Sensitizing Plant Protein Kinases to Specific Inhibition by ATP-Competitive Molecules
The highly conserved nature of the protein kinase catalytic domain and the low permeability of plant cell membranes pose a challenge to the development of specific inhibitors that target individual protein kinases in vivo. Here, we describe a chemical-genetic approach to specifically sensitize individual plant kinases to cell-permeable small molecules that do not inhibit wild-type kinases. In this approach, a single amino-acid substitution is introduced in the ATP-binding site of the enzyme enabling specific binding of ATP-competitive molecules. Cell-permeable molecules can then be used to specifically target the sensitized allele in transgenic Arabidopsis thaliana plants that do not express the wild-type form of the kinase. This strategy provides a useful tool for the functional characterization of protein kinases in planta and for the dissection of the signaling pathways in which they are involved.
Key wordsProtein kinase PP1 Gatekeeper ATP-competitive molecules Analog-sensitive Arabidopsis Kinase activity assay Transgenic plants
We thank Tal Sherman for his advice on Arabidopsis plant transformations. This work was supported by the US–Israel Binational Agricultural Research and Development Fund (BARD; grant no. IS-4159-08C to G.S.) and by the Binational Science Foundation (BSF; grant no. 2007091 to G.S. and K.M.S.).
- 2.Chang, C. (2003) Ethylene signaling: the MAPK module has finally landed Trends Plant Sci 8, 365–8.Google Scholar
- 4.Morillo, S. A., and Tax, F. E. (2006) Functional analysis of receptor-like kinases in monocots and dicots Curr Opin Plant Biol 9, 460–9.Google Scholar
- 5.To, J. P. C., and Kieber, J. J. (2008) Cytokinin signaling: two-components and more Trends Plant Sci 13, 85–92.Google Scholar
- 6.Zhu, J. K. (2002) Salt and drought stress signal transduction in plants Annu Rev Plant Biol 53, 247–73.Google Scholar
- 8.Bishop, A.C., Ubersax, J.A., Petsch, D.T., Matheos, D.P., Gray, N.S., Blethrow, J., Shimizu, E., Tsien, J. Z., Schultz, P. G., Rose, M. D., Wood, J.L., Morgan, D. O., and Shokat, K.M. (2000) A chemical switch for inhibitor-sensitive alleles of any protein kinase Nature 407, 395–401.Google Scholar
- 9.Bohmer, M., and Romeis, T. (2007) A chemical-genetic approach to elucidate protein kinase function in planta Plant Mol Biol 65, 817–27.Google Scholar
- 10.Brodersen, P., Petersen, M., Nielsen, H. B., Zhu, S. J., Newman, M. A., Shokat, K. M., Rietz, S., Parker, J., and Mundy, J. (2006) Arabidopsis MAP kinase 4 regulates salicylic acid- and jasmonic acid/ethylene-dependent responses via EDS1 and PAD4 Plant J 47, 532–46.Google Scholar
- 11.Lin, J. H., Li, H., Yasumura, D., Cohen, H. R., Zhang, C., Panning, B., Shokat, K. M., LaVail, M. M., and Walter, P. (2007) IRE1 signaling affects cell fate during the unfolded protein response Science 318, 944–9.Google Scholar
- 12.Bishop, A. C., Kung, C.-y., Shah, K., Witucki, L., Shokat, K. M., and Liu, Y. (1999) Generation of monospecific nanomolar tyrosine kinase inhibitors via a chemical genetic approach. J Am Chem Soc 121, 627–31.Google Scholar
- 13.Benjamin, K. R., Zhang, C., Shokat, K. M., and Herskowitz, I. (2003) Control of landmark events in meiosis by the CDK Cdc28 and the meiosis-specific kinase Ime2 Genes Dev 17, 1524–39.Google Scholar
- 14.Blethrow, J., Zhang, C., Shokat, K. M., and Weiss, E. L. (2004) Design and use of analog-sensitive protein kinases Curr Protoc Mol Biol Chapter 18, Unit 18 11.Google Scholar
- 15.Zhang, C., Kenski, D. M., Paulson, J. L., Bonshtien, A., Sessa, G., Cross, J. V., Templeton, D. J., and Shokat, K. M. (2005) A second-site suppressor strategy for chemical genetic analysis of diverse protein kinases Nat Methods 2, 435–41.Google Scholar