Proteome-wide survey of phosphorylation patterns affected by nuclear DNA polymorphisms in Arabidopsis thaliana

Abstract

BackgroundProtein phosphorylation is an important post-translational modification influencing many aspects of dynamic cellular behavior. Site-specific phosphorylation of amino acid residues serine, threonine, and tyrosine can have profound effects on protein structure, activity, stability, and interaction with other biomolecules. Phosphorylation sites can be affected in diverse ways in members of any species, one such way is through single nucleotide polymorphisms (SNPs). The availability of large numbers of experimentally identified phosphorylation sites, and of natural variation datasets in Arabidopsis thaliana prompted us to analyze the effect of non-synonymous SNPs (nsSNPs) onto phosphorylation sites.ResultsFrom the analyses of 7,178 experimentally identified phosphorylation sites we found that: (i) Proteins with multiple phosphorylation sites occur more often than expected by chance. (ii) Phosphorylation hotspots show a preference to be located outside conserved domains. (iii) nsSNPs affected experimental phosphorylation sites as much as the corresponding non-phosphorylated amino acid residues. (iv) Losses of experimental phosphorylation sites by nsSNPs were identified in 86 A. thaliana proteins, among them receptor proteins were overrepresented.These results were confirmed by similar analyses of predicted phosphorylation sites in A. thaliana. In addition, predicted threonine phosphorylation sites showed a significant enrichment of nsSNPs towards asparagines and a significant depletion of the synonymous substitution. Proteins in which predicted phosphorylation sites were affected by nsSNPs (loss and gain), were determined to be mainly receptor proteins, stress response proteins and proteins involved in nucleotide and protein binding. Proteins involved in metabolism, catalytic activity and biosynthesis were less affected.ConclusionsWe analyzed more than 7,100 experimentally identified phosphorylation sites in almost 4,300 protein-coding loci in silico, thus constituting the largest phosphoproteomics dataset for A. thaliana available to date. Our findings suggest a relatively high variability in the presence or absence of phosphorylation sites between different natural accessions in receptor and other proteins involved in signal transduction. Elucidating the effect of phosphorylation sites affected by nsSNPs on adaptive responses represents an exciting research goal for the future.

Phosphorylation in Protein-Protein Binding: Effect on Stability and Function

“…As one can see from Figure 1, the distribution of fractions of phosphosites in phosphoproteins is quite narrow with a large majority of all phosphocomplexes having about 5-10% of all Ser, Thr, or Tyr residues phosphorylated. The distribution has a long tail, which is consistent with the fact that proteins with multiple phosphorylation sites occur more often than expected by chance, in agreement with previous studies for Arabidopsis thaliana (Riano-Pachon et al, 2010). Overall, we observed the relative fractions of the types of phosphosites to be ~40% pSer, ~25% pThr, and ~35% pTyr in protein structural complexes, and this observation did not depend on whether the complexes represented hetero- or homooligomers.…”

Characterization and Prediction of Protein Phosphorylation Hotspots in Arabidopsis thaliana

“…In total, 79 P-hotspots were detected in 75 Arabidopsis proteins based on experimental P-sites (Table 1). As described similarly by Riano-Pachon et al (2010) using a P-hotspot definition based on comparisons to background distributions, P-hotspot proteins identified in this study were found to be involved primarily in RNA binding and splicing processes (GO term enrichment analysis yielding p FDR  = 0.001 for GO function term “RNA binding,” p FDR  = 4.2E−13 for GO process term “RNA splicing”), and are located in the “nuclear speck” ( p FDR  = 1.3E−09), the “plasma membrane” ( p FDR  = 4.7E−06), and the “splicosome” ( p FDR  = 0.05).…”

PhosPhAt goes kinases—searchable protein kinase target information in the plant phosphorylation site database PhosPhAt

“…Where the kinase that phosphorylates a particular residue is known, the residue is highlighted in bold orange. Phosphorylation hotspots (38) are highlighted in light green and Pfam (http://pfam.sanger.ac.uk) domains in yellow. Positioning the mouse cursor on any of the highlighted phosphorylation sites brings up additional information below the sequence, such as details of the experimentally identified peptide sequence, prediction scores or the phosphorylating kinase.…”

“…Phosphorylation hotspots are regions in a protein with clustered phosphorylation sites (38). These hotspots may have particular functions in integration of signals from different pathways and in redirecting proteins to a new intracellular location.…”