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Proc Natl Acad Sci U S A. 2015 May 12;112(19):5875-82. doi: 10.1073/pnas.1505787112. Epub 2015 Apr 16.

CDK1 substitutes for mTOR kinase to activate mitotic cap-dependent protein translation.

Author information

1
Cancer Virology Program, University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213.
2
Cancer Virology Program, University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213 psm9@pitt.edu yc70@pitt.edu.

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.

KEYWORDS:

4E-BP1; Merkel cell; cyclin-dependent kinase 1; mitosis; small T

PMID:
25883264
PMCID:
PMC4434708
DOI:
10.1073/pnas.1505787112
[Indexed for MEDLINE]
Free PMC Article

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