Specific Phosphatase Inhibitor Research Articles

Ectophosphatase activity in the early-diverging fungus Blastocladiella emersonii: Biochemical characterization and possible role on cell differentiation

Blastocladiella emersonii is an interesting model for studding the evolution of cell differentiation in eukaryotic cell because of its taxonomic position towards the base of the fungal phylogenetic tree and because it undergoes radical morphological and biochemical changes throughout its life cycle. In this work, we biochemically characterized a high alkaline phosphotyrosine phosphatase activity present on the cell surface (ectophosphatase) of B. emersonii. The ectophosphatase activity was strongly inhibited at acidic pH values as well as by specific phosphatase inhibitors, such as sodium orthovanadate and bpv-PHEN. In addition, the enzyme activity was modulated by the extracellular concentration of inorganic phosphate (Pi) present in both reaction mixture and culture medium. Phosphotyrosine was hydrolysed at the same extent of its analog, p-NPP, while the hydrolysis of phosphothreonine was 2-fold lower, suggesting that a phosphotyrosine ectophosphatase activity is present on the cell surface of B. emersonii. The ectophosphatase activity was also strongly inhibited by EGTA, indicating the participation of Ca2+ ions on catalysis. The hydrolysis of p-NPP was differentially regulated throughout the B. emersonii life cycle, suggesting that the ectophosphatase activity could be involved in cell differentiation processes. In support of this, the addition of bpv-PHEN or vanadate at the beginning of germination inhibited the differentiation of zoospores to germ cells, compared to control or tartrate-treated cells. On the other hand, if the inhibitors are added 15 or 30 min after initiation of germination the inhibitory effect on zoospore germination decreases significantly, suggesting that the phosphotyrosine ectophosphatase activity is important at the first minutes of germination. The addition of vanadate, molybdate and bpv-PHEN during vegetative growth inhibited the enlargement of the cells compared to control or tartrate-treated cells. Finally, vanadate or bpv-PHEN added during sporulation strongly inhibited zoospore biogenesis, indicating an important role of such ectophosphatases in this differentiation process. Taken together, these data show the existence of a high alkaline ectophosphotyrosine phosphatase activity in B. emersonii that is probably tied to cell differentiation processes of the fungus.

Inhibition of Setaria cervi protein tyrosine phosphatases by Phenylarsine oxide: A proteomic and biochemical study

Phenylarsine oxide (PAO), a specific protein tyrosine phosphatase (PTP) inhibitor significantly decreased the motility and viability of Setaria cervi ultimately leading to its death. The PTP activity present in the cytosolic and detergent soluble fractions as well as on surface of these parasites was significantly inhibited by PAO. A marked alteration in protein spots abundance after proteomic analysis showed 14 down-regulated and 9 upregulated spots in the treated parasites as compared to the control. The PTP inhibition led to increase in the cytosolic and mitochondrial calpain activity in these parasites. PAO also blocked the ATP generation in the parasite depicted by reduced activity of phosphoglycerate kinase and expression of enolase. An increased ROS level, induced lipid peroxidation/protein carbonyl formation and decreased activity of different antioxidant enzymes like thioredoxin reductase, glutathione reductase and glutathione transferases was also observed in the PAO treated parasites. PAO, thus disturbs the overall homeostasis of the filarial parasite by inhibiting PTPs. Thereby suggesting that these molecules could be used as a good chemotherapeutic target for lymphatic filariasis.

Receptor Protein Tyrosine Phosphatase Sigma (RPTP-σ) Increases pro-MMP Activity in a Trabecular Meshwork Cell Line Following Oxidative Stress Conditions.

To elucidate the role of phosphatases in the eye drainage system by overexpressing the receptor tyrosine phosphatase sigma (RPTP-σ) in a human normal trabecular meshwork (NTM) cell line. The efficacy, expression, and location of RPTP-σ were evaluated following its transfection in NTM cells (NTM(T)) and in NTM control cells. The cells were also analyzed for viability, matrix metalloproteinase (MMP) activity, and phosphatase activity following oxidative stress conditions. Assays were conducted in the presence or absence of a specific RPTP-σ inhibitor. Transfection efficacy measurements revealed that RPTP-σ expression measured via GFP fluorescence was significantly higher (×3.8) in NTM(T) cells than in control cells. Western blot analyses showed that RPTP-σ expression was significantly higher (×2.25) in NTM(T) cells than in control cells. No significant differences were observed in cell viability between NTM(T) and control cells after oxidative stress. We found that pro-MMP-2 and pro-MMP-9 showed a significantly higher activity (×2.18 and ×1.9; respectively) in NTM(T) cells than in control cells. Serine/threonine phosphatase activity in NTM(T) cells was significantly increased following oxidative stress. The specific phosphatase inhibitor PTP-IV inhibited 15% of the RPTP-σ expression in NTM cells and 31% in NTMT cells. The activity of pro-MMP-9, pro-MMP-2, and MMP-9 was significantly inhibited (48%, 35%, and 78% respectively). The findings indicate that RPTP-σ is expressed constituently in NTM cells and that oxidative stress changes the general phosphatase balance in NTM cells. In addition, the results show that expression levels of RPTP-σ affect the activity of various forms of MMP.

New potential eukaryotic substrates of the mycobacterial protein tyrosine phosphatase PtpA: hints of a bacterial modulation of macrophage bioenergetics state.

The bacterial protein tyrosine phosphatase PtpA is a key virulence factor released by Mycobacterium tuberculosis in the cytosol of infected macrophages. So far only two unrelated macrophage components (VPS33B, GSK3α) have been identified as PtpA substrates. As tyrosine phosphatases are capable of using multiple substrates, we developed an improved methodology to pull down novel PtpA substrates from an enriched P-Y macrophage extract using the mutant PtpA D126A. This methodology reduced non-specific protein interactions allowing the identification of four novel putative PtpA substrates by MALDI-TOF-MS and nano LC-MS: three mitochondrial proteins - the trifunctional enzyme (TFP), the ATP synthase, and the sulfide quinone oxidoreductase - and the cytosolic 6-phosphofructokinase. All these proteins play a relevant role in cell energy metabolism. Using surface plasmon resonance, PtpA was found to bind immunopurified human TFP through its catalytic site since TFP-PtpA association was inhibited by a specific phosphatase inhibitor. Moreover, PtpA wt was capable of dephosphorylating immunopurified human TFP in vitro supporting that TFP may be a bona fide PtpA susbtrate. Overall, these results suggest a novel scenario where PtpA-mediated dephosphorylation may affect pathways involved in cell energy metabolism, particularly the beta oxidation of fatty acids through modulation of TFP activity and/or cell distribution.

Abstract 469: The Role of PP2A in Angiotensin AT2R and Dopamine-1 Receptor Membrane Recruitment in Human Renal Proximal Tubule Cells

The renal dopamine-1 receptor (D1R) and angiotensin type 2 receptor (AT2R) are two important receptors that regulate renal sodium reabsorption through heterologous and autologous plasma membrane recruitment following stimulation of protein kinase A (PKA) via D1R and protein kinase G (PKG) via AT2R. Using the cell permeable analog of PKA (8Br-cAMP) along with the PKG analog 8Br-cGMP, we did not find additive effects, which led to our hypothesis that downstream protein phosphatase 2A (PP2A) is the common link in the D1R/AT2R interaction. To test our hypothesis, we used two human renal proximal tubule cell lines (RPTC): one with normal D1R coupling (nRPTC) to adenylyl cyclase (AC) and a 2nd cell line with a defect in D1R coupling to AC (uncoupled or uRPTC). A dose response curve for 8Br-cAMP and 8Br-cGMP using cell surface Dylight-649-AngIII binding for 1 hr in the presence of losartan (a measure of surface AT2R) showed maximal recruitment at 1 mM for both 8Br-cAMP and 8Br-cGMP. EC50 of 8Br-cAMP was 129.9±60.58 μM (log EC50 was -3.9). EC50 of 8Br-cGMP was 99.17±48.13 μM (log EC50 was -4.0) A 1 hr addition of 1 mM 8Br-cAMP or 8Br-cGMP resulted in increased PP2A activity. Specifically, PP2A activity increased by 1.59±0.24 fold (N=3, P<0.05 vs vehicle (VEH, baseline value of 1216 pmoles of phosphate at 650 nm) when 8Br-cAMP was added. The addition of 8Br-cGMP induced a 1.48±0.33 fold increase in PP2A (N=3, P< 0.05 vs VEH at 1216 pmoles of phosphate at 650 nm). Using flow cytometry for cell surface D1R and AT2R, recruitment of either receptor (AT2R or D1R) was completely blocked by okadaic acid (OA, a specific PP2A phosphatase inhibitor at 10 nM), whether stimulating D1R with fenoldopam (FEN), AT2R with AngIII, or using cell permeable downstream second messenger agonists 8Br-cAMP or 8Br-cGMP. Similarly, addition of the AC inhibitor SQ 22536 (10 μM) or the guanylyl cyclase (GC) inhibitor ODQ (10 μM) completely blocked FEN, AngIII, 8Br-cAMP and 8Br-cGMP-induced D1R and AT2R cell surface recruitment. In summary, we determined that PP2A is the common mediator of D1R and AT2R cell surface recruitment and may be an important target for regulating renal sodium homeostasis.

Effect of sorafenib on cathepsin B-dependent BID-mediated apoptosis in cancer cells.

e15515 Background: Several tyrosine kinase inhibitors (TKIs), have been developed and approved for clinical use in multi-targeted cancer therapy. Among these, sorafenib is an orally available multikinase inhibitor approved for the treatment of the advanced renal cell carcinoma (RCC) and hepatocellular carcinoma (HCC). Aim of our study was to evaluate the mechanisms responsible for the cytotoxic effects induced by in vitro use of µM doses of sorafenib in 5637 and J82 bladder cancer (BC) cell lines. Methods: The viability of BC cell lines were tested by MTT assay. Autophagy was evaluated by western blot analysis with the anti-LC3 and anti-p62 antibodies, acridine orange staining and cytofluorimetric analysis. Apoptosis, (ΔΨm) dissipation and ROS generation were determined by Annexin-V/PI, JC-1 and DCFDA staining, respectively and cytofluorimetric analysis. The cathepsin B activation was evaluated by western blot using an anti-cathepsin B antibody; the cathepsin B proteolytic activity was determined using the fluorogenic Z-Arg-Arg-AMC peptide and the fluorescence of the hydrolyzed 7-amino-4-methyl-coumarin was detected by a SpectraMax Gemini XPS microplate reader. Results: We found that sorafenib markedly reduced at µM dose the viability of BC cells. Treatment for 24h with 20µM of sorafenib, triggered “Incomplete autophagy”, that induced apoptosis of BC cells. Sorafenib by inducing an increased cathepsin B activity and pro-apoptotic protein BID activation, triggered a ROS-mediated-mitochondrial-dependent apoptosis of BC cells. Moreover, the increase of cathepsin B activity induced by sorafenib was inhibited by a specific tyrosine phosphatase inhibitor (e.g., orthovanadate) strongly suggesting for a contribute of tyrosine-phosphatases in sorafenib-induced apoptosis. Conclusions: Sorafenib by triggering incomplete autophagy, stimulates a cathepsin B-induced-BID-mediated-ROS- and mitochondrial-dependent apoptosis of BC cells, which is likely regulated by tyrosine-phosphatases.

Estrogens and PTP1B Function in a Novel Pathway to Regulate Aromatase Enzymatic Activity in Breast Cancer Cells

Local estrogen production by aromatase is an important mechanism of autocrine stimulation in hormone-dependent breast cancer. We have previously shown that 17-β estradiol (E(2)) rapidly enhances aromatase enzymatic activity through an increase of tyrosine protein phosphorylation controlled by the activity of the c-Src kinase in breast cancer cells. Here, we investigated the protein tyrosine phosphatase PTP1B (protein tyrosine phosphatase 1B) as a potential regulator of aromatase activity. We demonstrated a specific association between PTP1B and aromatase at protein-protein level and a reduction of aromatase activity in basal and E(2)-treated MCF-7 and ZR75 breast cancer cells when PTP1B was overexpressed. Indeed, a specific tyrosine phosphatase inhibitor increased basal and E(2)-induced enzymatic activity as well as tyrosine phosphorylation status of the purified aromatase protein. Moreover, E(2) through phosphatidylinositol 3 kinase/Akt activation caused a significant decrease of PTP1B catalytic activity along with an increase in its serine phosphorylation. Concomitantly, the phosphatidylinositol 3 kinase inhibitor LY294002 or a dominant negative of Akt was able to reduce the E(2) stimulatory effects on activity and tyrosine phosphorylation levels of aromatase. Taken together, our results suggest that E(2) can impair PTP1B ability to dephosphorylate aromatase, and thus it increases its enzymatic activity, creating a positive feedback mechanism for estradiol signaling in breast cancer.

Tyrosine phosphatase inhibition induces an ASC-dependent pyroptosis

Pyroptosis is a type of cell death in which danger associated molecular patterns (DAMPs) and pathogen associated molecular patterns (PAMPs) induce mononuclear phagocytes to activate caspase-1 and release mature IL-1β. Because the tyrosine kinase inhibitor AG126 can prevent DAMP/PAMP induced activation of caspase-1, we hypothesized that tipping the tyrosine kinase/phosphatase balance toward phosphorylation would promote caspase-1 activation and cell death. THP-1 derived macrophages were therefore treated with the potent specific tyrosine phosphatase inhibitor, sodium orthovanadate (OVN) and analyzed for caspase-1 activation and cell death. OVN induced generalized increase in phosphorylated proteins, IL-1β release and cell death in a time and dose dependent pattern. This OVN induced pyroptosis correlated with speck formations that contained the apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC). Culturing the cells in the presence of extracellular K+ (known to inhibit ATP dependent pyroptosis), a caspase inhibitor (ZVAD) or down regulating the expression of ASC with stable expression of siASC prevented the OVN induced pyroptosis. These data demonstrate that pyroptotic death is linked to tyrosine phosphatase activity providing novel targets for future pharmacologic interventions.

Endoplasmic reticulum stress causes EBV lytic replication

AbstractEndoplasmic reticulum (ER) stress triggers a homeostatic cellular response in mammalian cells to ensure efficient folding, sorting, and processing of client proteins. In lytic-permissive lymphoblastoid cell lines (LCLs), pulse exposure to the chemical ER-stress inducer thapsigargin (TG) followed by recovery resulted in the activation of the EBV immediate-early (BRLF1, BZLF1), early (BMRF1), and late (gp350) genes, gp350 surface expression, and virus release. The protein phosphatase 1 a (PP1a)–specific phosphatase inhibitor Salubrinal (SAL) synergized with TG to induce EBV lytic genes; however, TG treatment alone was sufficient to activate EBV lytic replication. SAL showed ER-stress–dependent and –independent antiviral effects, preventing virus release in human LCLs and abrogating gp350 expression in 12-O-tetradecanoylphorbol-13-acetate (TPA)–treated B95-8 cells. TG resulted in sustained BCL6 but not BLIMP1 or CD138 expression, which is consistent with maintenance of a germinal center B-cell, rather than plasma-cell, phenotype. Microarray analysis identified candidate genes governing lytic replication in LCLs undergoing ER stress.

Reactive oxygen species and p38 MAPK regulate Bax translocation and calcium redistribution in salubrinal-induced apoptosis of EBV-transformed B cells

Salubrinal is a specific eIF2α phosphatase inhibitor that inhibits ER stress-mediated apoptosis. However, maintaining hyper-phosphorylated eIF2α state with high doses of salubrinal treatment promotes apoptosis in some cancer cells. In this report, we found that salubrinal induced apoptosis of EBV-transformed B cells. Notably, salubrinal induced ROS generation and p38 MPAK activation, which then induced expression of FasL. Moreover, salubrinal subsequently led to activation of caspases, calcium redistribution, Bax translocation, cytochrome c release, and apoptosis. These findings suggest that salubrinal may be a novel therapeutic approach for EBV-associated malignant diseases.

Inhibition of MAPK by Prolactin Signaling through the Short Form of Its Receptor in the Ovary and Decidua: INVOLVEMENT OF A NOVEL PHOSPHATASE

Prolactin (PRL) is essential for normal reproduction and signals through two types of receptors, the short (PRL-RS) and long (PRL-RL) form. We have previously shown that transgenic mice expressing only PRL-RS (PRLR(-/-)RS) display abnormal follicular development and premature ovarian failure. Here, we report that MAPK, essential for normal follicular development, is critically inhibited by PRL in reproductive tissues of PRLR(-/-)RS mice. Consequently, the phosphorylation of MAPK downstream targets are also markedly inhibited by PRL without affecting immediate upstream kinases, suggesting involvement of MAPK specific phosphatase(s) in this inhibition. Similar results are obtained in a PRL-responsive ovary-derived cell line (GG-CL) that expresses only PRL-RS. However, we found the expression/activation of several known MAPK phosphatases not to be affected by PRL, suggesting a role of unidentified phosphatase(s). We detected a 27-kDa protein that binds to the intracellular domain of PRL-RS and identified it as dual specific phosphatase DUPD1. PRL does not induce expression of DUDP1 but represses its phosphorylation on Thr-155. We also show a physical association of this phosphatase with ERK1/2 and p38 MAPK. Using an in vitro phosphatase assay and overexpression studies, we established that DUPD1 is a MAPK phosphatase. Dual specific phosphatase inhibitors as well as siRNA to DUPD1, completely prevent PRL-mediated MAPK inhibition in ovarian cells. Our results strongly suggest that deactivation of MAPK by PRL/PRL-RS contributes to the severe ovarian defect in PRLR(-/-)RS mice and demonstrate the novel association of PRL-RS with DUPD1 and a role for this phosphatase in MAPK deactivation.

Endoplasmic reticulum stress inducers provide protection against 6-hydroxydopamine-induced cytotoxicity

6-Hydroxydopamine (6-OHDA) is a neurotoxin used to establish experimental models of Parkinson's disease. Exposure to 6-OHDA results in cell death associated with oxidative stress. Pretreatments with sublethal oxidative stress and some pharmacological drugs have been shown to exert preconditioning effects on cytotoxicity caused by 6-OHDA. In this study, we investigated whether endoplasmic reticulum (ER) stress exerts preconditioning effects on 6-OHDA-induced cytotoxicity in human neuroblastoma SH-SY5Y cells. Pretreatment with ER stress inducers, thapsigargin (Tg) and tunicamycin (Tm), promoted GRP78 mRNA induction and ATF4 translation, which are ER stress markers, under our experimental conditions and protected against the cytotoxicity. The protective effect of Tg was more potent than that of Tm. We also found that Tg induced the expression of the antioxidant gene heme oxygenase-1 (HO-1) in a dose-dependent manner, whereas Tm had a weak effect on HO-1 induction. Flow cytometric analysis revealed that reactive oxygen species generated by 6-OHDA were more effectively suppressed in cells pretreated with Tg than with Tm. Therefore, it is likely that Tg enhances antioxidative defenses in SH-SY5Y cells compared with Tm. Because actinomycin D inhibited HO-1 induction by Tg, the induction of HO-1 may be regulated at the transcriptional level. Moreover, the specific eIF2α phosphatase inhibitor salubrinal augmented Tg-induced HO-1 expression. Therefore, the downstream signaling pathway of eIF2α might be involved in Tg-induced HO-1 expression. On the other hand, the reporter assay revealed that Tg stimulated the antioxidant response element (ARE) that is located in regulatory regions of antioxidant genes such as HO-1. Taken together, our data suggest that preconditioning effects induced by Tg mediate an adaptive response to 6-OHDA-induced cytotoxicity via phosphorylation of eIF2α and activation of the ARE.

ChemInform Abstract: Generation of an Ugi Library of Phosphate Mimic-Containing Compounds and Identification of Novel Dual Specific Phosphatase Inhibitors.

The four-component Ugi reaction was utilized to prepare a library of dipeptidic compounds in order to explore the binding requirements of the key cell cycle phosphatase, Cdc25. Several phosphate surrogates were incorporated into the Ugi product to mimic either the mono- or bis-phosphorylated substrate.

Rga4 Modulates the Activity of the Fission Yeast Cell Integrity MAPK Pathway by Acting as a Rho2 GTPase-activating Protein

Rho GTPase-activating proteins (GAPs) are responsible for the inactivation of Rho GTPases, which are involved in the regulation of critical biological responses in eukaryotic cells, ranging from cell cycle control to cellular morphogenesis. The genome of fission yeast Schizosaccharomyces pombe contains six genes coding for putative Rho GTPases, whereas nine genes code for predicted Rho GAPs (Rga1 to Rga9). One of them, Rga4, has been recently described as a Cdc42 GAP, involved in the control of cell diameter and symmetry in fission yeast. In this work we show that Rga4 is also a Rho2 GAP that negatively modulates the activity of the cell integrity pathway and its main effector, MAPK Pmk1. The DYRK-type protein kinase Pom1, which regulates both the localization and phosphorylation state of Rga4, is also a negative regulator of the Pmk1 pathway, but this control is not dependent upon the Rga4 role as a Rho2-GAP. Hence, two subsets of Rga4 negatively regulate Cdc42 and Rho2 functions in a specific and unrelated way. Finally, we show that Rga7, another Rho2 GAP, down-regulates the Pmk1 pathway in addition to Rga4. These results reinforce the notion of the existence of complex mechanisms determining the selectivity of Rho GAPs toward Rho GTPases and their functions.

Synergistic Apoptosis Induction in Leukemic Cells by the Phosphatase Inhibitor Salubrinal and Proteasome Inhibitors

BackgroundCells adapt to endoplasmic reticulum (ER)-stress by arresting global protein synthesis while simultaneously activating specific transcription factors and their downstream targets. These processes are mediated in part by the phosphorylation-dependent inactivation of the translation initiation factor eIF2α. Following restoration of homeostasis protein synthesis is resumed when the serine/threonine-protein phosphatase PP1 dephosphorylates and reactivates eIF2α. Proteasome inhibitors, used to treat multiple myeloma patients evoke ER-stress and apoptosis by blocking the ER-associated degradation of misfolded proteins (ERAD), however, the role of eIF2α phosphorylation in leukemic cells under conditions of proteasome inhibitor-mediated ER stress is currently unclear.Methodology and Principal FindingsBcr-Abl-positive and negative leukemic cell lines were used to investigate the functional implications of PP1-related phosphatase activities on eIF2α phosphorylation in proteasome inhibitor-mediated ER stress and apoptosis. Rather unexpectedly, salubrinal, a recently identified PP1 inhibitor capable to protect against ER stress in various model systems, strongly synergized with proteasome inhibitors to augment apoptotic death of different leukemic cell lines. Salubrinal treatment did not affect the phosphorlyation status of eIF2α. Furthermore, the proapoptotic effect of salubrinal occurred independently from the chemical nature of the proteasome inhibitor, was recapitulated by a second unrelated phosphatase inhibitor and was unaffected by overexpression of a dominant negative eIF2α S51A variant that can not be phosphorylated. Salubrinal further aggravated ER-stress and proteotoxicity inflicted by the proteasome inhibitors on the leukemic cells since characteristic ER stress responses, such as ATF4 and CHOP synthesis, XBP1 splicing, activation of MAP kinases and eventually apoptosis were efficiently abrogated by the translational inhibitor cycloheximide.ConclusionsAlthough PP1 activity does not play a major role in regulating the ER stress response in leukemic cells, phosphatase signaling nevertheless significantly limits proteasome inhibitor-mediated ER-stress and apoptosis. Inclusion of specific phosphatase inhibitors might therefore represent an option to improve current proteasome inhibitor-based treatment modalities for hematological cancers.

Basal Levels of eIF2α Phosphorylation Determine Cellular Antioxidant Status by Regulating ATF4 and xCT Expression

eIF2alpha is part of a multimeric complex that regulates cap-dependent translation. Phosphorylation of eIF2alpha (phospho-eIF2alpha) is induced by various forms of cell stress, resulting in changes to the proteome of the cell with two diametrically opposed consequences, adaptation to stress or initiation of programmed cell death. In contrast to the robust eIF2alpha phosphorylation seen in response to acute insults, less is known about the functional role of basal levels of eIF2alpha phosphorylation. Here we show that mouse embryonic fibroblasts expressing a nonphosphorylatable eIF2alpha have enhanced sensitivity to diverse toxic insults, including amyloid beta-(1-42) peptide (Abeta), a key factor in the pathogenesis of Alzheimer disease. This correlates with impaired glutathione metabolism because of down-regulation of the light chain, xCT, of the cystine/glutamate antiporter system X(-)(c). The mechanistic link between the absence of phospho-eIF2alpha and xCT expression is nuclear factor ATF4. Consistent with these findings, long term activation of the phospho-eIF2alpha/ATF4/xCT signaling module by the specific eIF2alpha phosphatase inhibitor, salubrinal, induces resistance against oxidative glutamate toxicity in the hippocampal cell line HT22 and primary cortical neurons. Furthermore, in PC12 cells selected for resistance against Abeta, increased activity of the phospho-eIF2alpha/ATF4/xCT module contributes to the resistant phenotype. In wild-type PC12 cells, activation of this module by salubrinal ameliorates the response to Abeta. Furthermore, in human brains, ATF4 and phospho-eIF2alpha levels are tightly correlated and up-regulated in Alzheimer disease, most probably representing an adaptive response against disease-related cellular stress rather than a correlate of neurodegeneration.

Inhibition of protein tyrosine phosphatase 1B and alkaline phosphatase by bis(maltolato)oxovanadium (IV)

Vanadate has been recognized as a specific and potent phosphatase inhibitor since its structure is similar to that of phosphate. In this study, we measured the inhibition of glutathione S-transferase-tagged protein tyrosine phosphatase 1B (GST–PTP1B) and alkaline phosphatase (ALP) by the insulin enhancing compounds, bis(maltolato)oxovanadium(IV) (BMOV). The results showed that the activity of GST–PTP1B was reversibly inhibited by solutions of BMOV with an IC 50 value of 0.86 ± 0.02 μM. Steady state kinetic studies showed that inhibition of GST–PTP1B by BMOV was of a mixed competitive and noncompetitive type. In addition, incubation of GST–PTP1B with BMOV showed a time-dependent biphasic inactivation of the protein. On the other hand, the inhibitory behavior of BMOV on ALP activity was reversible and competitive with an IC 50 value of 32.1 ± 0.6 μM. Incubation with BMOV did not show biphasic inactivation of ALP. The reversible inhibition of GST–PTP1B by BMOV is more potent than that of ALP, but solutions of BMOV inhibited both enzymes. This data support the suggestion that mechanisms for the inhibitory effects of BMOV on GST–PTP1B and ALP are very different.

Studies of small molecule interactions with protein phosphatases using biosensor technology

Reversible protein phosphorylation of serine, threonine, and tyrosine residues by protein kinases and phosphatases is important for the regulation of cellular signal transduction and controls many cellular functions. Disturbances in this regulation have been implicated in a growing number of diseases, making kinases and phosphatases useful targets for therapeutic intervention. The suitability of surface plasmon resonance (SPR) technology has been widely demonstrated in many drug discovery applications. A novel and straightforward methodology is presented for analyzing small molecule binding to two serine/threonine phosphatases, PP1 and PP2B (calcineurin), and to the prototypic tyrosine phosphatase, PTP1B. Emphasis was placed on investigating the immobilization conditions of the phosphatases by using reducing conditions, inhibitors and metal ions. A comparison of inhibitor binding, either to phosphatase (PP2B) alone or in complex with the regulatory protein subunit calmodulin, revealed different kinetics. The methodology was also used to test inhibitor specificity toward different phosphatases. Inhibition of regulatory protein PP-inhibitor-2 binding to PP1 by a small molecule inhibitor was demonstrated. This type of information, together with data on compound binding that is independent of enzyme activity and in which affinities are resolved into kinetic rate constants, may be of great significance for the development of highly specific and high-affinity phosphatase inhibitors.

Protein Phosphatase2A Regulates Stromal Cell- Derived Factor-1 Mediated Responses of CD34+ Cord Blood Cells.

The chemokine stromal cell- derived factor-1(SDF-1) and its receptor, CXCR4, play a major role in migration, retention, and development of hematopoietic progenitors in the bone marrow. During steady-state hematopoiesis, CXCR4/SDF-1 interaction restricts hematopoietic stem progenitor cells in marrow and disruption of the SDF-1/CXCR4 axis leads to their egress into circulation. However, biological responses to SDF-1 are rapidly attenuated by overlapping mechanisms that include peptide degradation by cell surface proteases, receptor uncoupling from heterotrimeric G-proteins and receptor endocytosis. It is known that phosphorylation of G-protein coupled receptors (GPCRs) by several protein kinases including serine-threonine kinase, protein kinase C (PKC), can result in receptor desensitization. This prompted us to investigate whether the role of serine- threonine phosphatase in the regulation of SDF-1 induced responses of CD34+ cord blood (CB) cells. To investigate the role of serine- threonine phosphatase we evaluated the effect of okadaic acid, a specific serine-threonine phosphatase inhibitor, on SDF-1 induced chemotaxis and adhesion of CD34+ cells from cord blood. Pre-incubation of CD34+ CB cells with okadaic acid (100–1000nM) significantly reduced SDF-1 directed chemotaxis and adhesion of CD34+ CB cells. This correlated with an increase in PKC phosphorylation. Although primary SDF-1 induced calcium flux in CD34+CB cells was unaffected by okadaic acid, if the cells were pretreated with SDF-1 and then stimulated with SDF-1, calcium flux was significantly less in CD34+CB cells pretreated with okadaic acid compared to untreated cells. Further, confocal microscopy was used to demonstrate the co-localization of serine threonine phosphatase and PKC. Using antibodies specific to PP2A catalytic subunit (PP2Ac) and various classes of PKC super family, it was observed in unstimulated CD34+ CB cells PP2Ac was mainly localized in nucleus and some phosphor-PKC-delta was detected in the cytoplasm. However, upon SDF-1 stimulation, co-localization of PP2Ac and phospho-PKC-delta was observed on the membrane of CD34+CB cells. These results indicate that serine-threonine phosphatase regulate SDF-1 mediated responses in CD34+CB cells and may play an important role in the ability of hematopoietic stem progenitor cells to continually respond to SDF-1.

PTPL1 is a direct transcriptional target of EWS-FLI1 and modulates Ewing's Sarcoma tumorigenesis

Ewing's Sarcoma family tumors (ESFT) are characterized by a translocation t(11:22) forming an aberrant transcription factor EWS-FLI1. Protein tyrosine phosphatase L1 (PTPL1) was identified as a gene upregulated by EWS-FLI1 in transfected cells by microarray. Our results show that PTPL1 is a transcriptional target of EWS-FLI1 both by chromatin immunoprecipitation and promoter activation studies. We demonstrate that PTPL1 is highly expressed in ESFT cells and patient tumors compared with normal tissues, with a trend towards higher expression in metastatic versus primary tumors. Reduction of PTPL1 protein in ESFT cells correlated with a significant reduction in both monolayer and soft-agar cell growth. In addition, these PTPL1-reduced cells were more sensitive to etoposide-induced apoptosis than the controls. We therefore report a novel transcriptional activation of a phosphatase involved in the oncogenesis of ESFT. Increasing interest in specific phosphatase inhibitors would allow PTPL1 to be evaluated as a therapeutic target in ESFT.