Dataset Information

Regulation of the Met receptor-tyrosine kinase by the protein-tyrosine phosphatase 1B and T-cell phosphatase.

ABSTRACT: The non-receptor protein-tyrosine phosphatases (PTPs) 1B and T-cell phosphatase (TCPTP) have been implicated as negative regulators of multiple signaling pathways including receptor-tyrosine kinases. We have identified PTP1B and TCPTP as negative regulators of the hepatocyte growth factor receptor, the Met receptor-tyrosine kinase. In vivo, loss of PTP1B or TCPTP enhances hepatocyte growth factor-mediated phosphorylation of Met. Using substrate trapping mutants of PTP1B or TCPTP, we have demonstrated that both phosphatases interact with Met and that these interactions require phosphorylation of twin tyrosines (Tyr-1234/1235) in the activation loop of the Met kinase domain. Using confocal microscopy, we show that trapping mutants of both PTP1B and the endoplasmic reticulum-targeted TCPTP isoform, TC48, colocalize with Met and that activation of Met enables the nuclear-localized isoform of TCPTP, TC45, to exit the nucleus. Using small interfering RNA against PTP1B and TCPTP, we demonstrate that phosphorylation of Tyr-1234/1235 in the activation loop of the Met receptor is elevated in the absence of either PTP1B or TCPTP and further elevated upon loss of both phosphatases. This enhanced phosphorylation of Met corresponds to enhanced biological activity and cellular invasion. Our data demonstrate that PTP1B and TCPTP play distinct and non-redundant roles in the regulation of the Met receptor-tyrosine kinase.

SUBMITTER: Sangwan V

PROVIDER: S-EPMC2662243 | biostudies-literature | 2008 Dec

REPOSITORIES: biostudies-literature

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Regulation of the Met receptor-tyrosine kinase by the protein-tyrosine phosphatase 1B and T-cell phosphatase.

Sangwan Veena V Paliouras Grigorios N GN Abella Jasmine V JV Dubé Nadia N Monast Anie A Tremblay Michel L ML Park Morag M

The Journal of biological chemistry 20080926 49

The non-receptor protein-tyrosine phosphatases (PTPs) 1B and T-cell phosphatase (TCPTP) have been implicated as negative regulators of multiple signaling pathways including receptor-tyrosine kinases. We have identified PTP1B and TCPTP as negative regulators of the hepatocyte growth factor receptor, the Met receptor-tyrosine kinase. In vivo, loss of PTP1B or TCPTP enhances hepatocyte growth factor-mediated phosphorylation of Met. Using substrate trapping mutants of PTP1B or TCPTP, we have demonst ...[more]

PMID: 18819921

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Project description:Fms-like tyrosine kinase 3 (FLT3) plays an important role in hematopoietic differentiation, and constitutively active FLT3 mutant proteins contribute to the development of acute myeloid leukemia. Little is known about the protein-tyrosine phosphatases (PTP) affecting the signaling activity of FLT3. To identify such PTP, myeloid cells expressing wild type FLT3 were infected with a panel of lentiviral pseudotypes carrying shRNA expression cassettes targeting different PTP. Out of 20 PTP tested, expressed in hematopoietic cells, or presumed to be involved in oncogenesis or tumor suppression, DEP-1 (PTPRJ) was identified as a PTP negatively regulating FLT3 phosphorylation and signaling. Stable 32D myeloid cell lines with strongly reduced DEP-1 levels showed site-selective hyperphosphorylation of FLT3. In particular, the sites pTyr-589, pTyr-591, and pTyr-842 involved in the FLT3 ligand (FL)-mediated activation of FLT3 were hyperphosphorylated the most. Similarly, acute depletion of DEP-1 in the human AML cell line THP-1 caused elevated FLT3 phosphorylation. Direct interaction of DEP-1 and FLT3 was demonstrated by "substrate trapping" experiments showing association of DEP-1 D1205A or C1239S mutant proteins with FLT3 by co-immunoprecipitation. Moreover, activated FLT3 could be dephosphorylated by recombinant DEP-1 in vitro. Enhanced FLT3 phosphorylation in DEP-1-depleted cells was accompanied by enhanced FLT3-dependent activation of ERK and cell proliferation. Stable overexpression of DEP-1 in 32D cells and transient overexpression with FLT3 in HEK293 cells resulted in reduction of FL-mediated FLT3 signaling activity. Furthermore, FL-stimulated colony formation of 32D cells expressing FLT3 in methylcellulose was induced in response to shRNA-mediated DEP-1 knockdown. This transforming effect of DEP-1 knockdown was consistent with a moderately increased activation of STAT5 upon FL stimulation but did not translate into myeloproliferative disease formation in the 32D-C3H/HeJ mouse model. The data indicate that DEP-1 is negatively regulating FLT3 signaling activity and that its loss may contribute to but is not sufficient for leukemogenic cell transformation.

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.

Dataset Information

Regulation of the Met receptor-tyrosine kinase by the protein-tyrosine phosphatase 1B and T-cell phosphatase.

Publications

Regulation of the Met receptor-tyrosine kinase by the protein-tyrosine phosphatase 1B and T-cell phosphatase.

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