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Raptor is phosphorylated by cdc2 during mitosis.

PloS one | Feb 12, 2010

BACKGROUND: The appropriate control of mitotic entry and exit is reliant on a series of interlocking signaling events that coordinately drive the biological processes required for accurate cell division. Overlaid onto these signals that promote orchestrated cell division are checkpoints that ensure appropriate mitotic spindle formation, a lack of DNA damage, kinetochore attachment, and that each daughter cell has the appropriate complement of DNA. We recently discovered that AMP-activated protein kinase (AMPK) modulates the G2/M phase of cell cycle progression in part through its suppression of mammalian target of rapamycin (mTOR) signaling. AMPK directly phosphorylates the critical mTOR binding partner raptor inhibiting mTORC1 (mTOR-raptor rapamycin sensitive mTOR kinase complex 1). As mTOR has been previously tied to mitotic control, we examined further how raptor may contribute to this process. METHODOLOGY/PRINCIPAL FINDINGS: We have discovered that raptor becomes highly phosphorylated in cells in mitosis. Utilizing tandem mass spectrometry, we identified a number of novel phosphorylation sites in raptor, and using phospho-specific antibodies demonstrated that raptor becomes phosphorylated on phospho-serine/threonine-proline sites in mitosis. A combination of site-directed mutagenesis in a tagged raptor cDNA and analysis with a series of new phospho-specific antibodies generated against different sites in raptor revealed that Serine 696 and Threonine 706 represent two key sites in raptor phosphorylated in mitosis. We demonstrate that the mitotic cyclin-dependent kinase cdc2/CDK1 is the kinase responsible for phosphorylating these sites, and its mitotic partner Cyclin B efficiently coimmunoprecipitates with raptor in mitotic cells. CONCLUSIONS/SIGNIFICANCE: This study demonstrates that the key mTOR binding partner raptor is directly phosphorylated during mitosis by cdc2. This reinforces previous studies suggesting that mTOR activity is highly regulated and important for mitotic progression, and points to a direct modulation of the mTORC1 complex during mitosis.

Pubmed ID: 20169205 RIS Download

Mesh terms: Adaptor Proteins, Signal Transducing | Amino Acid Sequence | Binding Sites | CDC2 Protein Kinase | Cell Line | Cell Line, Tumor | Cyclin B | Cyclin-Dependent Kinases | HeLa Cells | Humans | Immunoblotting | Immunoprecipitation | Mass Spectrometry | Mitosis | Molecular Sequence Data | Mutagenesis, Site-Directed | Phosphorylation | Protein Binding | Sequence Homology, Amino Acid | Serine | Threonine | Transcription Factors | Transfection

Research resources used in this publication

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Data used in this publication

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Associated grants

  • Agency: NCI NIH HHS, Id: P01 CA120964
  • Agency: NIDDK NIH HHS, Id: R01 DK080425
  • Agency: Howard Hughes Medical Institute, Id:

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PhosphoSitePlus: Protein Modification Site

A freely accessible on-line systems biology resource devoted to all aspects of protein modification, as well as other post-translational modifications. It provides valuable and unique tools for both cell biologists and mass spectroscopists. PhosphoSite is a human- and mouse-centric database. It includes features such as: viewing the locations of modified residues on molecular models; browsing and searching MS2 records by disease, tissue, and cell line; submitting lists of peptides to identify previously reported genes; searching by sub-cellular localization, treatment, tissues, cell types, cell lines and diseases, and protein types and protein domains; searching for experimentally-verified kinase substrates and viewing preferred substrate motifs; and viewing MS2 spectra for peptides and sites not previously published.


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