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      The C-Terminal Extension Unique to the Long Isoform of the Shelterin Component TIN2 Enhances Its Interaction with TRF2 in a Phosphorylation- and Dyskeratosis Congenita Cluster-Dependent Fashion

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          ABSTRACT

          TIN2 is central to the shelterin complex, linking the telomeric proteins TRF1 and TRF2 with TPP1/POT1. Mutations in TINF2, which encodes TIN2, that are found in dyskeratosis congenita (DC) result in very short telomeres and cluster in a region shared by the two TIN2 isoforms, TIN2S (short) and TIN2L (long). Here we show that TIN2L, but not TIN2S, is phosphorylated. TRF2 interacts more with TIN2L than TIN2S, and both the DC cluster and phosphorylation promote this enhanced interaction. The binding of TIN2L, but not TIN2S, is affected by TRF2-F120, which is also required for TRF2's interaction with end processing factors such as Apollo. Conversely, TRF1 interacts more with TIN2S than with TIN2L. A DC-associated mutation further reduces TIN2L-TRF1, but not TIN2S-TRF1, interaction. Cells overexpressing TIN2L or phosphomimetic TIN2L are permissive to telomere elongation, whereas cells overexpressing TIN2S or phosphodead TIN2L are not. Telomere lengths are unchanged in cell lines in which TIN2L expression has been eliminated by clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-mediated mutation. These results indicate that TIN2 isoforms are biochemically and functionally distinguishable and that shelterin composition could be fundamentally altered in patients with TINF2 mutations.

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          Most cited references 33

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          Phosphate-binding tag, a new tool to visualize phosphorylated proteins.

          We introduce two methods for the visualization of phosphorylated proteins using alkoxide-bridged dinuclear metal (i.e. Zn(2+) or Mn(2+)) complexes as novel phosphate-binding tag (Phos-tag) molecules. Both Zn(2+)- and Mn(2+)-Phos-tag molecules preferentially capture phosphomonoester dianions bound to Ser, Thr, and Tyr residues. One method is based on an ECL system using biotin-pendant Zn(2+)-Phos-tag and horseradish peroxidase-conjugated streptavidin. We demonstrate the electroblotting analyses of protein phosphorylation status by the phosphate-selective ECL signals. Another method is based on the mobility shift of phosphorylated proteins in SDS-PAGE with polyacrylamide-bound Mn(2+)-Phos-tag. Phosphorylated proteins in the gel are visualized as slower migration bands compared with corresponding dephosphorylated proteins. We demonstrate the kinase and phosphatase assays by phosphate affinity electrophoresis (Mn(2+)-Phos-tag SDS-PAGE).
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            Phosphoproteomic analysis identifies Grb10 as an mTORC1 substrate that negatively regulates insulin signaling.

            The evolutionarily conserved serine-threonine kinase mammalian target of rapamycin (mTOR) plays a critical role in regulating many pathophysiological processes. Functional characterization of the mTOR signaling pathways, however, has been hampered by the paucity of known substrates. We used large-scale quantitative phosphoproteomics experiments to define the signaling networks downstream of mTORC1 and mTORC2. Characterization of one mTORC1 substrate, the growth factor receptor-bound protein 10 (Grb10), showed that mTORC1-mediated phosphorylation stabilized Grb10, leading to feedback inhibition of the phosphatidylinositol 3-kinase (PI3K) and extracellular signal-regulated, mitogen-activated protein kinase (ERK-MAPK) pathways. Grb10 expression is frequently down-regulated in various cancers, and loss of Grb10 and loss of the well-established tumor suppressor phosphatase PTEN appear to be mutually exclusive events, suggesting that Grb10 might be a tumor suppressor regulated by mTORC1.
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              The RNA component of telomerase is mutated in autosomal dominant dyskeratosis congenita.

              Dyskeratosis congenita is a progressive bone-marrow failure syndrome that is characterized by abnormal skin pigmentation, leukoplakia and nail dystrophy. X-linked, autosomal recessive and autosomal dominant inheritance have been found in different pedigrees. The X-linked form of the disease is due to mutations in the gene DKC1 in band 2, sub-band 8 of the long arm of the X chromosome (ref. 3). The affected protein, dyskerin, is a nucleolar protein that is found associated with the H/ACA class of small nucleolar RNAs and is involved in pseudo-uridylation of specific residues of ribosomal RNA. Dyskerin is also associated with telomerase RNA (hTR), which contains a H/ACA consensus sequence. Here we map the gene responsible for dyskeratosis congenita in a large pedigree with autosomal dominant inheritance. Affected members of this family have an 821-base-pair deletion on chromosome 3q that removes the 3' 74 bases of hTR. Mutations in hTR were found in two other families with autosomal dominant dyskeratosis congenita.
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                Author and article information

                Journal
                Mol Cell Biol
                Mol. Cell. Biol
                mcb
                mcb
                MCB
                Molecular and Cellular Biology
                American Society for Microbiology (1752 N St., N.W., Washington, DC )
                0270-7306
                1098-5549
                26 March 2018
                29 May 2018
                15 June 2018
                29 May 2018
                : 38
                : 12
                Affiliations
                [a ]Department of Molecular & Human Genetics, Baylor College of Medicine, Houston, Texas, USA
                [b ]Division of Hematology/Oncology, Department of Pediatrics, Baylor College of Medicine, Houston, Texas, USA
                [c ]Division of Cancer and Genetics, School of Medicine, Cardiff University, Cardiff, United Kingdom
                [d ]Bioinformatics Institute, Agency for Science Technology and Research, Singapore, Singapore
                Author notes
                Address correspondence to Alison A. Bertuch, abertuch@ 123456bcm.edu .
                [*]

                Present address: Nya D. Nelson, Department of Pathology and Laboratory Medicine, Hospital of the University of Pennsylvania, Philadelphia, Pennsylvania, USA; Laura Escudero, Translational Research Program, Vall d'Hebron Institute of Oncology, Barcelona, Spain.

                Citation Nelson ND, Dodson LM, Escudero L, Sukumar AT, Williams CL, Mihalek I, Baldan A, Baird DM, Bertuch AA. 2018. The C-terminal extension unique to the long isoform of the shelterin component TIN2 enhances its interaction with TRF2 in a phosphorylation- and dyskeratosis congenita cluster-dependent fashion. Mol Cell Biol 38:e00025-18. https://doi.org/10.1128/MCB.00025-18.

                Article
                00025-18
                10.1128/MCB.00025-18
                5974431
                29581185
                Copyright © 2018 Nelson et al.

                This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

                Page count
                supplementary-material: 1, Figures: 9, Tables: 0, Equations: 0, References: 58, Pages: 17, Words: 10578
                Product
                Funding
                Funded by: HHS | NIH | National Cancer Institute (NCI), https://doi.org/10.13039/100000054;
                Award ID: F30CA168144
                Award Recipient :
                Funded by: Cancer Prevention and Research Institute of Texas (CPRIT), https://doi.org/10.13039/100004917;
                Award ID: RP120076
                Award Recipient :
                Funded by: American Society of Hematology (ASH), https://doi.org/10.13039/100001422;
                Award ID: Bridge Award
                Award Recipient :
                Funded by: HHS | NIH | National Heart, Lung, and Blood Institute (NHLBI), https://doi.org/10.13039/100000050;
                Award ID: R01HL131744
                Award Recipient :
                Funded by: DOD | United States Army | MEDCOM | Congressionally Directed Medical Research Programs (CDMRP), https://doi.org/10.13039/100000090;
                Award ID: W81XWH-10-1-0389
                Award Recipient :
                Funded by: Cancer Research UK (CRUK), https://doi.org/10.13039/501100000289;
                Award ID: A18246
                Award Recipient :
                Funded by: Cancer Research Wales (CRW), https://doi.org/10.13039/100009828;
                Award ID: C17199
                Award Recipient :
                Categories
                Research Article
                Custom metadata
                June 2018

                Molecular biology

                telomere, shelterin, dyskeratosis congenita, casein kinase 2, trf2, trf1, tpp1, tin2

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