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. Author manuscript; available in PMC: 2022 Sep 1.
Published in final edited form as: Cell Stem Cell. 2020 May 7;26(5):755–765.e7. doi: 10.1016/j.stem.2019.12.006

Figure 4. Cyclin Dependent Kinase-1 (CDK1) mediates 4E-BP1 Ser-65 phosphorylation and activates eIF4E-(cap)-dependent translation in myeloid progenitors.

Figure 4.

(A) Expression of detectable cyclin-dependent kinase family members in LSK and MP cells as determined by mass spectrometry (PSM, peptide spectral matches) (n=3). (B) Immunoblot and quantitation of CDK1 expression in LSK and MP cells. Representative blot shown (n=3). (C) Immunoblot and quantitation of CDK1 and phosphorylated 4E-BP1 Ser-65 in MP cells treated with DMSO or RO-3306 (10uM) for 2 hours. Representative blot shown (n=3). (D) Immunoprecipitation experiment of CDK1 in MP cells treated with DMSO or RO-3066 for 2 hours. Representative blot shown (n=3). (E) Immunoprecipitation experiment of transfected WT and 4E-BP1 phosphorylation site mutants (T37A. T46A, and S65A) constructs in HEK-293T cells. Representative blot shown (n=2). (F) Immunoblot of cap-bead binding experiments performed with WT LSK and MP cells following 2-hour treatment with DMSO vehicle or RO-3306 (top panel). Quantitation of eIF4E bound to cap-beads in LSK and MP cells (bottom panel) Representative blot shown (n=3). (G) Poly/sub poly RNA ratios in WT LSK and MPs cells following 2-hour ex vivo treatment (n=4). (H) Colony forming activity of LSK and MP cells following treatment (n=3). (I) Absolute number of live LSK and MP cells in CFC assays on day 8, following treatment (n=3). (J) Model for translational reprogramming in early hematopoiesis. Despite lower total global translation, LSK cells exhibit mTOR signal activation and preferentially translate mTOR activated mRNAs as well as mRNAs required for HSC maintenance. In contrast, MP cells shows increased global translation despite proteasome-mediated mTOR protein degradation, stimulated by the E3 ubiquitin ligase activity of c-Cbl. Aberrant mTOR expression in MPs has significant consequences, resulting in increased mature myeloid cell output. In the absence of mTOR in MPs, eIF4E-cap-dependent translation is activated through the action of CDK1, which phosphorylates the S65 residue of 4E-BP1, allowing release of eIF4E.