(T. K.) Thomas K. Harris

Associate Professor of
Biochemistry and Molecular Biology

Ph.D. (1994) University of Mississippi
Medical Center
Postdoctorate (1994-2000)
Johns Hopkins University School of Medicine

NMR and Kinetic Studies of Multidomain
Protein Kinase Action and Regulation

 Tel:  (305) 243-3358, Fax:  (305) 243-3955

 tkharris@miami.edu

RESEARCH OBJECTIVES

In many cancers, oncogenic transformation has been found to correlate with increased activation of a number of signal transduction pathways mediated internally by members of the serine-threonine protein kinase family; and large efforts are being directed towards design of potent and selective inhibitors of well established protein kinase drug targets. Since the overwhelming majority of protein kinase inhibitors bind in or near the ATP binding pocket shared by the catalytic domain of all kinases, very few serine-threonine protein kinase inhibitors have been clinically approved due to their broad specificity and overall high toxicity. Therefore, we are pursuing the hypothesis that serine-threonine protein kinase inhibitor selectivity may be better achieved by designing compounds that target interfacial clefts and crevices formed between contiguous regulatory and catalytic kinase domains, thereby 'allosterically' stabilizing inactive or autoinhibited conformations of multi-domain protein kinases.

The idea of improving selectivity by targeting interfacial clefts is motivated by inspection of primary structure alignments of a number of well established serine-threonine protein kinase drug targets. While the catalytic kinase domains share a high degree of structural homology, the most distinguishing feature among these kinases is the distribution of various types of regulatory domains, many of which are contiguous with the catalytic kinase domain. We are employing two complementary approaches that seek to establish both the functional role and the structural basis by which regulatory domains affect kinase activity. The foregoing approach is determination of the kinetic mechanisms of target protein kinases, which focus on establishing the degree of activation or inhibition that a regulatory domain exerts on one or more specific elementary reaction steps such as substrate binding or chemical phosphorylation. For example in the PDK1 and S6K1 protein kinase drug targets, we have demonstrated (i) how contiguous regulatory domains stabilize inactive conformations of their respective catalytic kinase domain and (ii) how allosteric effectors such as second messenger binding or phosphorylation can lead to kinase activation. Subsequent elucidation of the structures entailing inactive conformations will facilitate design of the hypothesized highly selective 'allosteric' inhibitors.

While three-dimensional structures have been determined for individual regulatory and catalytic domain constructs of numerous oncogenic protein kinase targets, the dynamical nature of the majority of multi-domain protein kinases precludes formation of diffraction quality crystals so that their overall domain-domain orientations remain elusive. In order to detect the elusive but potentially vast frontier of new drug targeting sites that may exist in pockets formed between contiguous autoregulatory and catalytic kinase domains, we are developing a novel structure-based approach termed Magnetic Relaxation Enhancement Triangulation (MRET). For a given multi-domain protein kinase drug target, the MRET approach combines site-directed paramagnetic spin labeling of the catalytic domain with segmental ¹⁵N-isotopic labeling of a contiguous regulatory domain. NMR relaxation studies are performed to obtain distance restraints from the site-directed spin label of the catalytic domain to each backbone amide proton (¹⁵N-H) of the regulatory domain. Distances can be calculated from the amount that either the longitudinal (R1) or transverse (R2) relaxation rates of the amide protons are increased in the presence of the spin label. In addition, distance calculations require NMR or EPR measurement of the effective rotational correlation times of the individual electron-amide proton vectors (t_c). With such numerous distance restraints, distance geometry/simulated annealing protocols in CNS/XPLOR are used to calculate the overall orientation exhibited between the contiguous catalytic and regulatory domains.

Representative Publications

(34) Keshwani, M. M., and Harris, T. K. (2008) Kinetic mechanism of fully activated S6K1 protein kinase. J. Biol. Chem. 283, 11972-11980.

(33)  Keshwani, M. M., Ross, D. B., Ragan, T. J., and Harris, T. K. (2008) Baculovirus-mediated expression, purification, and characterization of a fully activated catalytic kinase domain of the 70 kDa 40S ribosomal protein S6 kinase-1 aII isoform (S6K1aII). Protein Expr. Purif. 58, 32-41.

(32) Ragan, T. J., Ross, D., Keshwani, M., and Harris, T. K. (2008) Expression, purification, and characterization of a structurally disordered but functional C-terminal autoinhibitory domain (AID) of the 70 kDa 40S ribosomal protein S6 kinase-1 (S6K1). Protein Expr. Purif. 57, 271-279.

(31) Yildiz, I., Gao, X., Harris, T. K., and Raymo, F. M. (2007) Fluoresence resonance energy transfer in quantum dot-protein kinase assemblies. J. Biomed. Biotech. 2007, 18081.

(30) Al-Ali, H., Ragan, T. J., Gao, X., and Harris, T. K. (2007) Reconstitution of modular PDK1 functions on trans-splicing of the regulatory PH and catalytic kinase domains. Bioconjugate Chem. 18, 1294-1302.

(29)  Harris, T. K. (2006) Discovering new drug targeting sites on flexible multi-domain protein kinases: combining segmental isotopic and site-directed spin labeling for NMR detection of interfacial clefts. Methods Mol. Biol. 316, 199-225.

(28)  Gao, X., and Harris, T. K. (2006) Steady-state kinetic mechanism of PDK1. J. Biol. Chem. 281, 21670-21681.

(27)  Gao, X., and Harris, T. K. (2006) Role of PH domain in regulating in vitro autophosphorylation events required for reconstitution of PDK1 catalytic activity. Bioorg. Chem. 34, 200-223.

(26)  Gao, X., Yo, P., and Harris, T. K. (2005) Improved yields for baculovirus-mediated expression of human His₆-PDK1 and His₆-PKBb/Akt2 and characterization of phospho-specific isoforms for design of inhibitors that stabilize inactive conformations. Protein Expr. Purif. 43, 44-56.

(25)  Gao, X., Yo, P., Keith, A., Ragan, T. J., and Harris, T. K. (2003) Thermodynamically balanced inside-out (TBIO) PCR-based gene synthesis: a novel method of primer design for high-fidelity assembly of longer gene sequences. Nuc. Acids Res. 31(22), e143.

(24)  Harris, T. K. (2003) PDK1 and PKB/Akt: ideal targets for development of new strategies to structure-based drug design. IUBMB Life 55, 117-126.

Honors and Professional Activities 

American Chemical Society (ACS)
American Society of Biochemistry and Molecular Biology (ASBMB)
Sylvester Comprehensive Cancer Center
Editorial Board, IUBMB Life,
Editorial Board, International Journal of Biological Chemistry
Albert L. Lehninger Award (2000)
NIH F32 GM017514 (1996-1997)
Florida Biomedical Research Program (2001-2003)
NIH R01 GM069868 (2004-2008)