Reactive oxygen species mediate phorbol ester-regulated tyrosine phosphorylation and phospholipase A2 activation: potentiation by vanadate.
ABSTRACT: We have previously shown that vanadate potentiates the activating effect of phorbol ester (TPA) on cellular phospholipase A2 (PLA2) in a pathway dependent on the formation of reactive oxygen species (ROS). Here we evaluate the chain of enzymes (protein kinases and phosphatases) that participate in this process. Treatment of macrophages with vanadate plus TPA led to activation of protein kinase C (PKC) and NADPH oxidase (O2- generation in intact cells), massive cellular protein tyrosine phosphorylation, suppression of protein tyrosine phosphatase (PTP) activity and a sustained activation of protein tyrosine kinase (PTK) and myelin basic protein kinase activity (the latter three enzyme activities were assessed in cell lysates). Inhibition of ROS formation by diphenyleneiodonium (DPI) prevented PTP inhibition, PTK activation and protein tyrosine phosphorylation by vanadate plus TPA. Vanadate plus H2O2 mimicked the effect of vanadate plus TPA on PKC activation, cellular protein tyrosine phosphorylation, PTP and PTK, but their effects were resistant to DPI. Suppression of PKC activity (down-regulation; selective inhibitors) prevented the above-mentioned effects of vanadate plus TPA, but not of vanadate plus H2O2. Collectively, the results show that ROS formation induced by TPA in association with vanadate is essential in the modulation of protein tyrosine phosphorylation and PLA2 activity.
Project description:The anion-exchange band 3 protein is the main erythrocyte protein that is phosphorylated by protein tyrosine kinase (PTK). We have previously identified a band 3-associated phosphotyrosine phosphatase (PTP) that is normally highly active and prevents the accumulation of band 3 phosphotyrosine. Band 3 tyrosine phosphorylation can be induced by inhibition of PTP (vanadate, thiol oxidation), activation of PTK (hypertonic NaCl) or intracellular increased Ca(2+) (mechanism unknown). We now show that there is inhibition of dephosphorylation of band 3 in Ca(2+)/ionophore-treated erythrocytes and in membranes isolated from the treated cells. These membranes exhibit phosphatase activity upon the addition of exogenous substrate. Dephosphorylation of the endogenous substrate (band 3) can be activated in these membranes by the addition of Mg(2+). Thus the inability of PTP to dephosphorylate the band 3 phosphotyrosine is not due to inhibition of the enzyme itself. Ca(2+) rise in the erythrocyte causes dissociation of PTP from band 3, thus leaving the kinase unopposed. This is shown by a significant diminution in band 3/PTP co-precipitation. Addition of Mg(2+) to these membranes leads to reassociation of band 3 with PTP. The Ca(2+)-induced inhibition of band 3 dephosphorylation may be due to Ca(2+)-dependent alterations in membrane components and structure, affecting the interaction of band 3 with PTP. The Ca(2+)-induced tyrosine phosphorylation, involving an apparent PTP inhibition via dissociation from the substrate, may play a role in signal transduction pathways and in certain pathological disorders associated with increased cell Ca(2+).
Project description:We investigated the mechanism by which the G-protein activators aluminium fluoride and vanadate stimulate arachidonic acid release in pig aortic endothelial cells. Our previous study demonstrated a novel Ca(2+)-independent pathway of phospholipase A2 (PLA2) activation stimulated by aluminium fluoride in this model. In the present study, we found that sodium metavanadate stimulated a rapid concentration-dependent release of [3H]arachidonic acid from prelabelled cells. A more than 3-fold enhancement of arachidonic acid release was achieved in cells treated with 1 mM vanadate for 20 min. Synthesis of prostaglandin products was similarly enhanced. The release of arachidonic acid was not dependent on the presence of extracellular Ca2+, but did require protein synthesis de novo. Both cycloheximide and actinomycin D completely blocked aluminium fluoride- and vanadate-stimulated arachidonic acid release. Because fluoride and vanadate are known protein tyrosine phosphatase inhibitors, it is possible that PLA2 activation occurred secondarily to changes in protein tyrosine phosphorylation. Both aluminium fluoride and vanadate stimulated the rapid phosphorylation of 58, 93 and 120 kDa tyrosine-containing protein substrates. However, in contrast with arachidonic acid release, this response was found to be sensitive to the presence of extracellular Ca2+ and insensitive to blockers of protein synthesis de novo. Furthermore H2O2 treatment resulted in rapid tyrosine phosphorylation of the same substrates without a concomitant increase in arachidonic acid release. These results suggest that the effects of aluminium fluoride and vanadate on PLA2 are not due to changes in protein tyrosine phosphorylation, but do require rapid protein synthesis de novo.
Project description:Protein phosphatase activity specific for Tyr(P) (phosphotyrosine) residues (PTP-phosphatase) was found in the cytosol and particulate fractions of human placenta. In the particulate fraction, half of the PTP-phosphatase activity could be extracted with 1% Triton X-100. The PTP-phosphatase remaining in the Triton-insoluble residue was solubilized with 0.6 M-KCl plus 1% CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]propane-1-sulphonate) and was purified 1850-fold by adsorption to DEAE-Sepharose, affinity chromatography on Zn2+-iminodiacetate-agarose, phosphocellulose adsorption, Fractogel filtration and Mono Q chromatography. The cytoskeleton-associated PTP-phosphatase was distinguished from acid, alkaline and other protein Ser(P) (phosphoserine)/Thr(P) (phosphothreonine) phosphatases by its neutral pH optimum, activity in the presence of EDTA, inhibition by Zn2+, vanadate, or molybdate, and low activity with either [Ser(P)]phosphorylase a or p-nitrophenyl phosphate. The PTP-phosphate displayed a Km of 0.15 microM with [Tyr(P)]serum albumin as substrate, 10-100-fold lower than the Km for previously described protein phosphatases. The cytoskeleton-associated PTP-phosphatase catalysed the dephosphorylation of receptors for insulin, insulin-like growth factor-1 and epidermal growth factor labelled by autophosphorylation. The properties of this PTP-phosphatase suggest that it plays a role in the regulation of hormone receptors and cytoskeleton proteins by reversible phosphorylation on tyrosine residues.
Project description:The families of protein tyrosine phosphatases (PTPs) and protein tyrosine kinases (PTKs) function in a coordinated manner to regulate signal transduction events that are critical for cellular homeostasis. Aberrant tyrosine phosphorylation, resulting from disruption of either PTP or PTK function, has been shown to be the cause of major human diseases, including cancer and diabetes. Consequently, the characterization of small-molecule inhibitors of these kinases and phosphatases may not only provide molecular probes with which to define the significance of particular signaling events, but also may have therapeutic implications. BAY-11-7082 is an anti-inflammatory compound that has been reported to inhibit I?B kinase activity. The compound has an ?,?-unsaturated electrophilic center, which confers the property of being a Michael acceptor; this suggests that it may react with nucleophilic cysteine-containing proteins, such as PTPs. In this study, we demonstrated that BAY-11-7082 was a potent, irreversible inhibitor of PTPs. Using mass spectrometry, we have shown that BAY-11-7082 inactivated PTPs by forming a covalent adduct with the active-site cysteine. Administration of the compound caused an increase in protein tyrosine phosphorylation in RAW 264 macrophages, similar to the effects of the generic PTP inhibitor sodium orthovanadate. These data illustrate that BAY-11-7082 is an effective pan-PTP inhibitor with cell permeability, revealing its potential as a new probe for chemical biology approaches to the study of PTP function. Furthermore, the data suggest that inhibition of PTP function may contribute to the many biological effects of BAY-11-7082 that have been reported to date.
Project description:Transactivation of the epidermal growth factor (EGF) receptor (EGFR) has been proposed to represent an essential link between G-protein-coupled receptors and the mitogen-activated protein kinase (MAPK) pathway in various cell types. In the present work we report, in contrast, that in A431 cells bradykinin transinactivates the EGFR and stimulates MAPK activity independently of EGFR tyrosine phosphorylation. Both effects of bradykinin are mediated by a pertussis-toxin-insensitive G-protein. Three lines of evidence suggest the activation of a protein tyrosine phosphatase (PTP) by bradykinin: (i) treatment of A431 cells with bradykinin decreases both basal and EGF-induced EGFR tyrosine phosphorylation, (ii) this effect of bradykinin can be blocked by two different PTP inhibitors, and (iii) bradykinin significantly increased the PTP activity in total A431 cell lysates when measured in vitro. The transmembrane receptor PTP sigma was identified as a putative mediator of bradykinin-induced downregulation of EGFR autophosphorylation. Activation of MAPK in response to bradykinin was insensitive towards AG 1478, a specific inhibitor of EGFR tyrosine kinase, but was blocked by wortmannin or bisindolylmaleimide, inhibitors of phosphatidylinositol 3-kinase (PI3-K) and protein kinase C (PKC) respectively. These results also suggest that the bradykinin-induced activation of MAPK is independent of EGFR and indicate a pathway involving PI3-K and PKC. In addition, bradykinin evokes a rapid and transient increase in Src kinase activity. Although Src does not participate in bradykinin-induced stimulation of PTP activity, inhibition of Src by 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo(3,4-d)pyrimidine leads to an increase in MAPK activation by bradykinin. Our results suggest that in A431 cells the G(q/11)-protein-coupled bradykinin B(2) receptor may stimulate PTP activity and thereby transinactivate the EGFR, and may simultaneously activate MAPK by an alternative signalling pathway which can bypass EGFR.
Project description:The introduction of second-generation tyrosine kinase inhibitors (TKIs) targeting the protein-tyrosine kinase (PTK) BCR-ABL1 has improved treatment response in chronic myeloid leukemia (CML). However, in some patients response still remains suboptimal. Protein-tyrosine phosphatases (PTPs) are natural counter-actors of PTK activity and can affect TKI sensitivity, but the impact of PTPs on treatment response to second-generation TKIs is unknown. We assessed the mRNA expression level of 38 PTPs in 66 newly diagnosed CML patients and analyzed the potential relation with treatment outcome after 9 months of nilotinib medication. A significantly positive association with response was observed for higher PTPN13, PTPRA, PTPRC (also known as CD45), PTPRG, and PTPRM expression. Selected PTPs were then subjected to a functional analysis in CML cell line models using PTP gene knockout by CRISPR/Cas9 technology or PTP overexpression. These analyses revealed PTPRG positively and PTPRC negatively modulating nilotinib response. Consistently, PTPRG negatively and PTPRC positively affected BCR-ABL1 dependent transformation. We identified BCR-ABL1 signaling events, which were affected by modulating PTP levels or nilotinib treatment in the same direction. In conclusion, the PTP status of CML cells is important for the response to second generation TKIs and may help in optimizing therapeutic strategies.
Project description:Transmitter release at synapses is regulated by preceding neuronal activity, which can give rise to short-term enhancement of release like post-tetanic potentiation (PTP). Diacylglycerol (DAG) and Protein-kinase C (PKC) signaling in the nerve terminal have been widely implicated in the short-term modulation of transmitter release, but the target protein of PKC phosphorylation during short-term enhancement has remained unknown. Here, we use a gene-replacement strategy at the calyx of Held, a large CNS model synapse that expresses robust PTP, to study the molecular mechanisms of PTP. We find that two PKC phosphorylation sites of Munc18-1 are critically important for PTP, which identifies the presynaptic target protein for the action of PKC during PTP. Pharmacological experiments show that a phosphatase normally limits the duration of PTP, and that PTP is initiated by the action of a 'conventional' PKC isoform. Thus, a dynamic PKC phosphorylation/de-phosphorylation cycle of Munc18-1 drives short-term enhancement of transmitter release during PTP. DOI: http://dx.doi.org/10.7554/eLife.01715.001.
Project description:Endothelial cells possess constitutive or inducible cyclo-oxygenase (COX) isoenzymes for prostacyclin production, but the mechanisms for their expression are largely unknown. We found that vanadate, an inhibitor of protein-tyrosine phosphatases, induced the expression of two COX isoenzyme mRNAs in human umbilical vein endothelial cells (HUVEC) in a time- and dose-dependent manner. Vanadate also stimulated an increase in COX-2 protein levels, but did not affect significantly the levels of constitutively expressed COX-1 protein. Synergistic enhancement of expression of the two COX isoenzyme mRNAs was observed on stimulation of HUVEC with vanadate plus interleukin-1alpha. Tyrphostin-47, which as an inhibitor of protein-tyrosine kinases abolished vanadate-induced protein-tyrosine phosphorylation, inhibited expression of the two COX isoenzyme mRNAs in HUVEC stimulated with vanadate or interleukin-1alpha. These data provide conclusive evidence that activation of protein-tyrosine kinases is causally linked to expression of the mRNAs for the two COX isoenzymes in HUVEC.
Project description:The guanine nucleotide exchange factor, C3G (RapGEF1), functions in multiple signaling pathways involved in cell adhesion, proliferation, apoptosis and actin reorganization. C3G is regulated by tyrosine phosphorylation on Y504, known to be mediated by c-Abl and Src family kinases. In the present study we explored the possibility of cellular phospho-C3G (pC3G) being a substrate of the intracellular T-cell protein tyrosine phosphatase TC-PTP (PTPN2) using the human neuroblastoma cell line, IMR-32. In vivo and in vitro binding assays demonstrated interaction between C3G and TC-PTP. Interaction is mediated through the Crk-binding region of C3G and C-terminal noncatalytic residues of TC-PTP. C3G interacted better with a substrate trap mutant of TC48 and this complex formation was inhibited by vanadate. Endogenous pC3G colocalized with catalytically inactive mutant TC48 in the Golgi. Expression of TC48 abrogated pervanadate and c-Src induced phosphorylation of C3G without affecting total cellular phospho-tyrosine. Insulin-like growth factor treatment of c-Src expressing cells resulted in dephosphorylation of C3G dependent on the activity of endogenous TC48. TC48 expression inhibited forskolin induced tyrosine phosphorylation of C3G and neurite outgrowth in IMR-32 cells. Our results identify a novel Golgi localized substrate of TC48 and delineate a role for TC48 in dephosphorylation of substrates required during differentiation of human neuroblastoma cells.
Project description:Protein tyrosine phosphatases (PTPs) are a diverse family of enzymes encoded by 107 genes in the human genome. Together with protein tyrosine kinases (PTKs), PTPs regulate various cellular activities essential for the initiation and maintenance of malignant phenotypes. While PTK inhibitors are now used routinely for cancer treatment, the PTP inhibitor development field is still in the discovery phase. In this article, the suitability of targeting PTPs for novel anticancer drug discovery is discussed. Examples are presented for PTPs that have been targeted for anticancer drug discovery as well as potential new PTP targets for novel anticancer drug discovery.