A chemical-genetic approach for functional analysis of plant protein kinases

Abstract

Plant genomes encode hundreds of protein kinases, yet only for a small fraction of them precise functions and phosphorylation targets have been identified. Recently, we applied a chemical-genetic approach to sensitize the tomato serine/ threonine kinase Pto to analogs of PP1, an ATP-competitive and cell-permeable small-molecule inhibitor. The Pto kinase confers resistance to Pst bacteria by activating immune responses upon specific recognition of bacterial effectors. By using PP1 analogs in combination with the analog-sensitive Pto, we shed new light on the role of Pto kinase activity in effector recognition and signal transduction. Here we broaden the use of this chemical-genetic approach to another defense-related plant protein kinase, the MAP kinase LeMPK3. In addition, we show that analog-sensitive but not wild-type kinases are able to use unnatural N 6 -modified ATP analogs as phosphodonors that can be exploited for tagging direct phosphorylation targets of the kinase of interest. Thus, sensitization of kinases to analogs of the small-molecule inhibitor PP1 and ATP can be an effective tool for the discovery of cellular functions and phosphorylation substrates of plant protein kinases. IntroductionProtein phosphorylation plays a central role in controlling a wide array of signal transduction pathways in plant cells. The large number of protein kinases present in plant proteomes (>1,000 in Arabidopsis, >1,500 in rice) makes it a very difficult challenge to assign precise roles to each individual kinase. 1,2 Understanding the function of any protein kinase requires identification of its direct substrates and evaluation of effects caused by its inactivation. Because knockout mutations and gene silencing strategies are often compromised by lethality, functional redundancy or cellular homeostasis, the development of cell-permeable inhibitors that are highly specific for individual protein kinases represents an invaluable tool for assisting in the definition of plant kinase function. We used a chemical-genetic strategy developed by Shokat and coworkers 3 to engineer plant kinases with sensitivity to small-molecule inhibitors that do not block the activity of wild-type kinases. These engineered kinases, which are referred to as "analog-sensitive" (as) alleles, also often accept analogs of ATP that can be exploited in their radiolabeled form for tagging direct phosphorylation targets of the analog-sensitive kinase in complex protein mixtures. 4 Here and in our recent paper in the Journal of Biological Chemistry, 5 we describe the application of this chemical-genetic approach to two defense-related protein kinases from tomato (Solanum lycopersicum): the serine/threonine kinase Pto, which confers resistance to Pseudomonas syringae pv. tomato (Pst) bacteria expressing the AvrPto and AvrPtoB effector proteins, 6 and the MAP kinase LeMPK3, which participates in signaling pathways activated by plant perception of pathogen molecules. 7-9 Sensitizing Plant Protein Kinases to Inhibition by Small-Molecule LigandsTo investigat...

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