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Reversible phosphorylation is a key mechanism for regulating protein function. Thus it is of high interest to know which kinase can phosphorylate which proteins. Comprehensive information about phosphorylation sites in Arabidopsis proteins is hosted within the PhosPhAt database (http://phosphat.mpimp-golm.mpg.de). However, our knowledge of the kinases that phosphorylate those sites is dispersed throughout the literature and very difficult to access, particularly for investigators seeking to interpret large scale and high-throughput experiments. Therefore, we aimed to compile information on kinase-substrate interactions and kinase-specific regulatory information and make this available via a new functionality embedded in PhosPhAt. Our approach involved systematic surveying of the literature for regulatory information on the members of the major kinase families in Arabidopsis thaliana, such as CDPKs, MPK(KK)s, AGC kinases and SnRKs, as well as individual kinases from other families. To date, we have researched more than 4450 kinase-related publications, which collectively contain information on about 289 kinases. Users can now query the PhosPhAt database not only for experimental and predicted phosphorylation sites of individual proteins, but also for known substrates for a given kinase or kinase family. Further developments include addition of new phosphorylation sites and visualization of clustered phosphorylation events, known as phosphorylation hotspots.

The PhosPhAt database provides a resource consolidating our current knowledge of mass spectrometry-based identified phosphorylation sites in Arabidopsis and combines it with phosphorylation site prediction specifically trained on experimentally identified Arabidopsis phosphorylation motifs. The database currently contains 1187 unique tryptic peptide sequences encompassing 1053 Arabidopsis proteins. Among the characterized phosphorylation sites, there are over 1000 with unambiguous site assignments, and nearly 500 for which the precise phosphorylation site could not be determined. The database is searchable by protein accession number, physical peptide characteristics, as well as by experimental conditions (tissue sampled, phosphopeptide enrichment method). For each protein, a phosphorylation site overview is presented in tabular form with detailed information on each identified phosphopeptide. We have utilized a set of 802 experimentally validated serine phosphorylation sites to develop a method for prediction of serine phosphorylation (pSer) in Arabidopsis. An analysis of the current annotated Arabidopsis proteome yielded in 27,782 predicted phosphoserine sites distributed across 17,035 proteins. These prediction results are summarized graphically in the database together with the experimental phosphorylation sites in a whole sequence context. The Arabidopsis Protein Phosphorylation Site Database (PhosPhAt) provides a valuable resource to the plant science community and can be accessed through the following link http://phosphat.mpimp-golm.mpg.de.