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      Grb2 monomer–dimer equilibrium determines normal versus oncogenic function

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          Abstract

          The adaptor protein growth factor receptor-bound protein 2 (Grb2) is ubiquitously expressed in eukaryotic cells and involved in a multitude of intracellular protein interactions. Grb2 plays a pivotal role in tyrosine kinase-mediated signal transduction including linking receptor tyrosine kinases to the Ras/mitogen-activated protein (MAP) kinase pathway, which is implicated in oncogenic outcome. Grb2 exists in a constitutive equilibrium between monomeric and dimeric states. Here we show that only monomeric Grb2 is capable of binding to SOS and upregulating MAP kinase signalling and that the dimeric state is inhibitory to this process. Phosphorylation of tyrosine 160 (Y160) on Grb2, or binding of a tyrosylphosphate-containing ligand to the SH2 domain of Grb2, results in dimer dissociation. Phosphorylation of Y160 on Grb2 is readily detectable in the malignant forms of human prostate, colon and breast cancers. The self-association/dissociation of Grb2 represents a switch that regulates MAP kinase activity and hence controls cancer progression.

          Abstract

          Grb2 is an adaptor protein that can exist as a dimer that dissociates on phosphorylation of Y160. Here, the authors show that only the monomeric protein is capable of activating mitogen-activated protein kinase signal transduction and hence control oncogenic outcome.

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          Most cited references33

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          The SH2 and SH3 domain-containing protein GRB2 links receptor tyrosine kinases to ras signaling.

          A cDNA clone encoding a novel, widely expressed protein (called growth factor receptor-bound protein 2 or GRB2) containing one src homology 2 (SH2) domain and two SH3 domains was isolated. Immunoblotting experiments indicate that GRB2 associates with tyrosine-phosphorylated epidermal growth factor receptors (EGFRs) and platelet-derived growth factor receptors (PDGFRs) via its SH2 domain. Interestingly, GRB2 exhibits striking structural and functional homology to the C. elegans protein sem-5. It has been shown that sem-5 and two other genes called let-23 (EGFR like) and let-60 (ras like) lie along the same signal transduction pathway controlling C. elegans vulval induction. To examine whether GRB2 is also a component of ras signaling in mammalian cells, microinjection studies were performed. While injection of GRB2 or H-ras proteins alone into quiescent rat fibroblasts did not have mitogenic effect, microinjection of GRB2 together with H-ras protein stimulated DNA synthesis. These results suggest that GRB2/sem-5 plays a crucial role in a highly conserved mechanism for growth factor control of ras signaling.
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            Microscale thermophoresis quantifies biomolecular interactions under previously challenging conditions.

            Microscale thermophoresis (MST) allows for quantitative analysis of protein interactions in free solution and with low sample consumption. The technique is based on thermophoresis, the directed motion of molecules in temperature gradients. Thermophoresis is highly sensitive to all types of binding-induced changes of molecular properties, be it in size, charge, hydration shell or conformation. In an all-optical approach, an infrared laser is used for local heating, and molecule mobility in the temperature gradient is analyzed via fluorescence. In standard MST one binding partner is fluorescently labeled. However, MST can also be performed label-free by exploiting intrinsic protein UV-fluorescence. Despite the high molecular weight ratio, the interaction of small molecules and peptides with proteins is readily accessible by MST. Furthermore, MST assays are highly adaptable to fit to the diverse requirements of different biomolecules, such as membrane proteins to be stabilized in solution. The type of buffer and additives can be chosen freely. Measuring is even possible in complex bioliquids like cell lysate allowing close to in vivo conditions without sample purification. Binding modes that are quantifiable via MST include dimerization, cooperativity and competition. Thus, its flexibility in assay design qualifies MST for analysis of biomolecular interactions in complex experimental settings, which we herein demonstrate by addressing typically challenging types of binding events from various fields of life science. Copyright © 2013 Elsevier Inc. All rights reserved.
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              Epidermal growth factor regulates p21ras through the formation of a complex of receptor, Grb2 adapter protein, and Sos nucleotide exchange factor.

              Antisera against murine Son of sevenless (Sos) recognize a protein of M(r) 155,000 in rat-1 fibroblasts with specific guanine nucleotide exchange activity toward p21c-Ha-ras. Epidermal growth factor (EGF) receptor coimmunoprecipitates with Sos from EGF-stimulated, but not quiescent, cells. The SH2 and SH3 domain-containing "adapter" protein Grb2 is also found in Sos immunoprecipitates in an EGF-inducible manner. In vitro reconstitution shows that Grb2 is required for the binding of activated EGF receptor to Sos. A phosphopeptide corresponding to tyrosine 1068 of the EGF receptor blocks both the assembly of the complex and EGF stimulation of nucleotide exchange on p21ras in a permeabilized cell system. These results suggest that EGF-induced activation of nucleotide exchange on p21ras proceeds through the recruitment of cytosolic Sos to a complex with EGF receptor and Grb2 at the plasma membrane.
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                Author and article information

                Journal
                Nat Commun
                Nat Commun
                Nature Communications
                Nature Pub. Group
                2041-1723
                24 June 2015
                2015
                : 6
                : 7354
                Affiliations
                [1 ]Department of Biochemistry and Molecular Biology, University of Texas, M.D. Anderson Cancer Center , Unit 1000, 1515 Holcombe Boulevard, Houston, Texas 77030, USA
                [2 ]Center for Biomolecular Structure and Function, University of Texas, M.D. Anderson Cancer Center , Unit 1000, 1515 Holcombe Boulevard, Houston, Texas 77030, USA
                [3 ]School of Molecular and Cellular Biology, University of Leeds , Leeds LS2 9JT, UK
                [4 ]Department of Chemistry, Rice University , Houston, Texas 77005, USA
                [5 ]Department of Veterinary Medicine and Surgery, University of Texas, M.D. Anderson Cancer Center , Unit 63, 1515 Holcombe Boulevard, Houston, Texas 77030, USA
                Author notes
                [*]

                These authors contributed equally to this work.

                [†]

                Present address: Laboratory of Cellular Dynamics, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany.

                Article
                ncomms8354
                10.1038/ncomms8354
                4491180
                26103942
                32e1d1cd-383f-49fe-8c02-6ffd45ef92f9
                Copyright © 2015, Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.

                This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

                History
                : 25 January 2015
                : 29 April 2015
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