PMID- 25883264 OWN - NLM STAT- MEDLINE DCOM- 20151214 LR - 20211203 IS - 1091-6490 (Electronic) IS - 0027-8424 (Print) IS - 0027-8424 (Linking) VI - 112 IP - 19 DP - 2015 May 12 TI - CDK1 substitutes for mTOR kinase to activate mitotic cap-dependent protein translation. PG - 5875-82 LID - 10.1073/pnas.1505787112 [doi] AB - Mitosis is commonly thought to be associated with reduced cap-dependent protein translation. Here we show an alternative control mechanism for maintaining cap-dependent translation during mitosis revealed by a viral oncoprotein, Merkel cell polyomavirus small T (MCV sT). We find MCV sT to be a promiscuous E3 ligase inhibitor targeting the anaphase-promoting complex, which increases cell mitogenesis. MCV sT binds through its Large T stabilization domain region to cell division cycle protein 20 (Cdc20) and, possibly, cdc20 homolog 1 (Cdh1) E3 ligase adapters. This activates cyclin-dependent kinase 1/cyclin B1 (CDK1/CYCB1) to directly hyperphosphorylate eukaryotic initiation factor 4E (eIF4E)-binding protein (4E-BP1) at authentic sites, generating a mitosis-specific, mechanistic target of rapamycin (mTOR) inhibitor-resistant delta phospho-isoform not present in G1-arrested cells. Recombinant 4E-BP1 inhibits capped mRNA reticulocyte translation, which is partially reversed by CDK1/CYCB1 phosphorylation of 4E-BP1. eIF4G binding to the eIF4E-m(7)GTP cap complex is resistant to mTOR inhibition during mitosis but sensitive during interphase. Flow cytometry, with and without sT, reveals an orthogonal pH3(S10+) mitotic cell population having higher inactive p4E-BP1(T37/T46+) saturation levels than pH3(S10-) interphase cells. Using a Click-iT flow cytometric assay to directly measure mitotic protein synthesis, we find that most new protein synthesis during mitosis is cap-dependent, a result confirmed using the eIF4E/4G inhibitor drug 4E1RCat. For most cell lines tested, cap-dependent translation levels were generally similar between mitotic and interphase cells, and the majority of new mitotic protein synthesis was cap-dependent. These findings suggest that mitotic cap-dependent translation is generally sustained during mitosis by CDK1 phosphorylation of 4E-BP1 even under conditions of reduced mTOR signaling. FAU - Shuda, Masahiro AU - Shuda M AD - Cancer Virology Program, University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213. FAU - Velasquez, Celestino AU - Velasquez C AD - Cancer Virology Program, University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213. FAU - Cheng, Erdong AU - Cheng E AD - Cancer Virology Program, University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213. FAU - Cordek, Daniel G AU - Cordek DG AD - Cancer Virology Program, University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213. FAU - Kwun, Hyun Jin AU - Kwun HJ AD - Cancer Virology Program, University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213. FAU - Chang, Yuan AU - Chang Y AD - Cancer Virology Program, University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213 psm9@pitt.edu yc70@pitt.edu. FAU - Moore, Patrick S AU - Moore PS AD - Cancer Virology Program, University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213 psm9@pitt.edu yc70@pitt.edu. LA - eng GR - R01 CA170354/CA/NCI NIH HHS/United States GR - CA170354/CA/NCI NIH HHS/United States GR - P30 CA047904/CA/NCI NIH HHS/United States GR - R01 CA136363/CA/NCI NIH HHS/United States GR - P30CA047904/CA/NCI NIH HHS/United States GR - CA12197305/CA/NCI NIH HHS/United States GR - R01 CA136806/CA/NCI NIH HHS/United States GR - R01CA136806/CA/NCI NIH HHS/United States GR - CA136363/CA/NCI NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural PT - Research Support, Non-U.S. Gov't DEP - 20150416 PL - United States TA - Proc Natl Acad Sci U S A JT - Proceedings of the National Academy of Sciences of the United States of America JID - 7505876 RN - 0 (Adaptor Proteins, Signal Transducing) RN - 0 (Antigens, Polyomavirus Transforming) RN - 0 (Cell Cycle Proteins) RN - 0 (EIF4EBP1 protein, human) RN - 0 (Phosphoproteins) RN - EC 2.7.1.1 (MTOR protein, human) RN - EC 2.7.11.1 (TOR Serine-Threonine Kinases) RN - EC 2.7.11.22 (CDC2 Protein Kinase) RN - EC 2.7.11.22 (CDK1 protein, human) RN - EC 2.7.11.22 (Cyclin-Dependent Kinases) RN - SH1WY3R615 (Nocodazole) SB - IM CIN - 5862 MH - Adaptor Proteins, Signal Transducing/*metabolism MH - Antigens, Polyomavirus Transforming/*metabolism MH - CDC2 Protein Kinase MH - Cell Cycle Proteins MH - Cell Proliferation MH - Cyclin-Dependent Kinases/*metabolism MH - HEK293 Cells MH - HeLa Cells MH - Humans MH - Interphase MH - Mitosis MH - Neoplasms/*metabolism MH - Nocodazole/chemistry MH - Phosphoproteins/*metabolism MH - Phosphorylation MH - Protein Binding MH - *Protein Biosynthesis MH - Protein Structure, Tertiary MH - TOR Serine-Threonine Kinases/*metabolism PMC - PMC4434708 OTO - NOTNLM OT - 4E-BP1 OT - Merkel cell OT - cyclin-dependent kinase 1 OT - mitosis OT - small T COIS- The authors declare no conflict of interest. EDAT- 2015/04/18 06:00 MHDA- 2015/12/15 06:00 PMCR- 2015/04/16 CRDT- 2015/04/18 06:00 PHST- 2015/04/18 06:00 [entrez] PHST- 2015/04/18 06:00 [pubmed] PHST- 2015/12/15 06:00 [medline] PHST- 2015/04/16 00:00 [pmc-release] AID - 1505787112 [pii] AID - 201505787 [pii] AID - 10.1073/pnas.1505787112 [doi] PST - ppublish SO - Proc Natl Acad Sci U S A. 2015 May 12;112(19):5875-82. doi: 10.1073/pnas.1505787112. Epub 2015 Apr 16.