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The role of protein tyrosine phosphatase 1B in Ras signaling.

ABSTRACT: Protein tyrosine phosphatase (PTP) 1B has been implicated as a negative regulator of multiple signaling pathways downstream of receptor tyrosine kinases. Inhibition of this enzyme was initially thought to potentially lead to increased oncogenic signaling and tumorigenesis. Surprisingly, we show that platelet-derived growth factor-stimulated extracellular-regulated kinase signaling in PTP1B-deficient cells is not significantly hyperactivated. Moreover, these cells exhibit decreased Ras activity and reduced proliferation by way of previously uncharacterized pathways. On immortalization, PTP1B-deficient fibroblasts display increased expression of Ras GTPase-activating protein (p120RasGAP). Furthermore, we demonstrate that p62Dok (downstream of tyrosine kinase) is a putative substrate of PTP1B and that tyrosine phosphorylation of p62Dok is indeed increased in PTP1B-deficient cells. Consistent with the decreased Ras activity in cells lacking PTP1B, introduction of constitutively activated Ras restored extracellular-regulated kinase signaling and their proliferative potential to those of WT cells. These results indicate that loss of PTP1B can lead to decreased Ras signaling, despite enhanced signaling of other pathways. This finding may in part explain the absence of increased tumor incidence in PTP1B-deficient mice. Thus, PTP1B can positively regulate Ras activity by acting on pathways distal to those of receptor tyrosine kinases.

SUBMITTER: Dube N

PROVIDER: S-EPMC357013 | biostudies-other | 2004 Feb

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:Vascular endothelial growth factor (VEGF) binding induces phosphorylation of VEGF receptor (VEGFR)2 in tyrosine, which is followed by disruption of VE-cadherin-mediated cell-cell contacts of endothelial cells (ECs), thereby stimulating EC proliferation and migration to promote angiogenesis. Tyrosine phosphorylation events are controlled by the balance of activation of protein tyrosine kinases and protein tyrosine phosphatases (PTPs). Little is known about the role of endogenous PTPs in VEGF signaling in ECs. In this study, we found that PTP1B expression and activity are markedly increased in mice hindlimb ischemia model of angiogenesis. In ECs, overexpression of PTP1B, but not catalytically inactive mutant PTP1B-C/S, inhibits VEGF-induced phosphorylation of VEGFR2 and extracellular signal-regulated kinase 1/2, as well as EC proliferation, whereas knockdown of PTP1B by small interfering RNA enhances these responses, suggesting that PTP1B negatively regulates VEGFR2 signaling in ECs. VEGF-induced p38 mitogen-activated protein kinase phosphorylation and EC migration are not affected by PTP1B overexpression or knockdown. In vivo dephosphorylation and cotransfection assays reveal that PTP1B binds to VEGFR2 cytoplasmic domain in vivo and directly dephosphorylates activated VEGFR2 immunoprecipitates from human umbilical vein endothelial cells. Overexpression of PTP1B stabilizes VE-cadherin-mediated cell-cell adhesions by reducing VE-cadherin tyrosine phosphorylation, whereas PTP1B small interfering RNA causes opposite effects with increasing endothelial permeability, as measured by transendothelial electric resistance. In summary, PTP1B negatively regulates VEGFR2 receptor activation via binding to the VEGFR2, as well as stabilizes cell-cell adhesions through reducing tyrosine phosphorylation of VE-cadherin. Induction of PTP1B by hindlimb ischemia may represent an important counterregulatory mechanism that blunts overactivation of VEGFR2 during angiogenesis in vivo.

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The role of protein tyrosine phosphatase 1B in Ras signaling.

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