Regulation of Signaling at Regions of Cell-Cell Contact byEndoplasmic Reticulum-Bound Protein-TyrosinePhosphatase 1BFawaz G. Haj 1,2 *, Ola Sabet 3 , Ali Kinkhabwala 3 , Sabine Wimmer-Kleikamp 3 , Vassilis Roukos 3 , Hong-Mei Han 3 , Markus Grabenbauer 3 , Martin Bierbaum 3 , Claude Antony 4 , Benjamin G. Neel 1,5 *,Philippe I. Bastiaens 3 *1Beth Israel Deaconess Medical Center, Harvard University, Boston, Massachusetts, United States of America, 2Nutrition Department, University of California Davis, Davis,California, United States of America, 3Department of Systemic Cell Biology, Max Planck Institute of Molecular Physiology, Dortmund, Germany, 4European MolecularBiology Laboratories, Heidelberg, Germany, 5Campbell Family Cancer Research Institute, Ontario Cancer Institute, Princess Margaret Hospital, University Health Network,and Department of Medical Biophysics, University of Toronto, Toronto, Ontario, CanadaAbstractProtein-tyrosine phosphatase 1B (PTP1B) is a ubiquitously expressed PTP that is anchored to the endoplasmic reticulum(ER). PTP1B dephosphorylates activated receptor tyrosine kinases after endocytosis, as they transit past the ER. However,PTP1B also can access some plasma membrane (PM)-bound substrates at points of cell-cell contact. To explore how PTP1Binteracts with such substrates, we utilized quantitative cellular imaging approaches and mathematical modeling of proteinmobility. We find that the ER network comes in close proximity to the PM at apparently specialized regions of cell-cellcontact, enabling PTP1B to engage substrate(s) at these sites. Studies using PTP1B mutants show that the ER anchor playsan important role in restricting its interactions with PM substrates mainly to regions of cell-cell contact. In addition,treatment with PTP1B inhibitor leads to increased tyrosine phosphorylation of EphA2, a PTP1B substrate, specifically atregions of cell-cell contact. Collectively, our results identify PM-proximal sub-regions of the ER as important sites of cellularsignaling regulation by PTP1B.Citation: Haj FG, Sabet O, Kinkhabwala A, Wimmer-Kleikamp S, Roukos V, et al. (2012) Regulation of Signaling at Regions of Cell-Cell Contact by EndoplasmicReticulum-Bound Protein-Tyrosine Phosphatase 1B. PLoS ONE 7(5): e36633. doi:10.1371/journal.pone.0036633Editor: F. Gisou van der Goot, Ecole Polytechnique Federale de Lausanne, SwitzerlandReceived December 13, 2011; Accepted April 4, 2012; Published May , 2012Copyright: ? 2012 Haj et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricteduse, distribution, and reproduction in any medium, provided the original author and source are credited.Funding: This work was funded by an EMBO short-term fellowship, a Research Grant from the Juvenile Diabetes Research Foundation (1-2009-337) and a JuniorFaculty Award from the American Diabetes Association (7-06-JF-28) to F.G.H., and R37 CA49152 to B.G.N. B.G.N. is a Canada Research Chair, Tier 1, and work in hislaboratory is partially supported by funds from the Ontario Ministry of Health and The Princess Margaret Hospital Foundation. S.W.K was supported by a CJ MartinFellowship (384352) of the National Health and Medical Research Council of Australia, and an Early Career Award of the Sydney Medical School. The funders hadno role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Competing Interests: The authors have declared that no competing interests exist.* E-mail: fghaj@ucdavis.edu (FGH); bneel@uhnresreach.ca (BGN); philippe.bastiaens@mpi-dortmund.mpg.de (PIB)IntroductionThe ubiquitously expressed non-receptor protein-tyrosinephosphatase (PTP1B) plays an important role in regulating diversecellular signaling pathways, including those initiated by receptortyrosine kinases (RTKs), cytokine receptors, integrins and cadher-ins [1]. Major insights into the physiological role of PTP1B weregained through the generation of knockout (KO) mice, whichshowed that PTP1B is a critical regulator of insulin sensitivity andenergy balance in vivo [2,3,4,5]. However, other functions forPTP1B have also been suggested, including roles in regulating cell-matrix [6] and cell-cell [7,8,9] interactions. Given the salutarymetabolic effects of PTP1B deletion, it has emerged as a potentialtarget for anti-diabetic and anti-obesity drug development [10,11].Consequently, it is important to understand its mechanism ofaction in detail.PTP1B is anchored to the cytosolic face of the endoplasmicreticulum (ER) via a hydrophobic C-terminal targeting sequence[12,13], which constrains its access to key substrates. Consistentwith this localization, PTP1B dephosphorylates the activatedepidermal growth factor receptor (EGFR), platelet-derived growthfactor receptor (PDGFR) and insulin receptor (IR) only afterendocytosis, as they transit past the ER [14,15,16]. PTP1B activityalso is spatially regulated in the cell, thus creating distinctmicroenvironments that enable RTK signal propagation, followedby signal termination [17]. Recently, PTP1B has been identified asa potential regulator of RTK endocytosis. Eden et al. reported thatPTP1B-EGFR interaction occurs through direct membranecontact between multivesicular bodies (MVB) and the ER, withPTP1B activity promoting the sequestration of EGFR to MVBinternal vesicles [18]. Consistent with these findings, Stuible et al.identified the endosomal protein STAM2, which regulates sortingof activated RTKs for degradation, as a PTP1B substrate [19].Collectively, these studies reveal that PTP1B is a major regulatorof RTK endocytosis and signaling.Although activated RTKs gain access to PTP1B only afterendocytosis, PTP1B also can interact with some plasma mem-brane (PM)-bound substrates [20,21]. For example, PTP1B targetsforming cell-matrix adhesion contacts and contributes to thestabilization of focal adhesions. This process appears to involvedynamic extension of the ER via a microtubule-dependent processPLoS ONE | www.plosone.org 1 May 2012 | Volume 7 | Issue 5 | e3663324
