end of the reaction, 40 μL aliquots were placed in a 96-well plate, 100 μL of Malachite Green Phosphatase reagent (Upstate Biotechnology) were added, and absorbance was measured at 630 nm.
Retinal Ex-vivo organ cultures
Retinal ex vivo organ cultures were carried out as previously described. Retinas were removed from Sprague–Dawley albino rats that were born and raised in dim cyclic light (5 lux; 12 h ON: 12 h OFF) and incubated for 5 min at 37°C in Dulbecco’s modified Eagle’s (DMEM) medium (Gibco BRL) in the presence or absence of 600 μM H2O2 or 100 μM PTP1B inhibitor (3-(3,5-Dibromo-4-hydroxy-benzoyl)-2-ethyl-benzofuran-6-sulfonicacid-(4-(thiazol-2- ylsulfamyl)-phenyl)-amide) or DMSO. At the indicated times, retinas were snap-frozen in liquid nitrogen and stored at −80°C until analyzed or lysed in lysis buffer [1% NP 40, 20 mM HEPES (pH 7.4), 2 mM EDTA, phosphatase inhibitors (100 mM NaF, 10 mM Na4P2O7, 1 mM NaVO3, and 1 mM molybdate), and protease inhibitors (10 μM leupeptin, 10 μg/ml aprotinin and 1 mM PMSF)].
Preparation of Rod outer segments
ROS were prepared from rat retinas using a discontinuous sucrose gradient as previously described. Retinas were homogenized in 4.0 ml of ice-cold 47% sucrose solution containing 100 mM NaCl, 1 mM EDTA, 1 mM PMSF, and 10 mM Tris–HCl (pH 7.4). Retinal homogenates were transferred to 15-ml centrifuge tubes and sequentially overlaid with 3.0 ml of 42%, 3.0 ml of 37%, and 4.0 ml of 32% sucrose dissolved in buffer A [10 mM Tris–HCl (pH 7.4) containing 100 mM NaCl and 1 mM EDTA]. The gradients were spun at 82,000 × g for 1 h at 4°C. The 32/37% interfacial sucrose band containing ROS membranes was harvested and diluted with buffer A, and centrifuged at 27,000 × g for 30 min. The ROS pellets were resuspended in buffer A, and stored at −20°C. All protein concentrations were determined by the BCA reagent following the manufacturer’s instructions.
Grb2-associated binding protein 1
Protein tyrosine phosphatase-1B
Src-homology phosphotyrosyl phosphatase 2
This work was supported by grants from the NIH (EY016507; EY00871; EY12190) and an unrestricted grant to the Department of Ophthalmology from the Research to Prevent Blindness, Inc. We thank Dr. Benjamin Neel (Ontario Cancer, Toronto, Canada) for providing global PTP1B knockout mice and Dr. Morris Birnbaum (University of Pennsylvania, Philadelphia, PA) for providing Akt2 knockout mice.
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