Biochemical characterization of plant protein kinases using a novel cholorimetric method

ROJAS, BRUNO E; PIATTONI CLAUDIA VANESA; HARTMAN, MATÍAS D.; FIGUEROA, CARLOS M.; IGLESIAS, ALBERTO ALVARO

Buenos Aires

Congreso; Reunión conjunta de sociedades de biociencias; 2017

SAIB & SAIC

Protein kinases (PKs, EC 2.7.11.-) constitute one of thelargest protein families and are important components of signalingcascades in eukaryotes. By modifying protein activity, they regulatenearly every aspect of cellular function. Due to their sessile lifestyle,plants developed new signaling systems to cope with the challengesimposed by an always-changing environment. Therefore, plantshave expanded PKs families, developing many that are unique totheir kingdom. Besides the major role that reversible phosphorylationplays in the regulation of carbon metabolism in plants, informationabout the biochemical properties of plant PKs is scarce. This ismainly due to the difficulties to assay their activity using radiolabeledATP and because the non-radioactive methods available to datelack the proper sensitivity and cannot be adapted to every kinase. Inthis work, we designed a straightforward protocol based on a cyclicenzymatic assay to measure PK activity using a 96-well microplatereader. Released ADP is used to oxidize NADH via pyruvate kinaseand lactate dehydrogenase. Afterwards, NAD+ is recycled using alcoholdehydrogenase/ethanol and phenazine ethosulfate/ThiazolylBlue Tetrazolium Bromide. The method measures released ADP afterthe phosphoryl transfer (linear range from 0 to 4 nmol); thus, itcan be used to analyze virtually all PKs. To validate the protocol, wecharacterized recombinant PKs with different catalytic mechanismsand from different plant species, including Arabidopsis thalianaSnRK1, Malus domestica SOS2 (a member of the SnRK3 subfamily),and Solanum tuberosum CDPK1. Assays run in parallel usingthe colorimetric method and radiolabeled ATP showed a correlationclose to 1. Overall, this tool represents a step forward in the functionalcharacterization of PKs, which is critical to expand our knowledgeabout signaling networks in plants.