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Protein tyrosine phosphatase PTP-S binds to the juxtamembrane region of the hepatocyte growth factor receptor Met.

ABSTRACT: We reported previously that a protein tyrosine phosphatase (PTP) activity is associated with the immunoprecipitated hepatocyte growth factor (HGF) receptor, also known as Met. The activity increased reversibly when Met was stimulated by HGF and decreased when Met was inactivated by PMA. To identify the PTP-binding region, we used deletion mutants of the receptor beta-subunit. The PTP activity did not associate with Tpr-Met, a construct containing residues 1010-1390 of Met fused to Tpr. In contrast, PTP activity was present when the expressed protein contained the full juxtamembrane region (residues 956-1390 of Met) or part of this region (residues 957-1390 or 995-1390), indicating that the PTP-binding region is between residues 995 and 1009. This region includes Tyr1003, a site involved in Met downstream signalling. Incubation of Met immunoprecipitated from GTL-16 cells with an 8-mer phosphopeptide derived from residues 1003-1010 induced a marked decrease in the associated PTP activity, suggesting that the peptide reproduced the PTP-binding region. Mutation of Glu, Asp or Arg at positions -4, -1 or +1 respectively relative to Tyr1003 in a 9-mer peptide (residues 999-1007) abolished the ability of the peptide to decrease the PTP activity associated with Met. Phosphorylation of Tyr1003 was not required for PTP binding, since: (1) both phospho- and dephospho-peptides on a solid bead bound PTP activity from a GTL-16 cell extract, and (2) PTP activity was associated with a Met deletion mutant lacking residues 1-955 in which Tyr1003 had been changed into Phe. In order to partially purify the PTP from the GTL-16 cell extract, an affinity column was prepared using the Met-derived peptide comprising residues 998-1007. Less than 0.1% of the total cellular PTP was retained by the column, and was eluted with low salt concentrations. Using antibodies, this PTP was identified as PTP-S, a soluble PTP present in epithelial cells and fibroblasts.

SUBMITTER: Villa-Moruzzi E

PROVIDER: S-EPMC1219863 | biostudies-other | 1998 Nov

REPOSITORIES: biostudies-other

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Project description:The tyrosine kinase Fyn has two regulatory tyrosine residues that when phosphorylated either activate (Tyr(420)) or inhibit (Tyr(531)) Fyn activity. Within the central nervous system, two protein tyrosine phosphatases (PTPs) target these regulatory tyrosines in Fyn. PTP? dephosphorylates Tyr(531) and activates Fyn, while STEP (STriatal-Enriched protein tyrosine Phosphatase) dephosphorylates Tyr(420) and inactivates Fyn. Thus, PTP? and STEP have opposing functions in the regulation of Fyn; however, whether there is cross talk between these two PTPs remains unclear. Here, we used molecular techniques in primary neuronal cultures and in vivo to demonstrate that STEP negatively regulates PTP? by directly dephosphorylating PTP? at its regulatory Tyr(789). Dephosphorylation of Tyr(789) prevents the translocation of PTP? to synaptic membranes, blocking its ability to interact with and activate Fyn. Genetic or pharmacologic reduction in STEP61 activity increased the phosphorylation of PTP? at Tyr(789), as well as increased translocation of PTP? to synaptic membranes. Activation of PTP? and Fyn and trafficking of GluN2B to synaptic membranes are necessary for ethanol (EtOH) intake behaviors in rodents. We tested the functional significance of STEP61 in this signaling pathway by EtOH administration to primary cultures as well as in vivo, and demonstrated that the inactivation of STEP61 by EtOH leads to the activation of PTP?, its translocation to synaptic membranes, and the activation of Fyn. These findings indicate a novel mechanism by which STEP61 regulates PTP? and suggest that STEP and PTP? coordinate the regulation of Fyn. STEP61 , PTP?, Fyn, and NMDA receptor (NMDAR) have been implicated in ethanol intake behaviors in the dorsomedial striatum (DMS) in rodents. Here, we report that PTP? is a novel substrate for STEP61. Upon ethanol exposure, STEP61 is phosphorylated and inactivated by protein kinase A (PKA) signaling in the DMS. As a result of STEP61 inhibition, there is an increase in the phosphorylation of PTP?, which translocates to lipid rafts and activates Fyn and subsequent NMDAR signaling. The results demonstrate a synergistic regulation of Fyn-NMDAR signaling by STEP61 and PTP?, which may contribute to the regulation of ethanol-related behaviors. NMDA, N-methyl-D-aspartate; PTP?, receptor-type protein tyrosine phosphatase alpha; STEP, STriatal-Enriched protein tyrosine Phosphatase.

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Protein tyrosine phosphatase PTP-S binds to the juxtamembrane region of the hepatocyte growth factor receptor Met.

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