Raptor is phosphorylated by cdc2 during mitosis

Abstract

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.

Figure 1. Raptor is phosphorylated on S/T*-P sites in cells treated with nocodazole.

(A) Raptor undergoes a mobility shift on SDS-PAGE following nocadazole which is collapsed by phosphatase treatment. Myc-tagged raptor was expressed in HEK293T cells and nocadazole treated for 16 h. Where indicated, immunoprecipitates were treated with or without calf-intestinal alkaline phosphatase (CIP) and then resolved in SDS-PAGE, and subjected to anti-myc immunoblotting. (B) Raptor is recognized by a phospho-threonine proline antibody in mitotic arrested cells. HEK293T cells transiently expressing myc-tagged raptor were treated for 16 h with taxol and immunoprecipitates were immunoblotted with an antibody that recognizes phospho-threonine followed by proline.

Figure 2. Mass spectrometry analysis of raptor reveals several novel phosphorylation sites.

(A) Phosphorylation sites in raptor after nocodazole treatment as detected by LC/MS/MS. The presence of a phosphate moiety is indicated by a magenta colored box. Note that four serine-or threonine sites followed by a proline were detected in this analysis. Sites of oxidation (green) and deamidation (blue) represent in vitro artifacts of the mass spectrometry experiment. (B). Schematic of human raptor domain structure with all known phosphorylation sites found in this and previous studies (for full details see Table S1). Note that most phosphorylation sites cluster in two regions of the protein. (C) Conservation of the indicated phosphorylation sites.

Figure 3. Raptor is phosphorylation on Ser696 and Thr706 during mitosis.

(A) The mitotic induced bandshift is collapsed by mutation of Ser696, Thr706, and Thr711. Indicated serine/threonine-to-alanine non-phosphorylatable raptor mutants were expressed in HEK293T cells treated with taxol as in Figure 1. (b, c) Wild-type or non-phosphorylatable raptor alleles were immunoprecipitated from nocadazol treated HEK293T cells and then immunoblotted with indicated site-specific phospho-raptor antibodies. Note specificity of each antisera and that Ser696 and Thr706, but not Ser863 or Ser877 are increased by nocadazole treatment.

Figure 4. Cdc2 is the raptor Ser696, Thr706 kinase.

(A) Purified cdc2 can directly phosphorylate raptor on Thr706 and Ser696 in vitro. Myc-raptor (wild-type or S696/T706AA) was immunoprecipitated from hydroxyurea treated HEK293T cells. Immunoprecipitates were incubated with or without active recombinant Cdc2/cyclin B and immunoblotted with phospho-raptor Ser696, Thr706 or total raptor. (B) Endogenous raptor is phosphorylated on Thr706 in synchronized cells undergoing mitosis, and this phosphorylation is blocked by the CDK inhibitor roscovitine. A549 cells were synchronized by double thymidine block and endogenous raptor was immunoprecipitated at the indicated times after release with an anti-raptor antibody and immunoblotted with phospho-raptor Thr706. Whole cell lysates taken from the same cells were immunoblotted for mitotic markers phospho-histone H3 Ser10 and phospho-Plk1 Thr210. (C) Raptor immunoprecipitates with endogenous cyclin B. Hela cells stably expressing myc-wt raptor with stable knockdown of endogenous raptor treated with or without taxol for 16 hours. myc-tagged raptor was immunoprecipitated and immunoblotted for Cyclin B.

Figure 5. Cdc2 phosphorylation of raptor does not change mTORC1 complexes or signaling.

(A) HEK293T cells were transiently transfected with myc-raptor (wild-type or Ser696/Thr706/Ser711AAA: “3A”), AU1-mTOR, HA-GbL and Flag-PRAS40 for 16 hours, followed by 16 hours of nocodazole treatment following addition of fresh media to plates. Cells were lysed and myc-raptor was immunoprecipitated using an antibody against the myc-tag. Immunoprecipitates were resolved by SDS-PAGE and probed with antibodies against the Flag- and HA- tags. (B,C) HEK293T cells were transiently transfected with myc-raptor (wild-type or 3A) and Flag-S6K or Flag-4EBP1. 16 hours later, media was changed and nocodazole was added for 16 hours. Cells were lysed and lysated were split in two; myc-raptor was immunoprecipitated with an antibody against the myc-tag, and Flag-S6K or 4EBP1 were immunoprecipitated with an antibody against the Flag-tag. Immunoprecipitates were resolved by SDS-PAGE and immunoblotted with indicated antibodies. (D) Hela cells stably expressing myc-raptor (wt or 3A) with stable knockdown of endogenous raptor were treated with nocodazole for 16 hours, then Torin1 or roscovitine were added for 4 hours. Cells were lysed and myc-raptor was immunoprecipitated with an antibody against the myc-tag. Immunoprecipitates were resolved by SDS-PAGE and immunoblotted with the indicated antibodies.