title: Chemical genetic analysis of protein kinase function in plants.
abstract: 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.
authors.affiliation: Institute for Biology and Biotechnology of Plants, University of Münster, Münster, Germany. mboehmer@ucsd.edu
authors.forename: Maik
authors.initials: M
authors.lastname: Böhmer
authors.forename: Michael
authors.initials: M
authors.lastname: Bölker
authors.forename: Tina
authors.initials: T
authors.lastname: Romeis
mesh_terms: D000255:Adenosine Triphosphate / Q000302:isolation & purification / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D002852:Chromatography, Ion Exchange; D015180:Electrophoresis, Gel, Two-Dimensional; D057075:Enzyme Assays; D018506:Gene Expression Regulation, Plant; D007553:Isotope Labeling; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D010944:Plants / Q000201:enzymology* / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*
chemical_list: D029681:Arabidopsis Proteins; D000255:Adenosine Triphosphate; D011494:Protein Kinases; C525986:CPK1 protein, Arabidopsis