A phosphotyrosine displacement mechanism for activation of Src by PTPalpha.
ABSTRACT Protein tyrosine phosphatase alpha (PTPalpha) is believed to dephosphorylate physiologically the Src proto-oncogene at phosphotyrosine (pTyr)527, a critical negative-regulatory residue. It thereby activates Src, and PTPalpha overexpression neoplastically transforms NIH 3T3 cells. pTyr789 in PTPalpha is constitutively phosphorylated and binds Grb2, an interaction that may inhibit PTPalpha activity. We show here that this phosphorylation also specifically enables PTPalpha to dephosphorylate pTyr527. Tyr789-->Phe mutation abrogates PTPalpha-Src binding, dephosphorylation of pTyr527 (although not of other substrates), and neoplastic transformation by overexpressed PTPalpha in vivo. We suggest that pTyr789 enables pTyr527 dephosphorylation by a pilot binding with the Src SH2 domain that displaces the intramolecular pTyr527-SH2 binding. Consistent with model predictions, we find that excess SH2 domains can disrupt PTPalpha-Src binding and can block PTPalpha-mediated dephosphorylation and activation in proportion to their affinity for pTyr789. Moreover, we show that, as predicted by the model, catalytically defective PTPalpha has reduced Src binding in vivo. The displacement mechanism provides another potential control point for physiological regulation of Src-family signal transduction pathways.
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ABSTRACT: Cell-cell adhesion couples the contractile cortices of epithelial cells together, generating tension to support a range of morphogenetic processes. E-cadherin adhesion plays an active role in generating junctional tension, by promoting actin assembly and cortical signaling pathways that regulate Myosin II. Multiple Myosin II paralogs accumulate at mammalian epithelial cell-cell junctions. Earlier we found that Myosin IIA responds to Rho-ROCK signaling to support junctional tension in MCF-7 cells. Although Myosin IIB is also found at the zonula adherens (ZA) in these cells, its role in junctional contractility, and its mode of regulation, are less well understood. We now demonstrate that Myosin IIB contributes to tension at the epithelial ZA. Further, we identify a RPTPα-SFK-Rap1 pathway as responsible for recruiting Myosin IIB to the ZA and supporting contractile tension. Overall, these findings reinforce the concept that orthogonal E-cadherin-based signaling pathways recruit distinct Myosin II paralogs to generate the contractile apparatus at apical epithelial junctions. © 2015 by The American Society for Cell Biology.
Dataset: Mol. Biol. Cell-2015-Gomez-1249-62
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ABSTRACT: The physiological Src proto-oncogene is a protein-tyrosine kinase that plays key roles in cell growth, division, migration, and survival signaling pathways. From the N- to C-terminus, Src contains a unique domain, an SH3 domain, an SH2 domain, a protein-tyrosine kinase domain, and a regulatory tail. The chief phosphorylation sites of human Src include an activating pTyr419 that results from phosphorylation in the kinase domain by an adjacent Src molecule and an inhibitory pTyr530 in the regulatory tail that results from phosphorylation by C-terminal Src kinase (Csk) or Chk (Csk homologous kinase). The oncogenic Rous sarcoma viral protein lacks the equivalent of Tyr530 and is constitutively activated. Inactive Src is stabilized by SH2 and SH3 domains on the rear of the kinase domain where they form an immobilizing and inhibitory clamp. Protein kinases including Src contain hydrophobic regulatory and catalytic spines and collateral shell residues that are required to assemble the active enzyme. In the inactive enzyme, the regulatory spine contains a kink or a discontinuity with a structure that is incompatible with catalysis. The conversion of inactive to active Src is accompanied by electrostatic exchanges involving the breaking and making of distinct sets of kinase domain salt bridges and hydrogen bonds. Src-catalyzed protein phosphorylation requires the participation of two Mg(2+) ions. Although nearly all protein kinases possess a common K/E/D/D signature, each enzyme exhibits its unique variations of the protein-kinase reaction template. Bosutinib, dasatinib, and ponatinib are Src/multikinase inhibitors that are approved by the FDA for the treatment of chronic myelogenous leukemia and vandetanib is approved for the treatment of medullary thyroid cancer. The Src and BCR-Abl inhibitors saracatinib and AZD0424, along with the previous four drugs, are in clinical trials for a variety of solid tumors including breast and lung cancers. Both ATP and targeted therapeutic Src protein kinase inhibitors such as dasatinib and ponatinib make hydrophobic contacts with catalytic spine residues and form hydrogen bonds with hinge residues connecting the small and large kinase lobes. Copyright © 2015. Published by Elsevier Ltd.Pharmacological Research 02/2015; 94. DOI:10.1016/j.phrs.2015.01.003 · 3.98 Impact Factor
Ross J Resnick