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Phosphoproteomic analysis reveals interconnected system-wide responses to perturbations of kinases and phosphatases in yeast.

ABSTRACT: The phosphorylation and dephosphorylation of proteins by kinases and phosphatases constitute an essential regulatory network in eukaryotic cells. This network supports the flow of information from sensors through signaling systems to effector molecules and ultimately drives the phenotype and function of cells, tissues, and organisms. Dysregulation of this process has severe consequences and is one of the main factors in the emergence and progression of diseases, including cancer. Thus, major efforts have been invested in developing specific inhibitors that modulate the activity of individual kinases or phosphatases; however, it has been difficult to assess how such pharmacological interventions would affect the cellular signaling network as a whole. Here, we used label-free, quantitative phosphoproteomics in a systematically perturbed model organism (Saccharomyces cerevisiae) to determine the relationships between 97 kinases, 27 phosphatases, and more than 1000 phosphoproteins. We identified 8814 regulated phosphorylation events, describing the first system-wide protein phosphorylation network in vivo. Our results show that, at steady state, inactivation of most kinases and phosphatases affected large parts of the phosphorylation-modulated signal transduction machinery-and not only the immediate downstream targets. The observed cellular growth phenotype was often well maintained despite the perturbations, arguing for considerable robustness in the system. Our results serve to constrain future models of cellular signaling and reinforce the idea that simple linear representations of signaling pathways might be insufficient for drug development and for describing organismal homeostasis.

SUBMITTER: Bodenmiller B

PROVIDER: S-EPMC3072779 | biostudies-literature | 2010 Dec

REPOSITORIES: biostudies-literature

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Phosphoproteomic analysis reveals interconnected system-wide responses to perturbations of kinases and phosphatases in yeast.

Bodenmiller Bernd B Wanka Stefanie S Kraft Claudine C Urban Jörg J Campbell David D Pedrioli Patrick G PG Gerrits Bertran B Picotti Paola P Lam Henry H Vitek Olga O Brusniak Mi-Youn MY Roschitzki Bernd B Zhang Chao C Shokat Kevan M KM Schlapbach Ralph R Colman-Lerner Alejandro A Nolan Garry P GP Nesvizhskii Alexey I AI Peter Matthias M Loewith Robbie R von Mering Christian C Aebersold Ruedi R

Science signaling 20101221 153

The phosphorylation and dephosphorylation of proteins by kinases and phosphatases constitute an essential regulatory network in eukaryotic cells. This network supports the flow of information from sensors through signaling systems to effector molecules and ultimately drives the phenotype and function of cells, tissues, and organisms. Dysregulation of this process has severe consequences and is one of the main factors in the emergence and progression of diseases, including cancer. Thus, major eff ...[more]

PMID: 21177495

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Project description:BackgroundA major goal in the discovery of cellular signaling networks is to identify regulated phosphorylation sites ("phosphosites") and map them to the responsible protein kinases. The V2 vasopressin receptor is a G-protein coupled receptor (GPCR) that is responsible for regulation of renal water excretion through control of aquaporin-2-mediated osmotic water transport in kidney collecting duct cells. Genome editing experiments have demonstrated that virtually all vasopressin-triggered phosphorylation changes are dependent on protein kinase A (PKA), but events downstream from PKA are still obscure.MethodsHere, we used: 1) Tandem mass tag-based quantitative phosphoproteomics to experimentally track phosphorylation changes over time in native collecting ducts isolated from rat kidneys; 2) a clustering algorithm to classify time course data based on abundance changes and the amino acid sequences surrounding the phosphosites; and 3) Bayes' Theorem to integrate the dynamic phosphorylation data with multiple prior "omic" data sets covering expression, subcellular location, known kinase activity, and characteristic surrounding sequences to identify a set of protein kinases that are regulated secondary to PKA activation.ResultsPhosphoproteomic studies revealed 185 phosphosites regulated by vasopressin over 15 min. The resulting groups from the cluster algorithm were integrated with Bayes' Theorem to produce corresponding ranked lists of kinases likely responsible for each group. The top kinases establish three PKA-dependent protein kinase modules whose regulation mediate the physiological effects of vasopressin at a cellular level. The three modules are 1) a pathway involving several Rho/Rac/Cdc42-dependent protein kinases that control actin cytoskeleton dynamics; 2) mitogen-activated protein kinase and cyclin-dependent kinase pathways that control cell proliferation; and 3) calcium/calmodulin-dependent signaling.ConclusionsOur findings identify a novel set of downstream small GTPase effectors and calcium/calmodulin-dependent kinases with potential roles in the regulation of water permeability through actin cytoskeleton rearrangement and aquaporin-2 trafficking. The proposed signaling network provides a stronger hypothesis for the kinases mediating V2 vasopressin receptor responses, encouraging future targeted examination via reductionist approaches. Furthermore, the Bayesian analysis described here provides a template for investigating signaling via other biological systems and GPCRs. Video abstract.

Project description:Breast cancer is the leading cause of cancer-related mortality in women worldwide, with an estimated 1.7 million new cases and 522,000 deaths around the world in 2012 alone. Cancer stem cells (CSCs) are essential for tumor reoccurrence and metastasis which is the major source of cancer lethality. G protein-coupled receptor chemokine (C-X-C motif) receptor 4 (CXCR4) is critical for tumor metastasis. However, stromal cell-derived factor 1 (SDF-1)/CXCR4-mediated signaling pathways in breast CSCs are largely unknown. Using isotope reductive dimethylation and large-scale MS-based quantitative phosphoproteome analysis, we examined protein phosphorylation induced by SDF-1/CXCR4 signaling in breast CSCs. We quantified more than 11,000 phosphorylation sites in 2,500 phosphoproteins. Of these phosphosites, 87% were statistically unchanged in abundance in response to SDF-1/CXCR4 stimulation. In contrast, 545 phosphosites in 266 phosphoproteins were significantly increased, whereas 113 phosphosites in 74 phosphoproteins were significantly decreased. SDF-1/CXCR4 increases phosphorylation in 60 cell migration- and invasion-related proteins, of them 43 (>70%) phosphoproteins are unrecognized. In addition, SDF-1/CXCR4 upregulates the phosphorylation of 44 previously uncharacterized kinases, 8 phosphatases, and 1 endogenous phosphatase inhibitor. Using computational approaches, we performed system-based analyses examining SDF-1/CXCR4-mediated phosphoproteome, including construction of kinase-substrate network and feedback regulation loops downstream of SDF-1/CXCR4 signaling in breast CSCs. We identified a previously unidentified SDF-1/CXCR4-PKA-MAP2K2-ERK signaling pathway and demonstrated the feedback regulation on MEK, ERK1/2, ?-catenin, and PPP1C? in SDF-1/CXCR4 signaling in breast CSCs. This study gives a system-wide view of phosphorylation events downstream of SDF-1/CXCR4 signaling in breast CSCs, providing a resource for the study of CSC-targeted cancer therapy.

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Phosphoproteomic analysis reveals interconnected system-wide responses to perturbations of kinases and phosphatases in yeast.

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Phosphoproteomic analysis reveals interconnected system-wide responses to perturbations of kinases and phosphatases in yeast.

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