A versatile strategy to define the phosphorylation preferences of plant protein kinases and screen for putative substrates

Abstract

SummaryMost 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.

Aquaporins Contribute to ABA-Triggered Stomatal Closure through OST1-Mediated Phosphorylation

“…First, OST1, a SnRK2 protein kinase closely linked to ABA receptors, was able to phosphorylate PIP2;1 peptides in vitro. In good agreement with its predicted site specificity (Vlad et al, 2008;Sirichandra et al, 2010), OST1 showed a large preference for loop B Ser-121, whereas two major C-terminal phosphorylation sites (Ser-280 and Ser-283) were not recognized. Second, coexpression of PIP2;1 with OST1 in oocytes enhanced PIP2;1 water transport activity and mutant analysis showed that Stomatal aperture was monitored in Col-0 (black circles), pip2;1-2 (red squares), or pip2;1-2 plants expressing S121A (S121A-1, dark-blue squares; S121A-2, light-blue squares) or S121D (S121D-1, dark-green squares; S121D-2, light-green squares) as described in Figure 1A, except that stomatal opening (t = 0 to 180 min) was induced in the light and in a solution depleted in CO 2 .…”

“…OST1 kinase activity was assayed essentially as described (Vlad et al, 2008) using the following synthetic peptides (Proteogenix) purified to >80% by HPLC and with the indicated sequences: rbohF, MALDRTRSSAQRKKK; loopB, MALARKVSLPRAKKK; loopB_S121A, MALARKVALPRAKKK; Cter, MASKSLGSFRSAANVKKK; Cter_pS280, MASKSLGpSFRSAANVKKK; and C-ter_S280AS283A, MASKSLGAFRAAANVKKK. In another series of experiments, longer peptides, containing 29 PIP2;1 residues and covering the entire loop B were used.…”

Feedback Inhibition of Ammonium Uptake by a Phospho-Dependent Allosteric Mechanism inArabidopsis

“…The amino acids surrounding T460 do not constitute a motif identified so far as a preferred sequence for a certain class of kinases nor is it conserved in other proteins known to be phosphorylated (Nü hse et al, 2004;Vlad et al, 2008).…”

Sucrose-induced Receptor Kinase SIRK1 Regulates a Plasma Membrane Aquaporin in Arabidopsis

“…However, we still know little about the kinases in plants that are responsible for phosphorylation of the large number of experimentally identified phosphorylation sites. Targets for recombinant expressed plant kinases have been experimentally screened for via either exposure to a substrate peptide mixture and subsequent mass spectrometric analysis of peptide phosphorylation (46) or protein or peptide arrays (47,48). In Arabidopsis, a number of kinase-substrate relationships have been identified through genetic interactions based on crosses of different mutants (see various examples in Ref.…”