title: CDK1 substitutes for mTOR kinase to activate mitotic cap-dependent protein translation.
abstract: 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 δ 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.
authors.affiliation: Cancer Virology Program, University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Shuda
authors.affiliation: Cancer Virology Program, University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213.
authors.forename: Celestino
authors.initials: C
authors.lastname: Velásquez
authors.affiliation: Cancer Virology Program, University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213.
authors.forename: Erdong
authors.initials: E
authors.lastname: Cheng
authors.affiliation: Cancer Virology Program, University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213.
authors.forename: Daniel G
authors.initials: DG
authors.lastname: Cordek
authors.affiliation: Cancer Virology Program, University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213.
authors.forename: Hyun Jin
authors.initials: HJ
authors.lastname: Kwun
authors.affiliation: Cancer Virology Program, University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213 psm9@pitt.edu yc70@pitt.edu.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Chang
authors.affiliation: Cancer Virology Program, University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213 psm9@pitt.edu yc70@pitt.edu.
authors.forename: Patrick S
authors.initials: PS
authors.lastname: Moore
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D000952:Antigens, Polyomavirus Transforming / Q000378:metabolism*; D016203:CDC2 Protein Kinase; D018797:Cell Cycle Proteins; D049109:Cell Proliferation; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D007399:Interphase; D008938:Mitosis; D009369:Neoplasms / Q000378:metabolism*; D015739:Nocodazole / Q000737:chemistry; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D014176:Protein Biosynthesis; D017434:Protein Structure, Tertiary; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D000952:Antigens, Polyomavirus Transforming; D018797:Cell Cycle Proteins; C087000:EIF4EBP1 protein, human; D010750:Phosphoproteins; C546842:MTOR protein, human; D058570:TOR Serine-Threonine Kinases; D016203:CDC2 Protein Kinase; C528899:CDK1 protein, human; D018844:Cyclin-Dependent Kinases; D015739:Nocodazole
keywords: 4E-BP1; Merkel cell; cyclin-dependent kinase 1; mitosis; small T