ABSTRACT
An important tenet of learning and memory is the notion of a molecular switch that promotes the formation of long-term memory1-4. The regulation of proteostasis is a critical and rate-limiting step in the consolidation of new memories5-10. One of the most effective and prevalent ways to enhance memory is by regulating the synthesis of proteins controlled by the translation initiation factor eIF211. Phosphorylation of the α-subunit of eIF2 (p-eIF2α), the central component of the integrated stress response (ISR), impairs long-term memory formation in rodents and birds11-13. By contrast, inhibiting the ISR by mutating the eIF2α phosphorylation site, genetically11 and pharmacologically inhibiting the ISR kinases14-17, or mimicking reduced p-eIF2α with the ISR inhibitor ISRIB11, enhances long-term memory in health and disease18. Here we used molecular genetics to dissect the neuronal circuits by which the ISR gates cognitive processing. We found that learning reduces eIF2α phosphorylation in hippocampal excitatory neurons and a subset of hippocampal inhibitory neurons (those that express somatostatin, but not parvalbumin). Moreover, ablation of p-eIF2α in either excitatory or somatostatin-expressing (but not parvalbumin-expressing) inhibitory neurons increased general mRNA translation, bolstered synaptic plasticity and enhanced long-term memory. Thus, eIF2α-dependent mRNA translation controls memory consolidation via autonomous mechanisms in excitatory and somatostatin-expressing inhibitory neurons.
Subject(s)
Eukaryotic Initiation Factor-2/metabolism , Hippocampus/cytology , Memory Consolidation , Neurons/metabolism , Somatostatin/metabolism , Animals , CA1 Region, Hippocampal/cytology , CA1 Region, Hippocampal/physiology , Eukaryotic Initiation Factor-2/deficiency , Eukaryotic Initiation Factor-2/genetics , Excitatory Postsynaptic Potentials , Hippocampus/physiology , Long-Term Potentiation , Male , Memory, Long-Term , Mice , Mice, Inbred C57BL , Neural Inhibition , Neuronal Plasticity , Parvalbumins , Phosphorylation , Pyramidal Cells/physiology , Synaptic TransmissionABSTRACT
Focal adhesion kinase (FAK) is an attractive drug target due to its overexpression in cancer. FAK functions as a non-receptor tyrosine kinase and scaffolding protein, coordinating several downstream signaling effectors and cellular processes. While drug discovery efforts have largely focused on targeting FAK kinase activity, FAK inhibitors have failed to show efficacy as single agents in clinical trials. Here, using structure-guided design, we report the development of a selective FAK inhibitor (BSJ-04-175) and degrader (BSJ-04-146) to evaluate the consequences and advantages of abolishing all FAK activity in cancer models. BSJ-04-146 achieves rapid and potent FAK degradation with high proteome-wide specificity in cancer cells and induces durable degradation in mice. Compared to kinase inhibition, targeted degradation of FAK exhibits pronounced improved activity on downstream signaling and cancer cell viability and migration. Together, BSJ-04-175 and BSJ-04-146 are valuable chemical tools to dissect the specific consequences of targeting FAK through small-molecule inhibition or degradation.
Subject(s)
Neoplasms , Proteolysis Targeting Chimera , Mice , Animals , Focal Adhesion Protein-Tyrosine Kinases/chemistry , Focal Adhesion Protein-Tyrosine Kinases/metabolism , Neoplasms/drug therapy , Signal Transduction , Phosphorylation , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/chemistryABSTRACT
The metabolite acetyl-coenzyme A (acetyl-CoA) serves as an essential element for a wide range of cellular functions including adenosine triphosphate (ATP) production, lipid synthesis, and protein acetylation. Intracellular acetyl-CoA concentrations are associated with nutrient availability, but the mechanisms by which a cell responds to fluctuations in acetyl-CoA levels remain elusive. Here, we generate a cell system to selectively manipulate the nucleo-cytoplasmic levels of acetyl-CoA using clustered regularly interspaced short palindromic repeat (CRISPR)-mediated gene editing and acetate supplementation of the culture media. Using this system and quantitative omics analyses, we demonstrate that acetyl-CoA depletion alters the integrity of the nucleolus, impairing ribosomal RNA synthesis and evoking the ribosomal protein-dependent activation of p53. This nucleolar remodeling appears to be mediated through the class IIa histone deacetylases (HDACs). Our findings highlight acetylation-mediated control of the nucleolus as an important hub linking acetyl-CoA fluctuations to cellular stress responses.
Subject(s)
Acetyl Coenzyme A/biosynthesis , Cell Nucleolus/metabolism , ATP Citrate (pro-S)-Lyase/deficiency , ATP Citrate (pro-S)-Lyase/genetics , ATP Citrate (pro-S)-Lyase/metabolism , Acetates/metabolism , Acetylation , Cell Line , Cell Nucleolus/ultrastructure , Gene Expression , Gene Knockout Techniques , HCT116 Cells , Histone Deacetylases/metabolism , Humans , Models, Biological , Nuclear Proteins/metabolism , Protein Processing, Post-Translational , Ribosomal Proteins/metabolism , Tumor Suppressor Protein p53/metabolismABSTRACT
Influenza A viruses (IAVs) quickly adapt to new environments and are well known to cross species barriers. To reveal a molecular basis for these phenomena, we compared the Ser/Thr and Tyr phosphoproteomes of murine lung epithelial cells early and late after infection with mouse-adapted SC35M virus or its nonadapted SC35 counterpart. With this analysis we identified a large set of upregulated Ser/Thr phosphorylations common to both viral genotypes, while Tyr phosphorylations showed little overlap. Most of the proteins undergoing massive changes of phosphorylation in response to both viruses regulate chromatin structure, RNA metabolism, and cell adhesion, including a focal adhesion kinase (FAK)-regulated network mediating the regulation of actin dynamics. IAV also affected phosphorylation of activation loops of 37 protein kinases, including FAK and several phosphatases, many of which were not previously implicated in influenza virus infection. Inhibition of FAK proved its contribution to IAV infection. Novel phosphorylation sites were found on IAV-encoded proteins, and the functional analysis of selected phosphorylation sites showed that they either support (NA Ser178) or inhibit (PB1 Thr223) virus propagation. Together, these data allow novel insights into IAV-triggered regulatory phosphorylation circuits and signaling networks.IMPORTANCE Infection with IAVs leads to the induction of complex signaling cascades, which apparently serve two opposing functions. On the one hand, the virus highjacks cellular signaling cascades in order to support its propagation; on the other hand, the host cell triggers antiviral signaling networks. Here we focused on IAV-triggered phosphorylation events in a systematic fashion by deep sequencing of the phosphoproteomes. This study revealed a plethora of newly phosphorylated proteins. We also identified 37 protein kinases and a range of phosphatases that are activated or inactivated following IAV infection. Moreover, we identified new phosphorylation sites on IAV-encoded proteins. Some of these phosphorylations support the enzymatic function of viral components, while other phosphorylations are inhibitory, as exemplified by PB1 Thr223 modification. Our global characterization of IAV-triggered patterns of phospho-proteins provides a rich resource to further understand host responses to infection at the level of phosphorylation-dependent signaling networks.
Subject(s)
Antiviral Agents/pharmacology , Influenza A virus/metabolism , Orthomyxoviridae Infections/metabolism , Proteome/analysis , Signal Transduction/drug effects , Animals , Cell Line , Focal Adhesion Protein-Tyrosine Kinases/metabolism , Genome , Host-Pathogen Interactions/physiology , Humans , Influenza A virus/genetics , Mice , Models, Molecular , Phosphorylation , Protein Conformation , Viral Proteins/chemistry , Viral Proteins/metabolismABSTRACT
The interconnected PI3K and MAPK signaling pathways are commonly perturbed in cancer. Dual inhibition of these pathways by the small-molecule PI3K inhibitor pictilisib (GDC-0941) and the MEK inhibitor cobimetinib (GDC-0973) suppresses cell proliferation and induces cell death better than either single agent in several preclinical models. Using mass spectrometry-based phosphoproteomics, we have identified the RING finger E3 ubiquitin ligase RNF157 as a target at the intersection of PI3K and MAPK signaling. We demonstrate that RNF157 phosphorylation downstream of the PI3K and MAPK pathways influences the ubiquitination and stability of RNF157 during the cell cycle in an anaphase-promoting complex/cyclosome-CDH1-dependent manner. Deletion of these phosphorylation-targeted residues on RNF157 disrupts binding to CDH1 and protects RNF157 from ubiquitination and degradation. Expression of the cyclin-dependent kinase 2 (CDK2), itself a downstream target of PI3K/MAPK signaling, leads to increased phosphorylation of RNF157 on the same residues modulated by PI3K and MAPK signaling. Inhibition of PI3K and MEK in combination or of CDK2 by their respective small-molecule inhibitors reduces RNF157 phosphorylation at these residues and attenuates RNF157 interaction with CDH1 and its subsequent degradation. Knockdown of endogenous RNF157 in melanoma cells leads to late S phase and G2/M arrest and induces apoptosis, the latter further potentiated by concurrent PI3K/MEK inhibition, consistent with a role for RNF157 in the cell cycle. We propose that RNF157 serves as a novel node integrating oncogenic signaling pathways with the cell cycle machinery and promoting optimal cell cycle progression in transformed cells.
Subject(s)
Apoptosis , MAP Kinase Signaling System , Melanoma/metabolism , Phosphatidylinositol 3-Kinase/metabolism , Protein Processing, Post-Translational , Signal Transduction , Ubiquitin-Protein Ligases/metabolism , Amino Acid Substitution , Antigens, CD , Apoptosis/drug effects , Cadherins/antagonists & inhibitors , Cadherins/genetics , Cadherins/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Cyclin-Dependent Kinase 2/antagonists & inhibitors , Cyclin-Dependent Kinase 2/genetics , Cyclin-Dependent Kinase 2/metabolism , Enzyme Inhibitors/pharmacology , Enzyme Stability/drug effects , Gene Deletion , Gene Expression Regulation, Neoplastic/drug effects , Humans , MAP Kinase Signaling System/drug effects , Melanoma/drug therapy , Melanoma/enzymology , Melanoma/pathology , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/chemistry , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Phosphoinositide-3 Kinase Inhibitors , Phosphorylation/drug effects , Point Mutation , Protein Processing, Post-Translational/drug effects , RNA Interference , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , S Phase/drug effects , Signal Transduction/drug effects , Ubiquitin-Protein Ligases/antagonists & inhibitors , Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/genetics , Ubiquitination/drug effectsABSTRACT
PURPOSE: The aims of the present study were 2-fold: first, to test the hypothesis that heat stress induces MET and EGFR signalling in hepatocellular carcinoma (HCC) cells and inhibition of this signalling decreases HCC clonogenic survival; and second, to identify signalling pathways associated with heat stress induced MET signalling. MATERIALS AND METHODS: MET+ and EGFR+ HCC cells were pre-treated with inhibitors to MET, EGFR, PI3K/mTOR or vehicle and subjected to heat stress or control ± HGF or EGF growth factors and assessed by colony formation assay, Western blotting and/or quantitative mass spectrometry. IACUC approved partial laser thermal or sham ablation was performed on orthotopic N1S1 and AS30D HCC tumours and liver/tumour assessed for phospho-MET and phospho-EGFR immunostaining. RESULTS: Heat-stress induced rapid MET and EGFR phosphorylation that is distinct from HGF or EGF in HCC cells and thermal ablation induced MET but not EGFR phosphorylation at the HCC tumour ablation margin. Inhibition of the MET and EGFR blocked both heat stress and growth factor induced MET and EGFR phosphorylation and inhibition of MET decreased HCC clonogenic survival following heat stress. Pathway analysis of quantitative phosphoproteomic data identified downstream pathways associated with heat stress induced MET signalling including AKT, ERK, Stat3 and JNK. However, inhibition of heat stress induced MET signalling did not block AKT signalling. CONCLUSIONS: Heat-stress induced MET and EGFR signalling is distinct from growth factor mediated signalling in HCC cells and MET inhibition enhances heat stress induced HCC cell killing via a PI3K/AKT/mTOR-independent mechanism.
Subject(s)
Carcinoma, Hepatocellular/genetics , Heat-Shock Response , Carcinoma, Hepatocellular/pathology , ErbB Receptors/genetics , ErbB Receptors/metabolism , Humans , Liver Neoplasms/genetics , Liver Neoplasms/pathology , Signal TransductionABSTRACT
A robust method was developed and optimized for enrichment and quantitative analysis of posttranslational modifications (PTMs) in serum/plasma samples by combining immunoaffinity purification and LC-MS/MS without depletion of abundant proteins. The method was used to survey serum samples of patients with acute myeloid leukemia (AML), breast cancer (BC), and nonsmall cell lung cancer (NSCLC). Peptides were identified from serum samples containing phosphorylation, acetylation, lysine methylation, and arginine methylation. Of the PTMs identified, lysine acetylation (AcK) and arginine mono-methylation (Rme) were more prevalent than other PTMs. Label-free quantitative analysis of AcK and Rme peptides was performed for sera from AML, BC, and NSCLC patients. Several AcK and Rme sites showed distinct abundance distribution patterns across the three cancer types. The identification and quantification of posttranslationally modified peptides in serum samples reported here can be used for patient profiling and biomarker discovery research.
Subject(s)
Breast Neoplasms/genetics , Carcinoma, Non-Small-Cell Lung/genetics , Leukemia, Myeloid, Acute/genetics , Neoplasm Proteins/biosynthesis , Acetylation , Breast Neoplasms/blood , Breast Neoplasms/pathology , Carcinoma, Non-Small-Cell Lung/blood , Carcinoma, Non-Small-Cell Lung/pathology , Cell Line, Tumor , Chromatography, Liquid , Female , Humans , Leukemia, Myeloid, Acute/blood , Leukemia, Myeloid, Acute/pathology , Methylation , Neoplasm Proteins/blood , Protein Processing, Post-Translational/genetics , Proteomics/methods , Tandem Mass SpectrometryABSTRACT
Reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) govern cellular homeostasis by inducing signaling. H2O2 modulates the activity of phosphatases and many other signaling molecules through oxidation of critical cysteine residues, which led to the notion that initiation of ROS signaling is broad and nonspecific, and thus fundamentally distinct from other signaling pathways. Here, we report that H2O2 signaling bears hallmarks of a regular signal transduction cascade. It is controlled by hierarchical signaling events resulting in a focused response as the results place the mitochondrial respiratory chain upstream of tyrosine-protein kinase Lyn, Lyn upstream of tyrosine-protein kinase SYK (Syk), and Syk upstream of numerous targets involved in signaling, transcription, translation, metabolism, and cell cycle regulation. The active mediators of H2O2 signaling colocalize as H2O2 induces mitochondria-associated Lyn and Syk phosphorylation, and a pool of Lyn and Syk reside in the mitochondrial intermembrane space. Finally, the same intermediaries control the signaling response in tissues and species responsive to H2O2 as the respiratory chain, Lyn, and Syk were similarly required for H2O2 signaling in mouse B cells, fibroblasts, and chicken DT40 B cells. Consistent with a broad role, the Syk pathway is coexpressed across tissues, is of early metazoan origin, and displays evidence of evolutionary constraint in the human. These results suggest that H2O2 signaling is under control of a signal transduction pathway that links the respiratory chain to the mitochondrial intermembrane space-localized, ubiquitous, and ancient Syk pathway in hematopoietic and nonhematopoietic cells.
Subject(s)
Electron Transport , Hydrogen Peroxide/metabolism , Mitochondrial Membranes/metabolism , Signal Transduction , Animals , Cells, Cultured , Chickens , Enzyme Activation , Intracellular Signaling Peptides and Proteins/metabolism , Mice , Phosphorylation , Protein-Tyrosine Kinases/metabolism , Reactive Oxygen Species/metabolism , Syk Kinase , Tyrosine/metabolismABSTRACT
The RAS-RAF-MEK-ERK (MAPK) pathway is prevalently perturbed in cancer. Recent large-scale sequencing initiatives profiled thousands of tumors providing insight into alterations at the DNA and RNA levels. These efforts confirmed that key nodes of the MAPK pathway, in particular KRAS and BRAF, are among the most frequently altered proteins in cancer. The establishment of targeted therapies, however, has proven difficult. To decipher the underlying challenges, it is essential to decrypt the phosphorylation network spanned by the MAPK core axis. Using mass spectrometry we identified 2241 phosphorylation sites on 1020 proteins, and measured their responses to inhibition of MEK or ERK. Multiple phosphorylation patterns revealed previously undetected feedback, as upstream signaling nodes, including receptor kinases, showed changes at the phosphorylation level. We provide a dataset rich in potential therapeutic targets downstream of the MAPK cascade. By integrating TCGA (The Cancer Genome Atlas) data, we highlight some downstream phosphoproteins that are frequently altered in cancer. All MS data have been deposited in the ProteomeXchange with identifier PXD003908 (http://proteomecentral.proteomexchange.org/dataset/PXD003908).
Subject(s)
Colonic Neoplasms/genetics , Feedback, Physiological , Gene Expression Regulation, Neoplastic , MAP Kinase Signaling System/drug effects , Mitogen-Activated Protein Kinase Kinases/genetics , Neoplasm Proteins/genetics , Phosphoproteins/genetics , Amino Acid Sequence , Antineoplastic Agents/pharmacology , Atlases as Topic , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , Gene Expression Profiling , HCT116 Cells , Humans , Internet , Mitogen-Activated Protein Kinase Kinases/antagonists & inhibitors , Mitogen-Activated Protein Kinase Kinases/metabolism , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/metabolism , Phosphoproteins/antagonists & inhibitors , Phosphoproteins/metabolism , Phosphorylation , Protein Kinase Inhibitors/pharmacology , Proteomics/methods , SoftwareABSTRACT
The PI3K pathway is commonly activated in cancer. Only a few studies have attempted to explore the spectrum of phosphorylation signaling downstream of the PI3K cascade. Such insight, however, is imperative to understand the mechanisms responsible for oncogenic phenotypes. By applying MS-based phosphoproteomics, we mapped 2509 phosphorylation sites on 1096 proteins, and quantified their responses to activation or inhibition of PIK3CA using isogenic knock-in derivatives and a series of targeted inhibitors. We uncovered phosphorylation changes in a wide variety of proteins involved in cell growth and proliferation, many of which have not been previously associated with PI3K signaling. A significant update of the posttranslational modification database PHOSIDA (http://www.phosida.com) allows efficient use of the data. All MS data have been deposited in the ProteomeXchange with identifier PXD003899 (http://proteomecentral.proteomexchange.org/dataset/PXD003899).
Subject(s)
Cell Transformation, Neoplastic/genetics , Epithelial Cells/metabolism , Gene Expression Regulation, Neoplastic , Neoplasm Proteins/genetics , Phosphatidylinositol 3-Kinases/genetics , Phosphoproteins/genetics , Protein Processing, Post-Translational , Antineoplastic Agents/pharmacology , Cell Line , Cell Transformation, Neoplastic/metabolism , Cell Transformation, Neoplastic/pathology , Class I Phosphatidylinositol 3-Kinases , Colon/cytology , Colon/drug effects , Colon/metabolism , Databases, Genetic , Epithelial Cells/cytology , Epithelial Cells/drug effects , Humans , Internet , Mutation , Neoplasm Proteins/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Phosphoproteins/metabolism , Phosphorylation , Protein Kinase Inhibitors/pharmacology , Proteomics/methods , Signal Transduction , SoftwareABSTRACT
Protein phosphorylation plays a central role in creating a highly dynamic network of interacting proteins that reads and responds to signals from growth factors in the cellular microenvironment. Cells of the neural crest employ multiple signaling mechanisms to control migration and differentiation during development. It is known that defects in these mechanisms cause neuroblastoma, but how multiple signaling pathways interact to govern cell behavior is unknown. In a phosphoproteomic study of neuroblastoma cell lines and cell fractions, including endosomes and detergent-resistant membranes, 1622 phosphorylated proteins were detected, including more than half of the receptor tyrosine kinases in the human genome. Data were analyzed using a combination of graph theory and pattern recognition techniques that resolve data structure into networks that incorporate statistical relationships and protein-protein interaction data. Clusters of proteins in these networks are indicative of functional signaling pathways. The analysis indicates that receptor tyrosine kinases are functionally compartmentalized into distinct collaborative groups distinguished by activation and intracellular localization of SRC-family kinases, especially FYN and LYN. Changes in intracellular localization of activated FYN and LYN were observed in response to stimulation of the receptor tyrosine kinases, ALK and KIT. The results suggest a mechanism to distinguish signaling responses to activation of different receptors, or combinations of receptors, that govern the behavior of the neural crest, which gives rise to neuroblastoma.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Endosomes/metabolism , Neuroblastoma/metabolism , Protein-Tyrosine Kinases/metabolism , Signal Transduction , src-Family Kinases/metabolism , Cell Line, Tumor , Computer Simulation , Humans , Membrane Microdomains , Models, Biological , Neoplasm Proteins/metabolism , Phosphoproteins/metabolismABSTRACT
In the past decade, mass-spectrometry-based methods have emerged for the quantitative profiling of dynamic changes in protein phosphorylation, allowing the behavior of thousands of phosphorylation sites to be monitored in a single experiment. However, when one is interested in specific signaling pathways, such shotgun methodologies are not ideal because they lack selectivity and are not cost and time efficient with respect to instrument and data analysis time. Here we evaluate and explore a peptide-centric antibody generated to selectively enrich peptides containing the cAMP-dependent protein kinase (PKA) consensus motif. This targeted phosphoproteomic strategy is used to profile temporal quantitative changes of potential PKA substrates in Jurkat T lymphocytes upon prostaglandin E2 (PGE2) stimulation, which increases intracellular cAMP, activating PKA. Our method combines ultra-high-specificity motif-based immunoaffinity purification with cost-efficient stable isotope dimethyl labeling. We identified 655 phosphopeptides, of which 642 (i.e. 98%) contained the consensus motif [R/K][R/K/X]X[pS/pT]. When our data were compared with a large-scale Jurkat T-lymphocyte phosphoproteomics dataset containing more than 10,500 phosphosites, a minimal overlap of 0.2% was observed. This stresses the need for such targeted analyses when the interest is in a particular kinase. Our data provide a resource of likely substrates of PKA, and potentially some substrates of closely related kinases. Network analysis revealed that about half of the observed substrates have been implicated in cAMP-induced signaling. Still, the other half of the here-identified substrates have been less well characterized, representing a valuable resource for future research.
Subject(s)
Cyclic AMP-Dependent Protein Kinases/metabolism , Phosphoproteins/metabolism , Proteomics/methods , Amino Acid Motifs , Chromatography, Liquid/methods , Dinoprostone/metabolism , Dinoprostone/pharmacology , Humans , Immunoprecipitation/methods , Jurkat Cells , Phosphoproteins/chemistry , Phosphoproteins/genetics , Protein Interaction Maps , Signal Transduction/drug effects , Substrate Specificity , Tandem Mass Spectrometry/methodsABSTRACT
Although K-Ras, Cdc42, and PAK4 signaling are commonly deregulated in cancer, only a few studies have sought to comprehensively examine the spectrum of phosphorylation-mediated signaling downstream of each of these key signaling nodes. In this study, we completed a label-free quantitative analysis of oncogenic K-Ras, activated Cdc42, and PAK4-mediated phosphorylation signaling, and report relative quantitation of 2152 phosphorylated peptides on 1062 proteins. We define the overlap in phosphopeptides regulated by K-Ras, Cdc42, and PAK4, and find that perturbation of these signaling components affects phosphoproteins associated with microtubule depolymerization, cytoskeletal organization, and the cell cycle. These findings provide a resource for future studies to characterize novel targets of oncogenic K-Ras signaling and validate biomarkers of PAK4 inhibition.
Subject(s)
Proto-Oncogene Proteins p21(ras)/metabolism , cdc42 GTP-Binding Protein/metabolism , p21-Activated Kinases/metabolism , Animals , Mice , NIH 3T3 Cells , Phosphopeptides/metabolism , Phosphoproteins/metabolism , Phosphorylation , Proteomics , Signal Transduction , p21-Activated Kinases/geneticsABSTRACT
Proteomic studies of post-translational modifications by metal affinity or antibody-based methods often employ data-dependent analysis, providing rich data sets that consist of randomly sampled identified peptides because of the dynamic response of the mass spectrometer. This can complicate the primary goal of programs for drug development, mutational analysis, and kinase profiling studies, which is to monitor how multiple nodes of known, critical signaling pathways are affected by a variety of treatment conditions. Cell Signaling Technology has developed an immunoaffinity-based LC-MS/MS method called PTMScan Direct for multiplexed analysis of these important signaling proteins. PTMScan Direct enables the identification and quantification of hundreds of peptides derived from specific proteins in signaling pathways or specific protein types. Cell lines, tissues, or xenografts can be used as starting material. PTMScan Direct is compatible with both SILAC and label-free quantification. Current PTMScan Direct reagents target key nodes of many signaling pathways (PTMScan Direct: Multipathway), serine/threonine kinases, tyrosine kinases, and the Akt/PI3K pathway. Validation of each reagent includes score filtering of MS/MS assignments, filtering by identification of peptides derived from expected targets, identification of peptides homologous to expected targets, minimum signal intensity of peptide ions, and dependence upon the presence of the reagent itself compared with a negative control. The Multipathway reagent was used to study sensitivity of human cancer cell lines to receptor tyrosine kinase inhibitors and showed consistent results with previously published studies. The Ser/Thr kinase reagent was used to compare relative levels of kinase-derived phosphopeptides in mouse liver, brain, and embryo, showing tissue-specific activity of many kinases including Akt and PKC family members. PTMScan Direct will be a powerful quantitative method for elucidation of changes in signaling in a wide array of experimental systems, combining the specificity of traditional biochemical methods with the high number of data points and dynamic range of proteomic methods.
Subject(s)
Intracellular Signaling Peptides and Proteins/chemistry , Peptide Fragments/chemistry , Protein Processing, Post-Translational , Animals , Brain/metabolism , Cell Line , Chromatography, Affinity , Chromatography, Liquid , Embryo, Mammalian/metabolism , Humans , Immunoprecipitation , Intracellular Signaling Peptides and Proteins/isolation & purification , Intracellular Signaling Peptides and Proteins/metabolism , Liver/metabolism , Mice , Mice, Inbred BALB C , Peptide Fragments/isolation & purification , Peptide Mapping/methods , Phosphoproteins/chemistry , Phosphoproteins/isolation & purification , Phosphoproteins/metabolism , Phosphorylation , Protein Interaction Maps , Receptor Protein-Tyrosine Kinases/chemistry , Receptor Protein-Tyrosine Kinases/isolation & purification , Receptor Protein-Tyrosine Kinases/metabolism , Signal Transduction , Tandem Mass SpectrometryABSTRACT
Demyelination is a common pathological feature in a wide range of diseases, characterized by the loss of myelin sheath and myelin-supporting oligodendrocytes. These losses lead to impaired axonal function, increased vulnerability of axons to damage, and result in significant brain atrophy and neuro-axonal degeneration. Multiple pathomolecular processes contribute to neuroinflammation, oligodendrocyte cell death, and progressive neuronal dysfunction. In this study, we use the cuprizone mouse model of demyelination to investigate long-term non-invasive gamma entrainment using sensory stimulation as a potential therapeutic intervention for promoting myelination and reducing neuroinflammation in male mice. Here, we show that multisensory gamma stimulation mitigates demyelination, promotes oligodendrogenesis, preserves functional integrity and synaptic plasticity, attenuates oligodendrocyte ferroptosis-induced cell death, and reduces brain inflammation. Thus, the protective effects of multisensory gamma stimulation on myelin and anti-neuroinflammatory properties support its potential as a therapeutic approach for demyelinating disorders.
Subject(s)
Cuprizone , Demyelinating Diseases , Disease Models, Animal , Myelin Sheath , Oligodendroglia , Animals , Cuprizone/toxicity , Male , Demyelinating Diseases/chemically induced , Demyelinating Diseases/therapy , Demyelinating Diseases/pathology , Mice , Oligodendroglia/metabolism , Oligodendroglia/pathology , Myelin Sheath/metabolism , Mice, Inbred C57BL , Ferroptosis , Neuronal Plasticity , Brain/pathology , Brain/metabolism , Neuroinflammatory Diseases/chemically induced , Neuroinflammatory Diseases/pathologyABSTRACT
The integrated stress response (ISR) is critical for cell survival under stress. In response to diverse environmental cues, eIF2α becomes phosphorylated, engendering a dramatic change in mRNA translation. The activation of ISR plays a pivotal role in the early embryogenesis, but the eIF2-dependent translational landscape in pluripotent embryonic stem cells (ESCs) is largely unexplored. We employ a multi-omics approach consisting of ribosome profiling, proteomics, and metabolomics in wild-type (eIF2α+/+) and phosphorylation-deficient mutant eIF2α (eIF2αA/A) mouse ESCs (mESCs) to investigate phosphorylated (p)-eIF2α-dependent translational control of naive pluripotency. We show a transient increase in p-eIF2α in the naive epiblast layer of E4.5 embryos. Absence of eIF2α phosphorylation engenders an exit from naive pluripotency following 2i (two chemical inhibitors of MEK1/2 and GSK3α/ß) withdrawal. p-eIF2α controls translation of mRNAs encoding proteins that govern pluripotency, chromatin organization, and glutathione synthesis. Thus, p-eIF2α acts as a key regulator of the naive pluripotency gene regulatory network.
Subject(s)
Mouse Embryonic Stem Cells , Pluripotent Stem Cells , Animals , Mice , Embryonic Stem Cells/metabolism , Mouse Embryonic Stem Cells/metabolism , Phosphorylation , Pluripotent Stem Cells/metabolism , RNA, Messenger/metabolism , Eukaryotic Initiation Factor-2/metabolismABSTRACT
c-Src tyrosine kinase is a renowned key intracellular signaling molecule and a potential target for cancer therapy. Secreted c-Src is a recent observation, but how it contributes to extracellular phosphorylation remains elusive. Using a series of domain deletion mutants, we show that the N-proximal region of c-Src is essential for its secretion. The tissue inhibitor of metalloproteinases 2 (TIMP2) is an extracellular substrate of c-Src. Limited proteolysis-coupled mass spectrometry and mutagenesis studies verify that the Src homology 3 (SH3) domain of c-Src and the P31VHP34 motif of TIMP2 are critical for their interaction. Comparative phosphoproteomic analyses identify an enrichment of PxxP motifs in phosY-containing secretomes from c-Src-expressing cells with cancer-promoting roles. Inhibition of extracellular c-Src using custom SH3-targeting antibodies disrupt kinase-substrate complexes and inhibit cancer cell proliferation. These findings point toward an intricate role for c-Src in generating phosphosecretomes, which will likely influence cell-cell communication, particularly in c-Src-overexpressing cancers.
Subject(s)
Protein-Tyrosine Kinases , Secretome , Protein-Tyrosine Kinases/metabolism , Signal Transduction , Phosphotransferases , Phosphorylation , src Homology Domains , Cell Communication , src-Family KinasesABSTRACT
The autosomal recessive disorder Ataxia-Telangiectasia is caused by a dysfunction of the stress response protein, ATM. In the nucleus of proliferating cells, ATM senses DNA double-strand breaks and coordinates their repair. This role explains T-cell dysfunction and tumour risk. However, it remains unclear whether this function is relevant for postmitotic neurons and underlies cerebellar atrophy, since ATM is cytoplasmic in postmitotic neurons. Here, we used ATM-null mice that survived early immune deficits via bone-marrow transplantation, and that reached initial neurodegeneration stages at 12 months of age. Global cerebellar transcriptomics demonstrated that ATM depletion triggered upregulations in most neurotransmission and neuropeptide systems. Downregulated transcripts were found for the ATM interactome component Usp2, many non-coding RNAs, ataxia genes Itpr1, Grid2, immediate early genes and immunity factors. Allelic splice changes affected prominently the neuropeptide machinery, e.g., Oprm1. Validation experiments with stressors were performed in human neuroblastoma cells, where ATM was localised only to cytoplasm, similar to the brain. Effect confirmation in SH-SY5Y cells occurred after ATM depletion and osmotic stress better than nutrient/oxidative stress, but not after ATM kinase inhibition or DNA stressor bleomycin. Overall, we provide pioneer observations from a faithful A-T mouse model, which suggest general changes in synaptic and dense-core vesicle stress adaptation.
Subject(s)
Neuroblastoma , Neurodegenerative Diseases , Neuropeptides , Mice , Animals , Humans , Infant , Ataxia Telangiectasia Mutated Proteins/genetics , Ataxia Telangiectasia Mutated Proteins/metabolism , Down-Regulation , Up-Regulation , Transcriptome/genetics , Synaptic Transmission/genetics , Neurodegenerative Diseases/metabolism , Mice, Knockout , Neuropeptides/genetics , Neuropeptides/metabolism , DNA , RNA, Untranslated , Atrophy , Inositol 1,4,5-Trisphosphate Receptors/metabolismABSTRACT
Protein ubiquitination is a key regulatory process essential to life at a cellular level; significant efforts have been made to identify ubiquitinated proteins through proteomics studies, but the level of success has not reached that of heavily studied post-translational modifications, such as phosphorylation. HRD1, an E3 ubiquitin ligase, has been implicated in rheumatoid arthritis, but no disease-relevant substrates have been identified. To identify these substrates, we have taken both peptide and protein level approaches to enrich for ubiquitinated proteins in the presence and absence of HRD1. At the protein level, a two-step strategy was taken using cells expressing His(6)-tagged ubiquitin, enriching proteins first based on their ubiquitination and second based on the His tag with protein identification by LC-MS/MS. Application of this method resulted in identification and quantification of more than 400 ubiquitinated proteins, a fraction of which were found to be sensitive to HRD1 and were therefore deemed candidate substrates. In a second approach, ubiquitinated peptides were enriched after tryptic digestion by peptide immunoprecipitation using an antibody specific for the diglycine-labeled internal lysine residue indicative of protein ubiquitination, with peptides and ubiquitination sites identified by LC-MS/MS. Peptide immunoprecipitation resulted in identification of over 1800 ubiquitinated peptides on over 900 proteins in each study, with several proteins emerging as sensitive to HRD1 levels. Notably, significant overlap exists between the HRD1 substrates identified by the protein-based and the peptide-based strategies, with clear cross-validation apparent both qualitatively and quantitatively, demonstrating the effectiveness of both strategies and furthering our understanding of HRD1 biology.
Subject(s)
Protein Processing, Post-Translational/physiology , Proteome/metabolism , Ubiquitin-Protein Ligases/metabolism , Ubiquitin/metabolism , Ubiquitination/physiology , Arthritis, Rheumatoid/metabolism , HeLa Cells , Humans , Phosphorylation/physiology , Proteomics/methodsABSTRACT
Traditional methods for analysis of peptides using liquid chromatography and tandem mass spectrometry (LC-MS/MS) lack the specificity to comprehensively monitor specific biological processes due to the inherent duty cycle limitations of the MS instrument and the stochastic nature of the analytical platform. PTMScan Direct is a novel, antibody-based method that allows quantitative LC-MS/MS profiling of specific peptides from proteins that reside in the same signaling pathway. New PTMScan Direct reagents have been produced that target peptides from proteins involved in DNA Damage/Cell Cycle and Apoptosis/Autophagy pathways. Together, the reagents provide access to 438 sites on 237 proteins in these signaling cascades. These reagents have been used to profile the response to UV damage of DNA in human cell lines. UV damage was shown to activate canonical DNA damage response pathways through ATM/ATR-dependent signaling, stress response pathways and induce the initiation of apoptosis, as assessed by an increase in the abundance of peptides corresponding to cleaved, activated caspases. These data demonstrate the utility of PTMScan Direct as a multiplexed assay for profiling specific cellular responses to various stimuli, such as UV damage of DNA.