The coordinated regulation of protein kinases is a rapid mechanism that integrates diverse cues and swiftly determines appropriate cellular responses. However, our understanding of cellular decision‐making has been limited by the small number of simultaneously monitored phospho‐regulatory events. Here, we have estimated changes in activity in 215 human kinases in 399 conditions derived from a large compilation of phosphopeptide quantifications. This atlas identifies commonly regulated kinases as those that are central in the signaling network and defines the logic relationships between kinase pairs. Co‐regulation along the conditions predicts kinase–complex and kinase–substrate associations. Additionally, the kinase regulation profile acts as a molecular fingerprint to identify related and opposing signaling states. Using this atlas, we identified essential mediators of stem cell differentiation, modulators of Salmonella infection, and new targets of AKT1. This provides a global view of human phosphorylation‐based signaling and the necessary context to better understand kinase‐driven decision‐making.

SummaryThe loss of cone photoreceptors that mediate daylight vision represents a leading cause of blindness, for which cell replacement by transplantation offers a promising treatment strategy. Here, we characterize cone differentiation in retinas derived from mouse embryonic stem cells (mESCs). Similar to in vivo development, a temporal pattern of progenitor marker expression is followed by the differentiation of early thyroid hormone receptor β2-positive precursors and, subsequently, photoreceptors exhibiting cone-specific phototransduction-related proteins. We establish that stage-specific inhibition of the Notch pathway increases cone cell differentiation, while retinoic acid signaling regulates cone maturation, comparable with their actions in vivo. MESC-derived cones can be isolated in large numbers and transplanted into adult mouse eyes, showing capacity to survive and mature in the subretinal space of Aipl1−/− mice, a model of end-stage retinal degeneration. Together, this work identifies a robust, renewable cell source for cone replacement by purified cell suspension transplantation.

ObjectiveDefective mitochondrial function attributed to optic atrophy 1 (OPA1) mutations causes primarily optic atrophy and, less commonly, neurodegenerative syndromes. The pathomechanism by which OPA1 mutations trigger diffuse loss of neurons in some, but not all, patients is unknown. Here, we used a tractable induced pluripotent stem cell (iPSC)‐based model to capture the biology of OPA1 haploinsufficiency in cases presenting with classic eye disease versus syndromic parkinsonism.MethodsiPSCs were generated from 2 patients with OPA1 haploinsufficiency and 2 controls and differentiated into dopaminergic neurons. Metabolic profile was determined by extracellular flux analysis, respiratory complex levels using immunoblotting, and complex I activity by a colorimetric assay. Mitochondria were examined by transmission electron microscopy. Mitochondrial DNA copy number and deletions were assayed using long‐range PCR. Mitochondrial membrane potential was measured by tetramethylrhodamine methyl ester uptake, and mitochondrial fragmentation was assessed by confocal microscopy. Exome sequencing was used to screen for pathogenic variants.ResultsOPA1 haploinsufficient iPSCs differentiated into dopaminergic neurons and exhibited marked reduction in OPA1 protein levels. Loss of OPA1 caused a late defect in oxidative phosphorylation, reduced complex I levels, and activity without a significant change in the ultrastructure of mitochondria. Loss of neurons in culture recapitulated dopaminergic degeneration in syndromic disease and correlated with mitochondrial fragmentation.InterpretationOPA1 levels maintain oxidative phosphorylation in iPSC‐derived neurons, at least in part, by regulating the stability of complex I. Severity of OPA1 disease associates primarily with the extent of OPA1‐mediated fusion, suggesting that activation of this mechanism or identification of its genetic modifiers may have therapeutic or prognostic value. Ann Neurol 2018;83:915–925

The coordinated regulation of protein kinases is a rapid mechanism that integrates diverse cues and swiftly determines appropriate cellular responses. However, our understanding of cellular decision-making has been limited by the small number of simultaneously monitored phosphoregulatory events. Here, we have estimated changes in activity in 215 human kinases in 399 conditions from a compilation of nearly 3 million phosphopeptide quantifications. This atlas identifies commonly regulated kinases as those that are central in the signaling network and defines the logic relationships between kinase pairs. Co-regulation along the conditions predicts kinase-complex and kinase-substrate associations. Additionally, the kinase regulation profile acts as a molecular fingerprint to identify related and opposing signaling states. Using this atlas, we identified essential mediators of stem cell differentiation, modulators of Salmonella infection and new targets of AKT1. This provides a global view of human phosphorylation-based signaling and the necessary context to better understand kinase driven decision-making. . CC-BY 4.0 International license peer-reviewed) is the author/funder. It is made available under aThe copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/067900 doi: bioRxiv preprint first posted online Aug. 4, 2016; Cells need to constantly adapt to internal and external conditions in order to maintain homoeostasis. During cellular decision-making, signal-transduction networks dynamically change in response to cues, triggering cellular state-defining responses. Multiple mechanisms exist to transfer this information from sensors to the corresponding molecular responses, one of the fastest being the reversible post-translational modification of proteins (PTMs). Through these targeted modifications, such as phosphorylation, the cell can quickly alter enzymatic activities, protein interactions or sub-cellular localization in order to produce a coordinated response to a given stimulus (Pawson, 2004). Protein phospho-regulation constitutes a highly conserved regulatory mechanism relevant for a broad set of biological functions and diseases (Beltrao et al., 2012).Over the past decades, our view of cellular signaling has advanced from an idea of isolated and linear cascades to highly complex and cooperative regulatory networks (Jordan et al., 2000; Gibson, 2009).Different perturbations in cellular conditions often activate different sets of interconnected kinases, ultimately triggering appropriate cellular responses. The complete understanding of such cell-fate decisions would require the systematic measurement of changes in kinase activities under multiple perturbations, but the small number of quantified regulatory events (i.e. tens) that were possible to date has limited our knowledge of cellular decision making and its molecular consequences (Kim et al., 2011; Bendall et al., 2011; Niepel et al., 2013; Garmaroudi et al., 2010).Advances in mass-spectrometry and enrichment methods now all...