Construction of human activity-based phosphorylation networks.

The landscape of human phosphorylationnetworks has not been systematically explored, representing vast, unchartered territories within cellular signaling networks. Although a large number of in vivo phosphorylatedresidues have been identified by mass spectrometry (MS)-based approaches, assigning the upstream kinasesto these residues requires biochemical analysis of kinase-substrate relationships (KSRs). Here, we developed a new strategy, called CEASAR, based on functional protein microarraysand bioinformatics to experimentally identify substrates for 289 unique kinases, resulting in 3656 high-quality KSRs. We then generated consensus phosphorylationmotifs for each of the kinasesand integrated this information, along with information about in vivo phosphorylationsites determined by MS, to construct a high-resolution map of phosphorylationnetworks that connects 230 kinasesto 2591 in vivo phosphorylationsites in 652 substrates. The value of this data set is demonstrated through the discovery of a new role for PKA downstream of Btk ( Bruton's tyrosine kinase) during B-cell receptorsignaling. Overall, these studies provide global insights into kinase-mediated signaling pathways and promise to advance our understanding of cellular signaling processes in humans.