Systematic Discovery of In Vivo Phosphorylation Networks

Abstract

Protein kinases control cellular decision processes by phosphorylating specific substrates. Thousands of in vivo phosphorylation sites have been identified, mostly by proteome-wide mapping. However, systematically matching these sites to specific kinases is presently infeasible, due to limited specificity of consensus motifs, and the influence of contextual factors, such as protein scaffolds, localization, and expression, on cellular substrate specificity. We have developed an approach (NetworKIN) that augments motif-based predictions with the network context of kinases and phosphoproteins. The latter provides 60%-80% of the computational capability to assign in vivo substrate specificity. NetworKIN pinpoints kinases responsible for specific phosphorylations and yields a 2.5-fold improvement in the accuracy with which phosphorylation networks can be constructed. Applying this approach to DNA damage signaling, we show that 53BP1 and Rad50 are phosphorylated by CDK1 and ATM, respectively. We describe a scalable strategy to evaluate predictions, which suggests that BCLAF1 is a GSK-3 substrate.

Osteoblast-induced EGFR/ERBB2 signaling in androgen-sensitive prostate carcinoma cells characterized by multiplex kinase activity profiling

“…Correct network definition may be restricted by the influence of contextual factors, such as subcellular compartmentalization or temporal expression of the proteins involved [33]. Therefore, to predict pathway connectivity with sufficient accuracy, the use of more than one computational technique on the dataset of interest has been recommended [20].…”

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.…”

ATM and the Mre11 complex combine to recognize and signal DNA double-strand breaks

“…There is also some evidence that a second member of the MRN complex, Mre11, is phosphorylated in response to DNA damage (Dong et al, 1999;Yuan et al, 2002;Costanzo et al, 2004). More recently, the use of large-scale and systematic proteomic analysis has identified a site on Rad50 that is phosphorylated in response to DNA damage (Linding et al, 2007;Matsuoka et al, 2007). However, at this stage there is no evidence that these phosphorylations are functionally significant.…”