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. 2015 Apr 16;112(19):5875–5882. doi: 10.1073/pnas.1505787112

Fig. 3.

Fig. 3.

CDK1/CYCB1 phosphorylates 4E-BP1 during mitosis. (A) CDK1 inhibition in mitotic lysates reduces 4E-BP1 phosphorylation. Mitotic HeLa cell lysates (10 µg) enriched by nocodazole arrest were mixed with 0.2 µg GST–4E-BP1, reacted for 30 min at 30 °C in the presence or absence of 5 µM mTOR (PP242), CDK1 (RO-3306), or AURK (VX-680) kinase inhibitors and then immunoblotted with antibodies as shown. ATP-dependent 4E-BP1 phosphorylation was sensitive to CDK1 inhibitor but resistant to mTOR and AURK inhibitors. Equal loading of total 4E-BP1, CYCB1, and α-tubulin is shown. Representative results are shown from three independent experiments. (B) Recombinant CDK1/CYCB1 kinase phosphorylates GST–4E-BP1 at the known regulatory residues T70, S65/S101, and T37/T46. CDK1/CYCB1 (20 units) was mixed with bacterial-expressed GST–4E-BP1 in kinase reaction buffer for 30 min at 30 °C and immunoblotted with phospho-specific antibodies. ATP-dependent 4E-BP1 phosphorylation by CDK1/CYCB1 occurred at phospho-specific sites and was sensitive to the CDK1 active site inhibitor RO-3306. Representative results are shown from two independent experiments. (C) δ-4E-BP1 is induced during mitosis and inhibited by a CDK1 inhibitor. The 293 cells were transfected with empty vector or MCV sT and arrested for 20 h with DMSO (asynchronous), nocodazole (prometaphase), and mimosine (late G1). Cells were treated at 16 h with kinase inhibitors (5 µM PP242 or 10 µM RO-3306 + 10 µM MG132) as indicated. MCV sT induces δ-4E-BP1 in asynchronous cells sensitive to RO-3306 but not PP242. Nocodazole arrest induces similarly RO-3306–sensitive and PP242-resistant δ-4E-BP1 even in the absence of sT, whereas δ-4E-BP1 is only weakly induced by sT in mimosine-arrested cells. Markers for mitosis (pH3S10, CYCB1), a CDK1 substrate (cdc25C), and an mTORC1 downstream substrate (pS6S235/S236) showed active drug treatments. Representative results are shown from two independent experiments. (D) δ-4E-BP1 phosphorylation during mitosis occurs in the absence of active mTOR. U2OS cells were arrested at the G2/M boundary with 10 µM RO-3306 for 24 h, released by washing, and harvested at the time points shown. Cells were treated for 3 h prerelease with DMSO or 5 µM PP242. In the absence of mTOR inhibition, no δ-4E-BP1 is found at 0 h but accumulates, together with β and γ isoforms, during mitotic transit. During PP242 inhibition, δ-4E-BP1 still accumulates during mitosis, but lower molecular mass (β–γ) isoforms are reduced. Results shown are from a single experiment.