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.

Processed N-termini of mature proteins in higher eukaryotes and their major contribution to dynamic proteomics.

N-terminal-ubiquitinylation (NTU) is a newly discovered protein degradation pathway initiated by ubiquitin-tagging of the N-terminal alpha-amino group. We have used data from recent genomic studies, especially those on humans, to up-date and re-interpret biochemical data to identify the sequence features associated with NTU. We compared a mini-proteome for which experimental protein sequence is available with large-scale genomic data. We conclude that N-alpha-acetylation involves less than 30%, and not the widely assumed 90%, of the proteins encoded by any higher eukaryote genome, greatly increasing thereby the number of possible targets for NTU-mediated degradation. Next, straightforward rules linking the first N-terminal residues of any nascent polypeptides to the nature of their processed N-termini are established and dedicated prediction tool is made available at . We provide strong arguments indicating that the nature of the processed N-terminus is a major determinant factor of the half-life of the protein. We finally reveal that one third of the nuclear-encoded proteins starting with an unprocessed and unblocked methionine are at least one order of magnitude less stable than is average in higher eukaryotes. This appears to be the first common feature of proteins undergoing N-terminal ubiquitinylation. Hence, a pool of about 3000 proteins in each proteome could be unstable per se and tagged for rapid degradation via NTU.

The proteomics of N-terminal methionine cleavage.

Methionine aminopeptidase (MAP) is a ubiquitous, essential enzyme involved in protein N-terminal methionine excision. According to the generally accepted cleavage rules for MAP, this enzyme cleaves all proteins with small side chains on the residue in the second position (P1'), but many exceptions are known. The substrate specificity of Escherichia coli MAP1 was studied in vitro with a large (>120) coherent array of peptides mimicking the natural substrates and kinetically analyzed in detail. Peptides with Val or Thr at P1' were much less efficiently cleaved than those with Ala, Cys, Gly, Pro, or Ser in this position. Certain residues at P2', P3', and P4' strongly slowed the reaction, and some proteins with Val and Thr at P1' could not undergo Met cleavage. These in vitro data were fully consistent with data for 862 E. coli proteins with known N-terminal sequences in vivo. The specificity sites were found to be identical to those for the other type of MAPs, MAP2s, and a dedicated prediction tool for Met cleavage is now available. Taking into account the rules of MAP cleavage and leader peptide removal, the N termini of all proteins were predicted from the annotated genome and compared with data obtained in vivo. This analysis showed that proteins displaying N-Met cleavage are overrepresented in vivo. We conclude that protein secretion involving leader peptide cleavage is more frequent than generally thought.