A versatile strategy to define the phosphorylation preferences of plant protein kinases and screen for putative substrates.
Most signaling networks are regulated by reversible protein phosphorylation. The specificity of this regulation depends in part on the capacity of protein kinases to recognize and efficiently phosphorylate particular sequence motifs in their substrates. Sequenced plant genomes potentially encode over than 1000 protein kinases, representing 4% of the proteins, twice the proportion found in humans. This plethora of plant kinases requires the development of high-throughput strategies to identify their substrates. In this study, we have implemented a semi-degenerate peptide array screen to define the phosphorylation preferences of four kinases from Arabidopsis thaliana that are representative of the plant calcium-dependent protein kinase and Snf1-related kinase superfamily. We converted these quantitative data into position-specific scoring matrices to identify putative substrates of these kinases in silico in protein sequence databases. Our data show that these kinases display related but nevertheless distinct phosphorylation motif preferences, suggesting that they might share common targets but are likely to have specific substrates. Our analysis also reveals that a conserved motif found in the stress-related dehydrin protein family may be targeted by the SnRK2-10 kinase. Our results indicate that semi-degenerate peptide array screening is a versatile strategy that can be used on numerous plant kinases to facilitate identification of their substrates, and therefore represents a valuable tool to decipher phosphorylation-regulated signaling networks in plants.
Vlad
Florina
F
Centre National de la Recherche Scientifique, Institut des Sciences du Végétal, UPR 2355, 1 avenue de la Terrasse, Bât. 23, 91198 Gif-sur-Yvette Cedex, France.
Turk
Benjamin E
BE
Peynot
Philippe
P
Leung
Jeffrey
J
Merlot
Sylvain
S
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology*; D029681:Arabidopsis Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D008969:Molecular Sequence Data; D010766:Phosphorylation; D040081:Protein Array Analysis; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D013379:Substrate Specificity
D029681:Arabidopsis Proteins; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; C492978:SnRK2 protein, Arabidopsis; C406738:SOS2 protein, Arabidopsis; C531469:At3g01090 protein, Arabidopsis; C518589:CDPK32 protein, Arabidopsis