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Protein tyrosine phosphatase 1B participates in the down-regulation of erythropoietin receptor signalling.

ABSTRACT: Erythropoietin (EPO) is the principal hormone regulating the proliferation of erythroid precursors and their differentiation into erythrocytes. Binding of ligand to the cell-surface EPO-R (EPO receptor) induces dimerization and JAK2 (Janus kinase 2)-mediated tyrosine phosphorylation of the receptor. Less than 1% of the EPO-Rs are displayed on the cell surface; most of the receptor molecules are retained in intracellular compartments, including the ER (endoplasmic reticulum). Using pervanadate (PV) as a potent tool to inhibit cellular PTPs (protein tyrosine phosphatases), we demonstrated previously the accumulation of mature (endoglycosidase H-resistant) tyrosine-phosphorylated EPO-R [Cohen, Altaratz, Zick, Klingmuller and Neumann (1997) Biochem. J. 327, 391-397]. In the present study, we investigated the participation of the ER-associated PTP1B in the dephosphorylation of intracellular EPO-R. We demonstrate tyrosine phosphorylation of EPO-R in BOSC-23T cells co-expressing EPO-R and the 'substrate-trapping' mutant form of PTP1B, PTP1B D181A (referred to as PTP1BD). In vivo interaction between EPO-R and PTP1B suggested that PTP1B dephosphorylates the EPO-R intracellularly. Endoglycosidase H resistance of tyrosine-phosphorylated EPO-R in cells expressing PTP1BD suggested that mature EPO-R is dephosphorylated by PTP1B. Stimulation with EPO of cells co-expressing EPO-R and either PTP1BD or PTP1B resulted in an increase or decrease respectively in phosphotyrosine EPO-R. We thus suggest that PTP1B dephosphorylates EPO-stimulated EPO-R and participates in the down-regulation cascade of EPO-mediated signal transduction.

SUBMITTER: Cohen J

PROVIDER: S-EPMC1223869 | biostudies-other | 2004 Jan

REPOSITORIES: biostudies-other

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Project description:A new paradigm in metallobiochemistry describes the activation of inactive metalloenzymes by metal ion removal. Protein tyrosine phosphatases (PTPs) do not seem to require a metal ion for enzymatic activity. However, both metal cations and metal anions modulate their enzymatic activity. One binding site is the phosphate binding site at the catalytic cysteine residue. Oxyanions with structural similarity to phosphate, such as vanadate, inhibit the enzyme with nanomolar to micromolar affinities. In addition, zinc ions (Zn2+) inhibit with picomolar to nanomolar affinities. We mapped the cation binding site close to the anion binding site and established a specific mechanism of inhibition occurring only in the closed conformation of the enzyme when the catalytic cysteine is phosphorylated and the catalytic aspartate moves into the active site. We discuss this dual inhibition by anions and cations here for PTP1B, the most thoroughly investigated protein tyrosine phosphatase. The significance of the inhibition in phosphorylation signaling is becoming apparent only from the functions of PTP1B in the biological context of metal cations as cellular signaling ions. Zinc ion signals complement redox signals but provide a different type of control and longer lasting inhibition on a biological time scale owing to the specificity and affinity of zinc ions for coordination environments. Inhibitor design for PTP1B and other PTPs is a major area of research activity and interest owing to their prominent roles in metabolic regulation in health and disease, in particular cancer and diabetes. Our results explain the apparent dichotomy of both cations (Zn2+) and oxyanions such as vanadate inhibiting PTP1B and having insulin-enhancing ("anti-diabetic") effects and suggest different approaches, namely targeting PTPs in the cell by affecting their physiological modulators and considering a metallodrug approach that builds on the knowledge of the insulin-enhancing effects of both zinc and vanadium compounds.

Project description:Because of their antagonistic catalytic functions, protein-tyrosine phosphatases (PTPs) and protein-tyrosine kinases act together to control phosphotyrosine-mediated signaling processes in mammalian cells. However, unlike for protein-tyrosine kinases, little is known about the cellular substrate specificity of many PTPs because of the lack of appropriate methods for the systematic and detailed analysis of cellular PTP function. Even for the most intensely studied, prototypic family member PTP1B many of its physiological functions cannot be explained by its known substrates. To gain better insights into cellular PTP1B function, we used quantitative MS to monitor alterations in the global tyrosine phosphorylation of PTP1B-deficient mouse embryonic fibroblasts in comparison with their wild-type counterparts. In total, we quantified 124 proteins containing 301 phosphotyrosine sites under basal, epidermal growth factor-, or platelet-derived growth factor-stimulated conditions. A subset of 18 proteins was found to harbor hyperphosphorylated phosphotyrosine sites in knock-out cells and was functionally linked to PTP1B. Among these proteins, regulators of cell motility and adhesion are overrepresented, such as cortactin, lipoma-preferred partner, ZO-1, or p120ctn. In addition, regulators of proliferation like p62DOK or p120RasGAP also showed increased cellular tyrosine phosphorylation. Physical interactions of these proteins with PTP1B were further demonstrated by using phosphatase-inactive substrate-trapping mutants in a parallel MS-based analysis. Our results correlate well with the described phenotype of PTP1B-deficient fibroblasts that is characterized by an increase in motility and reduced cell proliferation. The presented study provides a broad overview about phosphotyrosine signaling processes in mouse fibroblasts and, supported by the identification of various new potential substrate proteins, indicates a central role of PTP1B within cellular signaling networks. Importantly the MS-based strategies described here are entirely generic and can be used to address the poorly understood aspects of cellular PTP function.

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Protein tyrosine phosphatase 1B participates in the down-regulation of erythropoietin receptor signalling.

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