Mitotic MELK-eIF4B signaling controls protein synthesis and tumor cell survival Yubao Wanga,b, Michael Begleyc,d, Qing Lia,b, Hai-Tsang Huanga,b, Ana Lakoa,b, Michael J. Ecka,b, Nathanael S. Graya,b, Timothy J. Mitchisond, Lewis C. Cantleyc,d,e,1, and Jean J. Zhaoa,b,1 aDepartment of Cancer Biology, Dana–Farber Cancer Institute, Boston, MA 02115; bDepartment of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115; cDepartment of Medicine, Beth Israel Deaconess Medical Center, Boston, MA 02215; dDepartment of Systems Biology, Harvard Medical School, Boston, MA 02115; and eMeyer Cancer Center, Department of Medicine, Weill Cornell Medical College, New York, NY 10065 Contributed by Lewis C. Cantley, July 7, 2016 (sent for review May 2, 2016; reviewed by Kun-Liang Guan and Sean J. Morrison) The protein kinase maternal and embryonic leucine zipper kinase complement the posttranslational inactivation of specific proteins (MELK) is critical for mitotic progression of cancer cells; however, (8). Nevertheless, protein synthesis still occurs during mitosis, al- its mechanisms of action remain largely unknown. By combined though at an overall rate that is 30–65% of the overall rate in in- approaches of immunoprecipitation/mass spectrometry and pep- terphase cells (6, 8, 9). Moreover, the translation of certain mRNAs, tide library profiling, we identified the eukaryotic translation such as c-Myc, MCL1, and ornithine decarboxylase (ODC), is even initiation factor 4B (eIF4B) as a MELK-interacting protein during elevated during mitosis (8, 9). Together, these studies suggest mitosis and a bona fide substrate of MELK. MELK phosphorylates that protein synthesis may be functionally important for mi- eIF4B at Ser406, a modification found to be most robust in the totic cells and might be finely regulated by as yet unidentified mitotic phase of the cell cycle. We further show that the MELK– signaling pathways. eIF4B signaling axis regulates protein synthesis during mitosis. In this study, we aimed to identify downstream effectors of the Specifically, synthesis of myeloid cell leukemia 1 (MCL1), an anti- mitotic kinase MELK. Intriguingly, we found that MELK phos- apoptotic protein known to play a role in cancer cell survival dur- phorylates the eukaryotic translation initiation factor 4B (eIF4B). – ing cell division, depends on the function of MELK-elF4B. Our data implicate the MELK eIF4B pathway as a previously Inactivation of MELK or eIF4B results in reduced protein synthesis unrecognized signaling mechanism regulating mitotic protein CELL BIOLOGY of MCL1, which, in turn, induces apoptotic cell death of cancer synthesis and tumor cell survival. cells. Our study thus defines a MELK–eIF4B signaling axis that reg- Results ulates protein synthesis during mitosis, and consequently influ- ences cancer cell survival. Peptide Library Screen Identifies the Optimal Substrate Motif for MELK. To identify kinase substrates of MELK that mediate its MELK | eIF4B | MCL1 | protein synthesis | mitosis role in the regulation of mitosis and cell survival, we determined the consensus phosphorylation motif for MELK. Proteins carrying the optimal substrate motif are likely phosphorylated by MELK, aternal and embryonic leucine zipper kinase (MELK) is a and thus represent potential in vivo substrates. We expressed ac- Mserine/threonine kinase with potential roles in mitosis. tive full-length human MELK in insect cells and subjected the Similar to other established mitotic factors, such as Aurora ki- purified kinase to positional scanning peptide library screening, a nases and cyclin B1, MELK demonstrates increased protein technique that has been used extensively to identify optimal sub- abundance during mitosis and is degraded when cells progress strate motifs for kinases (10, 11). The profiling demonstrated that into G1 phase (1, 2). Our recent study proposed an essential role MELK is highly selective for its substrate, with a strong preference of MELK in the mitotic progression of specific cancer cell types, for arginine at the −3 position relative to the phosphoacceptor with MELK knockdown resulting in multiple mitotic defects, including G2/M arrest and mitotic cell death (2). Despite these Significance advances, there is a lack of mechanistic understanding of the role of MELK during cell division. An immediate question is the The work identifies the eukaryotic translation initiation factor identity of the MELK substrates that mediate its role in mitosis, 4B (eIF4B) as a substrate of maternal and embryonic leucine such as promoting mitotic cell survival. zipper kinase (MELK), a mitotic kinase known to be essential Myeloid cell leukemia 1 (MCL1) is an important negative reg- for aggressive types of malignancy. The MELK–eIF4B axis thus ulator of apoptosis. Uniquely among the Bcl-2 family, it is turned represents a previously unidentified signaling pathway that over rapidly by ubiquitin-mediated proteolysis and must be regulates protein synthesis during mitosis and, consequently, continuously resupplied by translation (3, 4). Protein abun- dance of MCL1 decreases during prolonged mitotic arrest in- the survival of cancer cells. duced by antimicrotubule drugs, rendering arrested cells highly Author contributions: Y.W., L.C.C., and J.J.Z. designed research; Y.W., M.B., and A.L. sensitive to inhibitors of other Bcl-2 family members (5). We performed research; Y.W., M.B., Q.L., H.-T.H., M.J.E., N.S.G., and L.C.C. contributed new thus speculate that synthesis of MCL1 is important for cell reagents/analytic tools; Y.W., N.S.G., T.J.M., L.C.C., and J.J.Z. analyzed data; and Y.W., T.J.M., survival during normal and drug-arrested mitosis and, con- and J.J.Z. wrote the paper. versely, that a drug that decreases MCL1 synthesis during mi- Reviewers: K.-L.G., University of California, San Diego; and S.J.M., University of Texas tosis might have anticancer potential. Southwestern Medical Center. The rate of protein synthesis and other basic biological processes, Conflict of interest statement: L.C.C. is a member of the Board of Directors of, and holds such as DNA or RNA biogenesis, fluctuates throughout the cell equity in Agios Pharmaceuticals, a company that is developing drugs that target cancer cycle. Overall protein synthesis significantly decreases when cells metabolism. L.C.C. is also a founder of and holds equity in Petra Pharmaceuticals. The data presented in this manuscript are unrelated to research at Agios Pharmaceuticals or Petra enter mitosis (6, 7), consistent with the notion that macromolecule Pharmaceuticals. The phosphorylation of eIF4B and related methods of use reported in this synthesis predominantly occurs in interphase before the segregation study are covered in the following published patent application: WO 2015073509 A2. of cellular mass in mitosis. 1To whom correspondence may be addressed. Email: [email protected] or A recent study based on ribosome profiling identified a set of [email protected]. genes that are translationally repressed in mitosis, and proposed that This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. suppressed protein synthesis might provide a unique mechanism to 1073/pnas.1606862113/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1606862113 PNAS Early Edition | 1of6 Downloaded by guest on September 24, 2021 – A Fixed residue D regulating cell survival (12). The MELK eIF4B interaction was further confirmed by ectopic expression of MELK or eIF4B in Fixed position A p-eIF4B HEK293T cells (Fig. S1 ). (S406) We proceeded to examine whether eIF4B carries the optimal substrate motif for MELK. Two serine residues of eIF4B, Ser406 p-eIF4B and Ser422, conformed to the consensus phosphorylation se- (S422) quence (Fig. 1C and Fig. S1B). In addition, S406/S422 and their flanking residues are evolutionarily conserved across vertebrates eIF4B (Fig. 1C and Fig. S1B), indicating that these residues might be subject to posttranslational modifications. Together, these find- p-Akt (T308) ings suggest that eIF4B might be a protein substrate of MELK. p-Akt (S473) EIF4B Phosphorylation During Mitosis. Given that MELK is a mi- totic kinase, its potential role in regulating eIF4B phosphoryla- Akt tion drove us to investigate first how eIF4B phosphorylation occurs at different stages of the cell cycle. We harvested mitotic MDA-MB- -5 -4 -3 -2 -1 +1 +2 +3 +4 p-MAPK 468 cells at prometaphase through nocodazole- or paclitaxel-induced B Input IP Akt / MAPK signaling (T202/Y204) Flag-MELK IP_MS/MS cell cycle arrest. As expected, the cells were enriched for mitotic - + - + Dox factors, including MELK, cyclin B1, and Aurora kinase A, and, in- # peptides Protein MAPK recovered name MELK terestingly, exhibited strong phosphorylation of eIF4B at Ser406, but not at Ser422 (Fig. 1D and Fig. S1C). In contrast, phosphorylation of 108 MELK eIF4B p-Histone H3 91 eIF4B (S10) some of the known upstream signaling molecules, such as Akt or MAPK (13), was apparently decreased in mitotic cells (Fig. 1D and C eIF4B S406 Cyclin B1 Fig. S1C). Human S406 RERHPSWRSE R S Chimpanzee S406 RE HP WRSE Aurora A Mitotic markers MELK Directly Phosphorylates eIF4B at Ser406. Monkey S402 RERHPSWRSE To investigate Cattle S406 RERHPSWRSE whether MELK phosphorylates eIF4B, we first performed R S Dog S406 RE HP WRSE MELK in vitro kinase assays using purified recombinant MELK and Mouse S406 RERHPSWRSE Rat S406 RERHPSWRSE eIF4B. MELK potently induced phosphorylation of eIF4B at Zebrafish S403 RERHPSWRSE Ser406, and to a lesser extent at Ser422, as indicated by immu- R S -tubulin Xenopus S423 RE HP WRSD noblot analysis with phospho-specific antibodies (Fig. 2A). We Fig. 1. Consensus phosphorylation motif for MELK. (A) Positional scanning also examined the effect of chemical inhibition of MELK kinase peptide library (PSPL) screen identified the optimal phosphorylation motif activity using OTSSP167 (14), and found that the inhibitor effi- for MELK. (Top) Phosphor imaging of a PSPL screen after in vitro reaction ciently suppressed MELK-driven eIF4B phosphorylation at with recombinant full-length MELK and radiolabeled ATP.