Bypassing Kinase Activity of the Tomato Pto Resistance Protein with Small Molecule Ligands

Salomon, Dor; Bonshtien, Arale; Mayrose, Maya; Zhang, Chao; Shokat, Kevan M.; Sessa, Guido

doi:10.1074/jbc.m809724200

Cited by 12 publications

(9 citation statements)

References 53 publications

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“…Cdk2 as appears most similar to a PKC gatekeeper mutant with weakened nucleotide binding and reduced T-loop phosphorylation, which was complemented by ATP analogs (Cameron et al, 2009), but may be unique in its impaired binding to an allosteric regulator, cyclin A. This might be analogous to effects of a gatekeeper mutation on interactions between the plant kinase Pto and its bacterial effector proteins, which were also corrected by PP1-based analogs (Salomon et al, 2009). …”

Section: Discussionmentioning

confidence: 99%

Switching Cdk2 On or Off with Small Molecules to Reveal Requirements in Human Cell Proliferation

et al. 2011

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Summary Multiple cyclin-dependent kinases (CDKs) control eukaryotic cell division, but assigning specific functions to individual CDKs remains a challenge. During the mammalian cell cycle, Cdk2 forms active complexes before Cdk1, but lack of Cdk2 protein does not block cell-cycle progression. To detect requirements and define functions for Cdk2 activity in human cells when normal expression levels are preserved, and non-physiologic compensation by other CDKs is prevented, we replaced the wild-type kinase with a version sensitized to specific inhibition by bulky adenine analogs. The sensitizing mutation also impaired a non-catalytic function of Cdk2 in restricting assembly of cyclin A with Cdk1, but this defect could be corrected by both inhibitory and non-inhibitory analogs. This allowed either chemical rescue or selective antagonism of Cdk2 activity in vivo, to uncover a requirement in cell proliferation, and non-redundant, rate-limiting roles in restriction point passage and S-phase entry.

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Section: Discussionmentioning

confidence: 99%

Switching Cdk2 On or Off with Small Molecules to Reveal Requirements in Human Cell Proliferation

et al. 2011

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“…Interestingly, in the presence of a PP1 analog a Pto as kinase deficient form was able to trigger the elicitation of an AvrPtodependent hypersensitive response in planta 5 suggesting that kinase activity is dispensable for Pto signaling. A similar effect of PP1 analogs was previously observed on the analog-sensitive allele of the stress sensor Ire1, which controls the activation of the unfolded protein response in yeast.…”

Section: Bypassing Kinase Activity With Pp1 Analogsmentioning

confidence: 99%

“…5 Unexpectedly, PP1 analogs rescued in yeast interactions of the kinase-deficient Pto as (K69R) with the AvrPto and AvrPtoB effectors, 5 and with a known Pto-interactor, the tomato transcription factor Pti4 14 (Fig. 2).…”

Section: Bypassing Kinase Activity With Pp1 Analogsmentioning

confidence: 99%

“…10 kinases, 3,11 substitution of the large gatekeeper residue with an Ala generated a unique pocket in the Pto as ATP binding site and resulted in sensitization of Pto as to specific inhibition by various PP1 analogs. 5 We applied a similar strategy to sensitize to smallmolecule inhibitors the tomato MAP kinase LeMPK3. To this aim, the LeMPK3 gatekeeper residue Thr123 was mutated to Ala or Gly yielding LeMPK3 as1 and LeMPK3 as2 , respectively.…”

Section: Sensitizing Plant Protein Kinases To Inhibition By Small-molmentioning

confidence: 99%

“…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.…”

Section: Introductionmentioning

confidence: 99%

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

Salomon

Bonshtien²,

Sessa³

2009

Plant Signaling & Behavior

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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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Chemical Genetic Analysis of Protein Kinase Function in Plants

Böhmer

Bölker

Romeis

2011

Methods in Molecular Biology

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Identification of protein kinase targets and specific inhibition of individual kinase isoforms on the protein level in planta are important techniques to elucidate signal transduction pathways. The use of ATP-binding pocket mutants, the so-called gatekeeper mutants, that accommodate N(6)-enlarged nucleotides and kinase inhibitors has allowed a dramatic increase in kinase isoform selectivity. In this chapter, we describe protocols for the identification and mutation of the gatekeeper residue, radiolabeling of N(6)-modified nucleotides, analysis of protein targets by using [(32)P]-labeled N(6)-modified nucleotides, and in vivo inhibition of kinase activity combined with subsequent molecular readouts. The chapter includes alternative approaches for the described techniques, considerations for other kinases and recommendations toward a setup of a substrate labeling experiment using total cell lysate.

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Bypassing Kinase Activity of the Tomato Pto Resistance Protein with Small Molecule Ligands

Cited by 12 publications

References 53 publications

Switching Cdk2 On or Off with Small Molecules to Reveal Requirements in Human Cell Proliferation

Switching Cdk2 On or Off with Small Molecules to Reveal Requirements in Human Cell Proliferation

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

Chemical Genetic Analysis of Protein Kinase Function in Plants

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