Scripps Research Logo

The Sauer Lab

Research

3. Multidisciplinary Analyses of Kinase Function and Small Molecule Inhibition

By transferring phosphate groups onto a plethora of diverse substrates in the cell, kinases are the "workhorses" of signal transduction. They are involved in and regulate almost any cellular process. Kinase malfunction or deregulation underlies a multitude of important diseases, in particular immune disorders and many cancers.

Within the "kinome"1,2, Src family protein tyrosine kinases (SFKs) are critical regulators of signaling downstream of many different membrane receptors in many different cell types3,4. They can regulate as diverse processes as responses to UV light, heat, osmotic shock, sensitivity to ethanol consumption, β-adrenergic signaling or growth factor responses. SFK hyperactivity is observed in many cancers4. In immune cells, SFKs play important roles in signaling from antigen receptors, cytokine receptors, GPCRs or integrins3. Physiologically, SFKs have been implicated in development and function of several components of the immune system, in particular T, B and myeloid cells3,5.

In this project, we combine functional genomics, molecular modeling, molecular dynamics simulations, genetics, biochemistry, enzymology and immunology into a multidisciplinary, integrated approach to analyze structural features which are critical for ATP binding, catalysis, interactions with ATP-competitive small molecule inhibitors and function of kinases both in vitro and in vivo. Through these studies, we hope to contribute to a better understanding of how kinases work mechanistically and physiologically, and to the development of novel and improved kinase inhibitor therapeutics.

In one example, the glycine-rich G-loop controls ATP binding and phosphate transfer in protein kinases. We recently showed that the functions of Src family and Abl protein tyrosine kinases require an electrostatic interaction between oppositely charged amino acids within their G loops that is conserved in multiple other phylogenetically distinct protein kinases from plants to humans (Fig. 1)6. By limiting G-loop flexibility, it controls ATP binding, catalysis and inhibition by ATP-competitive compounds such as Imatinib/Gleevec, exemplified by Abl. In WeeB mice, mutational disruption of the interaction results in expression of a Lyn protein with reduced catalytic activity, and in perturbed B cell receptor signaling. Like Lyn-/- mice, WeeB mice show profound defects in B cell development and function and succumb to autoimmune glomerulonephritis. This demonstrates the physiological importance of the conserved G-loop salt bridge and at the same time distinguishes the in vivo requirement for the Lyn kinase activity from other potential functions of the protein.

A conserved salt bridge in the G-loop of multiple phylogenetically diverse kinase domains is essential for catalysis

Fig. 1. A conserved salt bridge in the G-loop of multiple phylogenetically diverse kinase domains is essential for catalysis6. (A) Amino acid sequence alignment of the G-loop region of all SFK family members. h, human; m, murine; z, zebrafish; t, torpedo. β1, β2, β-strands flanking the G-loop. An extended SFK G-loop consensus sequence is shown below the alignment. Green, conserved Gs forming the characteristic protein kinase G-loop motif GxG0xxGxV. Black, deviations from the consensus. Blue, a conserved basic residue (K/R) at position -4 N-terminally of the invariant G0. Red, a conserved acidic E or D at position +4, corresponding to E260 in murine Lyn A. (B) Top, ribbon diagrams of the kinase domain structures of human Hck (1AD5), Lck (2OF2), c-Src (1Y57), Abl (1IEP), zea mays CK2A1 (1LP4) and human SLK (2JFL) bound to ATP analogs. Orange, G-loop; colored, ATP-analog. Bottom, G-loop blowups showing electrostatic interactions between the conserved basic and acidic side chains flanking the LGxG0xFG motif at positions -4 and +4. The polarity of the SLK interaction is reversed. (C) Phylogenetic tree of all eukaryotic protein kinase domains from kinase.com/human/kinome/phylogeny.html, rendered with HyperTree. Red dots indicate the kinases in (B), listing major family_subfamily_member. TK, tyrosine kinase; Src, Src family; STE20, Ste20-family kinase; Other, no major group. (D) Disruption of the conserved SFK G-loop salt bridge abrogates catalytic activity. Recombinant full-length mouse Lyn or the G-loop mutants E260G (LynWeeB), K252G, K252G/E260G, K252E/E260K or K252E were expressed in Sf21 cells to the levels shown via IB (upper panel). Lower panel, catalytic activity in vitro. Error bars, standard deviations of triplicates. Salt bridge disrupting mutations strongly reduce Lyn catalytic activity, indicating the importance of this structural "bridge". For details, see6.

Further Reading

1. Manning, G., Whyte, D.B., Martinez, R., Hunter, T. & Sudarsanam, S. The Protein Kinase Complement of the Human Genome. Science 298, 1912-1934 (2002).
2. Caenepeel, S., Charydczak, G., Sudarsanam, S., Hunter, T. & Manning, G. The mouse kinome: Discovery and comparative genomics of all mouse protein kinases. Proceedings of the National Academy of Sciences 101, 11707-11712 (2004).
3. Lowell, C.A. Src-family kinases: rheostats of immune cell signaling. Molecular Immunology 41, 631-643 (2004).
4. Yeatman, T.J. A renaissance for SRC. Nature Reviews 4, 470-480 (2004).
5. Xu, Y., Harder, K.W., Huntington, N.D., Hibbs, M.L. & Tarlinton, D.M. Lyn tyrosine kinase: accentuating the positive and the negative. Immunity 22, 9-18 (2005).
6. Barouch-Bentov, R., Che, J., Lee, C.C., Yang, Y., Herman, A., Jia, Y., Velentza, A., Watson, J., Sternberg, L., Kim, S., Ziaee, N., Miller, A., Jackson, C., Fujimoto, M., Young, M., Batalov, S., Liu, Y., Warmuth, M., Wiltshire, T., Cooke, M.P., and Sauer, K. A Conserved Salt Bridge in the G Loop of Multiple Protein Kinases Is Important for Catalysis and for in Vivo Lyn Function. Molecular Cell 33, 43-52 (2009).