Pharmacological Sciences

Protein kinases mediate many cell signaling events, and their tight control is essential for regulating vital processes ranging from cell division to energy metabolism. Thus, it is not surprising that protein kinases are directly or indirectly involved in many diseases and that kinases are key drug targets. For example, Src kinase was the first identified proto-oncogene and the formation of a de-regulated Abl fusion protein (BCRAbl) is the cause of disease in 95% of patients with chronic myeloid leukemia. X-ray crystal structures have shown that the same kinases can attain an active and various inactive conformations, implying that kinases are inherently flexible. How the active and inactive states are stabilized and how these states interconvert are key questions in understanding kinase regulation. Because X-ray crystal structures provide only static snapshots, we will use nuclear magnetic resonance (NMR) experiments and ligand binding kinetics to study the timescales and amplitudes of structural interconversions in Abl and Src kinase domains. BCR-Abl is the target of the clinically highly successful drug imatinib (Gleevec®, Novartis) in the treatment of chronic myelogenous leukemia (CML). The clinical success of imatinib is due to its excellent specificity, binding only to the inactive conformation of the kinase. Therefore drug binding is intimately related to the interconversion between active and inactive states. The goal of this study is to examine timescales and pathways of these interconversions between active and inactive conformations, how dynamics of structural elements relate to catalytic turnover of the kinase and how drug resistance mutations affect these dynamics. Therefore, we will compare the timescales and amplitudes of backbone motions between Src and Abl kinases in the presence of drugs by NMR experiments. Ligand binding kinetics will be used to address the role of the regulatory domains on kinase dynamics and the binding mechanisms of different classes of kinase inhibitors. The role of protein plasticity and dynamics on inhibitor promiscuity will be addressed by structural studies on kinase•inhibitor complexes, inhibitor binding kinetics and biochemical assays.

Selected Publications

Shan, Y., Seeliger, M.A., Eastwood, M.P., Frank, F., Xu, H., Jensen, M.O., Dror, R.O., Kuriyan, J., and Shaw, D.E., A conserved protonation-dependent switch controls drug binding in the Abl kinase. Proc Natl Acad Sci U S A, 2009. 106(1): p. 139-44.

Seeliger, M.A., Ranjitkar, P., Kasap, C., Shan, Y., Shaw, D.E., Shah, N.P., Kuriyan, J., and Maly, D.J., Equally potent inhibition of c-Src and Abl by compounds that recognize inactive kinase conformations. Cancer Res, 2009. 69(6): p. 2384-92. (Cover)

Seeliger, M.A. and Kuriyan, J., A MAPK scaffold lends a helping hand. Cell, 2009. 136(6): p. 994-6. (Preview)

Muratore, K.E., Seeliger, M.A., Wang, Z., Fomina, D., Neiswinger, J., Havranek, J.J., Baker, D., Kuriyan, J., and Cole, P.A., Comparative analysis of mutant tyrosine kinase chemical rescue. Biochemistry, 2009. 48(15): p. 3378-86.

Choi, Y., Seeliger, M., Panjarian, S.B., Kim, H., Deng, X., Sim, T., Couch, B., Koleske, A.J., Smithgall, T.E., and Gray, N.S., N-myristoylated c-ABL tyrosine kinase localizes to the endoplasmic reticulum upon binding to an allosteric inhibitor. 10.1074/jbc.M109.026633. J. Biol. Chem., 2009: p. M109.026633.

Statsuk, A.V., Maly, D.J., Seeliger, M.A., Fabian, M.A., Biggs, W.H., 3rd, Lockhart, D.J., Zarrinkar, P.P., Kuriyan, J., and Shokat, K.M., Tuning a three-component reaction for trapping kinase substrate complexes. J Am Chem Soc, 2008. 130(51): p. 17568-74.

Levinson, N.M., Seeliger, M.A., Cole, P.A., and Kuriyan, J., Structural basis for the recognition of c-Src by its inactivator Csk. Cell, 2008. 134(1): p. 124-34.

Azam, M., Seeliger, M.A., Gray, N.S., Kuriyan, J., and Daley, G.Q., Activation of tyrosine kinases by mutation of the gatekeeper threonine. Nat Struct Mol Biol, 2008. 15(10): p. 1109-18.

Seeliger, M.A., Nagar, B., Frank, F., Cao, X., Henderson, M.N., and Kuriyan, J., c-Src binds to the cancer drug imatinib with an inactive Abl/c-Kit conformation and a distributed thermodynamic penalty. Structure, 2007. 15(3): p. 299-311.

Young, M.A., Shah, N.P., Chao, L.H., Seeliger, M., Milanov, Z.V., Biggs, W.H., 3rd, Treiber, D.K., Patel, H.K., Zarrinkar, P.P., Lockhart, D.J., Sawyers, C.L., and Kuriyan, J., Structure of the kinase domain of an imatinib-resistant Abl mutant in complex with the Aurora kinase inhibitor VX-680. Cancer Res, 2006. 66(2): p. 1007-14.

Yao, Z.P., Zhou, M., Kelly, S.E., Seeliger, M.A., Robinson, C.V., and Itzhaki, L.S., Activation of ubiquitin ligase SCF(Skp2) by Cks1: insights from hydrogen exchange mass spectrometry. J Mol Biol, 2006. 363(3): p. 673-86.

Nagar, B., Hantschel, O., Seeliger, M., Davies, J.M., Weis, W.I., Superti-Furga, G., and Kuriyan, J., Organization of the SH3-SH2 unit in active and inactive forms of the c-Abl tyrosine kinase. Mol Cell, 2006. 21(6): p. 787-98.

Bader, R., Seeliger, M.A., Kelly, S.E., Ilag, L.L., Meersman, F., Limones, A., Luisi, B.F., Dobson, C.M., and Itzhaki, L.S., Folding and fibril formation of the cell cycle protein Cks1. J Biol Chem, 2006. 281(27): p. 18816-24.

Seeliger, M.A., Spichty, M., Kelly, S.E., Bycroft, M., Freund, S.M., Karplus, M., and Itzhaki, L.S., Role of conformational heterogeneity in domain swapping and adapter function of the Cks proteins. J Biol Chem, 2005. 280(34): p. 30448-59.

Seeliger, M.A., Young, M., Henderson, M.N., Pellicena, P., King, D.S., Falick, A.M., and Kuriyan, J., High yield bacterial expression of active c-Abl and c-Src tyrosine kinases. Protein Sci, 2005. 14(12): p. 3135-9.

Mapelli, M., Massimiliano, L., Crovace, C., Seeliger, M.A., Tsai, L.H., Meijer, L., and Musacchio, A., Mechanism of CDK5/p25 binding by CDK inhibitors. J Med Chem, 2005. 48(3): p. 671-9.

Seeliger, M.A., Breward, S.E. and Itzhaki, L.S., Weak cooperativity in the core causes a switch in folding mechanism between two proteins of the cks family. J Mol Biol, 2003. 325(1): p. 189-99.

Seeliger, M.A., Breward, S.E., Friedler, A., Schon, O., and Itzhaki, L.S., Cooperative organization in a macromolecular complex. Nat Struct Biol, 2003. 10(9): p. 718-24.

Sitry, D., Seeliger, M.A., Ko, T.K., Ganoth, D., Breward, S.E., Itzhaki, L.S., Pagano, M., and Hershko, A., Three different binding sites of Cks1 are required for p27-ubiquitin ligation. J Biol Chem, 2002. 277(44): p. 42233-40.

Seeliger, M.A., Schymkowitz, J.W., Rousseau, F., Wilkinson, H.R., and Itzhaki, L.S., Folding and association of the human cell cycle regulatory proteins ckshs1 and ckshs2. Biochemistry, 2002. 41(4): p. 1202-10.