Structural and functional analysis of transcription regulating kinases Cdk10 and Cdk11
Final Report Abstract
For the first topic of our research grant, we were able to provide a detailed analysis of Cdk10 substrate specificity and function. Using recombinant, full length Cdk10/CycQ protein complexes including T-loop mutants we show that Cdk10 phosphorylation at Thr196 is dispensable for complex formation with CycQ but critical for kinase activity. We identify RNA pol II CTD, c-MYC and Rb1 as in vitro substrates of Cdk10/CycQ and show that Cdk10 exhibits a strong site-specific activity for Ser2 within the CTD repeats. Based on our data and previous studies we speculate that Cdk10 obtains a hybrid position between cell cycle and transcriptional CDKs and acts as an integrator of external and internal stimuli such as cell stress, growth factors or DNA damage. We still follow up crystallization trials of Cdk10/CycQ, including a specific nanobody and inhibiting small molecular compounds. The work on the Cdk11 kinases with their cyclin partners L1 and L2 is still ongoing. We are currently following several paths to gain a stable Cdk11/CycL complex, characterize the ability for LLPS formation and generate nanobodies to stabilize the kinase for crystallization trials. A couple of contributions that remained from the first funding period or demanded our expertise in recombinant protein purification turned out to be scientifically very fruitful. This included the production of bacterial polymerases and a reverse transcriptase as well as kinase dose response measurements.
Publications
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(2019). Development of a selective CDK7 covalent inhibitor reveals predominant cell-cycle phenotype. Cell Chem. Biol. 26, 792–803
Olson, C.M., Liang, Y., Leggett, A., Park, W.D., Li, L., Mills, C.E., Elsarrag, S.Z., Ficarro, S.B., Zhang, T., Düster, R., Geyer, M., Sim, T., Marto, J.A., Sorger, P.K., Westover, K.D., Lin, C.Y., Kwiatkowski, N., and Gray, N.S.
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(2019). MIR sequences recruit zinc finger protein ZNF768 to expressed genes. Nucleic Acids Res. 47, 700–715
Rohrmoser, M., Kluge, M., Yahia, Y., Gruber-Eber, A., Maqbool, M.A., Forné, I., Krebs, S., Blum, H., Greifenberg, A.K., Geyer, M., Descostes, N., Imhof, A., Andrau, J.C., Friedel, C.C., and Eick, D.
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(2019). P-TEFb activation by RBM7 shapes a pro-survival transcriptional response to genotoxic stress. Mol. Cell 74, 254–267
Bugai, A., Quaresma, A.J.C., Friedel, C.C., Lenasi, T., Düster, R., Sibley, C.R., Kukanja, P., Fujinaga, K., Blasius, M., Hennig, T., Geyer, M., Ule, J., Dölken, L., and Barboric, M.
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(2020). Selective inhibition of CDK7 reveals high-confidence targets and new models for TFIIH function in transcription. Genes Dev. 34, 1452–1473
Rimel, J.K., Poss, Z.C., Erickson, B., Maas, Z.L., Ebmeier, C.C., Johnson, J.L., Decker, T.M., Yaron, T.M., Bradley, M.J., Hamman, K.B., Hu, S., Malojcic, G., Marineau, J.J., White, P.W., Breault, M., Tao, L., DeRoy, P., Clavette, C., Nayak, S., Damon, L.J., Kaltheuner, I.H., Bunch, H., Cantley, L.C., Geyer, M., Iwasa, J., Dowell, R.D., Bentley, D.L., Old, W.M., and Taatjes, D.J.
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(2021). 1,6-Hexanediol, commonly used to dissolve liquid-liquid phase separated condensates, directly impairs kinase and phosphatase activities. J. Biol. Chem. 296:100260
Düster, R., Kaltheuner, I.H., Schmitz, M., and Geyer, M.
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(2021). Abemaciclib is a potent inhibitor of DYRK1A and HIP kinases involved in transcriptional regulation. Nat. Commun. 12, 6607
Kaltheuner, I.H., Anand, K., Moecking, J., Düster, R., Wang, J., Gray, N.S., and Geyer, M.
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(2021). Discovery and resistance mechanism of a selective CDK12 degrader. Nat. Chem. Biol. 17, 675–683
Jiang, B., Gao, Y., Che, J., Lu, W., Kaltheuner, I.H., Dries, R., Kalocsay, M., Berberich, M.J., Jiang, J., You, I., Kwiatkowski, N.P., Riching, K.M., Daniels, D.L., Sorger, P.K., Geyer, M., Zhang, T., and Gray, N.S.
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(2021). LAMP-Seq enables sensitive, multiplexed COVID-19 diagnostics using molecular barcoding. Nat. Biotechnol. 39, 1556–1562
Ludwig, K.U., Schmithausen, R.M., Li, D., Jacobs, M.L., Hollstein, R., Blumenstock, K., Liebing, J., Słabicki, M., Ben-Shmuel, A., Israeli, O., Weiss, S., Ebert, T.S., Paran, N., Rüdiger, W., Wilbring, G., Feldman, D., Lippke, B., Ishorst, N., Hochfeld, L.M., Beins, E.C., Kaltheuner, I.H., Schmitz, M., Wöhler, A., Döhla, M., Sib, E., Jentzsch, M., Borrajo, J.D., Strecker, J., Reinhardt, J., Cleary, B., Geyer, M., Hölzel, M., Macrae, R., Nöthen, M.M., Hoffmann, P., Exner, M., Regev, A., Zhang, F., and Schmid-Burgk, J.L.
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(2021). Structure-activity relationship study of THZ531 derivatives enables the discovery of BSJ-01-175 as a dual CDK12/13 covalent inhibitor with efficacy in Ewing sarcoma. Eur. J. Med. Chem. 221:113481
Jiang, B., Jiang, J., Kaltheuner, I.H., Iniguez, A.B., Anand, K., Ferguson, F.M., Ficarro, S.B., Seong, B.K.A., Greifenberg, A.K., Dust, S., Kwiatkowski, N.P., Marto, J.A., Stegmaier, K., Zhang, T., Geyer, M., and Gray, N.S.
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(2021). Structure-guided multivalent nanobodies block SARS-CoV-2 infection and suppress mutational escape. Science 371:eabe6230
Koenig, P.A., Das, H., Liu, H., Kümmerer, B.M., Gohr, F.N., Jenster, L.M., Schiffelers, L.D.J., Tesfamariam, Y.M., Uchima, M., Wuerth, J.D., Gatterdam, K., Ruetalo, N., Christensen, M.H., Fandrey, C.I., Normann, S., Tödtmann, J.M.P., Pritzl, S., Hanke, L., Boos, J., Yuan, M., Zhu, X., Schmid-Burgk, J.L., Kato, H., Schindler, M., Wilson, I.A., Geyer, M., Ludwig, K.U., Hällberg, B.M., Wu, N.C., and Schmidt, F.I.
