Identification of Novel Phosphorylation Motifs Through an Integrative Computational and Experimental Analysis of the Human Phosphoproteome

Protein phosphorylationoccurs in certain sequence/structural contexts that are still incompletely understood. The amino acids surrounding the phosphorylatedresidues are important in determining the binding of the kinase to the protein sequence. Upon phosphorylationthese sequences also determine the binding of certain domains that specifically bind to phosphorylatedsequences. Thus far, such 'motifs' have been identified through alignment of a limited number of well identified kinase substrates. Results: Experimentally determined phosphorylationsites from Human Protein Reference Databasewere used to identify 1,167 novel serine/ threonineor tyrosine phosphorylationmotifs using a computational approach. We were able to statistically validate a number of these novel motifs based on their enrichment in known phosphopeptidesdatasets over phosphoserine/ threonine/tyrosine peptides in the human proteome. There were 299 novel serine/ threonineor tyrosine phosphorylationmotifs that were found to be statistically significant. Several of the novel motifs that we identified computationally have subsequently appeared in large datasets of experimentally determined phosphorylationsites since we initiated our analysis. Using a peptide microarrayplatform, we have experimentally evaluated the ability of casein kinaseI to phosphorylatea subset of the novel motifs discovered in this study. Our results demonstrate that it is feasible to identify novel phosphorylationmotifs through large phosphorylationdatasets. Our study also establishes peptide microarraysas a novel platform for high throughput kinase assays and for the validation of consensus motifs. Finally, this extended catalog of phosphorylationmotifs should assist in a systematic study of phosphorylationnetworks in signal transduction pathways.