title: Substrate analysis of Arabidopsis PP2C-type protein phosphatases.
abstract: Protein phosphorylation by protein kinases can be reversed by the action of protein phosphatases. In plants, the Ser/Thr-specific phosphatases dominate among the protein phosphatase families with the type 2C protein phosphatases (PP2Cs) being the most abundant among them. PP2Cs are monomeric enzymes that require metal cations for their activity and are insensitive to known phosphatase inhibitors. PP2Cs were shown to counteract the mitogen-activated protein kinase (MAP kinase/MAPK) activities in plants and to regulate developmental and stress signaling pathways. Studies of PP2C activities can be performed in vitro using recombinant proteins. The potential substrates of PP2Cs can be tested for dephosphorylation by the phosphatase in vitro. We have found that the stress-induced PP2Cs from alfalfa and Arabidopsis interact with stress-activated MAPKs in yeast two-hybrid (Y2H) screens. Consequently, recombinant MAPKs were employed as substrates for dephosphorylation by selected PP2Cs from different family clusters. The members of the PP2C phosphatase family demonstrated specificity toward the substrate already in vitro, supporting the notion that protein phosphatases are specific enzymes. The PP2C from Arabidopsis thaliana cluster B, Arabidopsis PP2C-type phosphatase (AP2C1), and its homolog from Medicago sativa, Medicago PP2C-type phosphatase (MP2C), were able to dephosphorylate and inactivate MAPKs, whereas the ABSCISIC ACID (ABA)-INSENSITIVE 2 (ABI2) and HOMOLOGY TO ABI1 (HAB1) PP2Cs from the distinct Arabidopsis cluster A were not able to do so. The method described here can be used for the determination of PP2C protein activity and for studying the effect of mutations introduced into their catalytic domains.
authors.affiliation: Max F. Perutz Laboratories, University of Vienna, Wien, Austria.
authors.forename: Julija
authors.initials: J
authors.lastname: Umbrasaite
authors.forename: Alois
authors.initials: A
authors.lastname: Schweighofer
authors.forename: Irute
authors.initials: I
authors.lastname: Meskiene
mesh_terms: D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D002364:Caseins / Q000378:metabolism; D057075:Enzyme Assays / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D002364:Caseins; D011994:Recombinant Proteins; D020928:Mitogen-Activated Protein Kinases; D020929:Mitogen-Activated Protein Kinase Kinases; D010749:Phosphoprotein Phosphatases