Deciphering Protein Kinase Specificity Through Large-Scale Analysis of Yeast Phosphorylation Site Motifs

Abstract

Phosphorylation is a universal mechanism for regulating cell behavior in eukaryotes. Although protein kinases are known to target short linear sequence motifs on their substrates, the rules for kinase substrate recognition are not completely understood. We used a rapid peptide screening approach to determine consensus phosphorylation site motifs targeted by 61 of the 122 kinases in Saccharomyces cerevisae. Correlation of these motifs with kinase primary sequence has uncovered previously unappreciated rules for determining specificity within the kinase family, including a residue determining P−3 Arg specificity among members of the CMGC group of kinases. Furthermore, computational scanning of the yeast proteome enabled the prediction of thousands of new kinase-substrate relationships. We experimentally verified several candidate substrates of the Prk1 family of kinases in vitro and in vivo, and we identified a protein substrate of the kinase Vhs1. Together, these results elucidate how kinase catalytic domains recognize their phosphorylation targets and suggest general avenues for the identification of new kinase substrates across eukaryotes.

Phosphorylation of the Hippo Pathway Component AMOTL2 by the mTORC2 Kinase Promotes YAP Signaling, Resulting in Enhanced Glioblastoma Growth and Invasiveness

“…This site also encompassed a conserved leucine in the ϩ1 position, which is unique to mTORs among previously profiled kinases (21,22). As shown in Fig.…”

Modulation of the Chromatin Phosphoproteome by the Haspin Protein Kinase

“…Computational Prediction of Haspin Substrates with the NetPhorest Algorithm-For the computational predictions of possible Haspin targets, we deployed an updated version of the NetPhorest algorithm, which can predict substrates for 222 kinases in the human kinome (17). As input data for the predictions, we used an in-house curated database (KinomeXplorer-DB), which collects known phosphorylation sites from public resources such as Phospho.ELM (18), PhosphoSitePlus (19), and PhosphoGRID (http://www.phosphogrid.org/).…”

Navigating cancer network attractors for tumor-specific therapy

“…In line with their importance, network-attacking mutations have attracted more attention in recent years [43][44][45][46][47][48] . Moreover, information has been accumulating steadily about how specificity in signaling networks and modular protein domains emerges [49][50][51] , leading to the definition of determinants of specificity in protein domains 52,53 . These determinants, sometimes referred to as specificitydetermining residues, are residues that can lead to substrate specificity changes after mutation.…”