NetworKIN: a resource for exploring cellular phosphorylation networks

Protein kinases control cellular responses by phosphorylating specific substrates. Recent proteome-wide mapping of protein phosphorylation sites by mass spectrometry has discovered thousands of in vivo sites. Systematically assigning all 518 human kinases to all these sites is a challenging problem. The NetworKIN database (http://networkin.info) integrates consensus substrate motifs with context modelling for improved prediction of cellular kinase–substrate relations. Based on the latest human phosphoproteome from the Phospho.ELM and PhosphoSite databases, the resource offers insight into phosphorylation-modulated interaction networks. Here, we describe how NetworKIN can be used for both global and targeted molecular studies. Via the web interface users can query the database of precomputed kinase–substrate relations or obtain predictions on novel phosphoproteins. The database currently contains a predicted phosphorylation network with 20 224 site-specific interactions involving 3978 phosphoproteins and 73 human kinases from 20 families.Genome Canada (through Ontario Genomics Institute)National Institutes of Health (U.S.) (U54-CA112967)National Institutes of Health (U.S.) (GM60594)European Community’s Human Potential Programme (BioSapiens Network of Excellence (contract number LSHG-CT-2003-503265))European Community’s Human Potential Programme (ADIT Integrated Project (contract number LSHB-CT-2005511065)

Positive Selection of Tyrosine Loss in Metazoan Evolution

John Nash showed that within a complex system, individuals are best off if they make the best decision that they can, taking into account the decisions of the other individuals. Here, we investigate whether similar principles influence the evolution of signaling networks in multicellular animals. Specifically, by analyzing a set of metazoan species we observed a striking negative correlation of genomically encoded tyrosine content with biological complexity (as measured by the number of cell types in each organism). We discuss how this observed tyrosine loss correlates with the expansion of tyrosine kinases in the evolution of the metazoan lineage and how it may relate to the optimization of signaling systems in multicellular animals. We propose that this phenomenon illustrates genome-wide adaptive evolution to accommodate beneficial genetic perturbation

The Adaptor Protein p66Shc Inhibits mTOR-Dependent Anabolic Metabolism

Adaptor proteins link surface receptors to intracellular signaling pathways and potentially control the way cells respond to nutrient availability. Mice deficient in p66Shc, the most recently evolved isoform of the Shc1 adaptor proteins and a mediator of receptor tyrosine kinase signaling, display resistance to diabetes and obesity. Using quantitative mass spectrometry, we found that p66Shc inhibited glucose metabolism. Depletion of p66Shc enhanced glycolysis and increased the allocation of glucose-derived carbon into anabolic metabolism, characteristics of a metabolic shift called the Warburg effect. This change in metabolism was mediated by the mammalian target of rapamycin (mTOR) because inhibition of mTOR with rapamycin reversed the glycolytic phenotype caused by p66Shc deficiency. Thus, unlike the other isoforms of Shc1, p66Shc appears to antagonize insulin and mTOR signaling, which limits glucose uptake and metabolism. Our results identify a critical inhibitory role for p66Shc in anabolic metabolism.National Institutes of Health (U.S.)United States. Dept. of DefenseW. M. Keck FoundationLAM FoundationNational Institutes of Health (U.S.) (Ruth L. Kirschstein National Research Service Award)National Institutes of Health (U.S.) (K99 Award

The Adaptor Protein p66Shc Inhibits mTOR-Dependent Anabolic Metabolism

Adaptor proteins link surface receptors to intracellular signaling pathways and potentially control the way cells respond to nutrient availability. Mice deficient in p66Shc, the most recently evolved isoform of the Shc1 adaptor proteins and a mediator of receptor tyrosine kinase signaling, display resistance to diabetes and obesity. Using quantitative mass spectrometry, we found that p66Shc inhibited glucose metabolism. Depletion of p66Shc enhanced glycolysis and increased the allocation of glucose-derived carbon into anabolic metabolism, characteristics of a metabolic shift called the Warburg effect. This change in metabolism was mediated by the mammalian target of rapamycin (mTOR) because inhibition of mTOR with rapamycin reversed the glycolytic phenotype caused by p66Shc deficiency. Thus, unlike the other isoforms of Shc1, p66Shc appears to antagonize insulin and mTOR signaling, which limits glucose uptake and metabolism. Our results identify a critical inhibitory role for p66Shc in anabolic metabolism.National Institutes of Health (U.S.)United States. Dept. of DefenseW. M. Keck FoundationLAM FoundationNational Institutes of Health (U.S.) (Ruth L. Kirschstein National Research Service Award)National Institutes of Health (U.S.) (K99 Award

PKA and PKC are required for long-term but not short-term in vivo operant memory in Aplysia

We investigated the involvement of PKA and PKC signaling in a negatively reinforced operant learning paradigm in Aplysia, learning that food is inedible (LFI). In vivo injection of PKA or PKC inhibitors blocked long-term LFI memory formation. Moreover, a persistent phase of PKA activity, although not PKC activity, was necessary for long-term memory. Surprisingly, neither PKA nor PKC activity was required for associative short-term LFI memory. Additionally, PKA and PKC were not required for the retrieval of short- or long-term memory (STM and LTM, respectively). These studies have identified key differences between the mechanisms underlying nonassociative sensitization, operant reward learning, and LFI memory in Aplysia

Systematic discovery of in vivo phosphorylation networks

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