RESUMEN
Compartmentalization is a defining characteristic of eukaryotic cells, and partitions distinct biochemical processes into discrete subcellular locations. Microscopy1 and biochemical fractionation coupled with mass spectrometry2-4 have defined the proteomes of a variety of different organelles, but many intracellular compartments have remained refractory to such approaches. Proximity-dependent biotinylation techniques such as BioID provide an alternative approach to define the composition of cellular compartments in living cells5-7. Here we present a BioID-based map of a human cell on the basis of 192 subcellular markers, and define the intracellular locations of 4,145 unique proteins in HEK293 cells. Our localization predictions exceed the specificity of previous approaches, and enabled the discovery of proteins at the interface between the mitochondrial outer membrane and the endoplasmic reticulum that are crucial for mitochondrial homeostasis. On the basis of this dataset, we created humancellmap.org as a community resource that provides online tools for localization analysis of user BioID data, and demonstrate how this resource can be used to understand BioID results better.
Asunto(s)
Biotinilación , Compartimento Celular , Transporte de Proteínas , Proteoma/análisis , Proteoma/química , Células Cultivadas , Conjuntos de Datos como Asunto , Retículo Endoplásmico/química , Retículo Endoplásmico/metabolismo , Células HEK293 , Células HeLa , Homeostasis , Humanos , Espectrometría de Masas , Mitocondrias/química , Mitocondrias/metabolismo , Orgánulos/química , Orgánulos/metabolismo , Proteoma/metabolismo , Reproducibilidad de los ResultadosRESUMEN
The mechanistic target of rapamycin complex 1 (mTORC1) couples nutrient sufficiency to cell growth. mTORC1 is activated by exogenously acquired amino acids sensed through the GATOR-Rag guanosine triphosphatase (GTPase) pathway, or by amino acids derived through lysosomal degradation of protein by a poorly defined mechanism. Here, we revealed that amino acids derived from the degradation of protein (acquired through oncogenic Ras-driven macropinocytosis) activate mTORC1 by a Rag GTPase-independent mechanism. mTORC1 stimulation through this pathway required the HOPS complex and was negatively regulated by activation of the GATOR-Rag GTPase pathway. Therefore, distinct but functionally coordinated pathways control mTORC1 activity on late endocytic organelles in response to distinct sources of amino acids.
Asunto(s)
Aminoácidos/metabolismo , GTP Fosfohidrolasas/metabolismo , Lisosomas/enzimología , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Proteínas R-SNARE/metabolismo , Activación Enzimática , Células HEK293 , Humanos , Pinocitosis , ProteolisisRESUMEN
Stress granules and P-bodies are cytosolic biomolecular condensates that dynamically form by the phase separation of RNAs and proteins. They participate in translational control and buffer the proteome. Upon stress, global translation halts and mRNAs bound to the translational machinery and other proteins coalesce to form stress granules (SGs). Similarly, translationally stalled mRNAs devoid of translation initiation factors shuttle to P-bodies (PBs). Here, we review the cumulative progress made in defining the protein components that associate with mammalian SGs and PBs. We discuss the composition of SG and PB proteomes, supported by a new user-friendly database (http://rnagranuledb.lunenfeld.ca/) that curates current literature evidence for genes or proteins associated with SGs or PBs. As previously observed, the SG and PB proteomes are biased toward intrinsically disordered regions and have a high propensity to contain primary sequence features favoring phase separation. We also provide an outlook on how the various components of SGs and PBs may cooperate to organize and form membraneless organelles.
Asunto(s)
Gránulos Citoplasmáticos/metabolismo , Proteoma/metabolismo , ARN Mensajero/metabolismo , Animales , HumanosRESUMEN
Targeting bromodomains (BRDs) of the bromo-and-extra-terminal (BET) family offers opportunities for therapeutic intervention in cancer and other diseases. Here, we profile the interactomes of BRD2, BRD3, BRD4, and BRDT following treatment with the pan-BET BRD inhibitor JQ1, revealing broad rewiring of the interaction landscape, with three distinct classes of behavior for the 603 unique interactors identified. A group of proteins associate in a JQ1-sensitive manner with BET BRDs through canonical and new binding modes, while two classes of extra-terminal (ET)-domain binding motifs mediate acetylation-independent interactions. Last, we identify an unexpected increase in several interactions following JQ1 treatment that define negative functions for BRD3 in the regulation of rRNA synthesis and potentially RNAPII-dependent gene expression that result in decreased cell proliferation. Together, our data highlight the contributions of BET protein modules to their interactomes allowing for a better understanding of pharmacological rewiring in response to JQ1.
Asunto(s)
Antineoplásicos/farmacología , Azepinas/farmacología , Terapia Molecular Dirigida/métodos , Neoplasias/tratamiento farmacológico , Proteínas Nucleares/antagonistas & inhibidores , Mapas de Interacción de Proteínas/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas de Unión al ARN/antagonistas & inhibidores , Factores de Transcripción/antagonistas & inhibidores , Triazoles/farmacología , Antineoplásicos/química , Azepinas/química , Proteínas de Ciclo Celular , Proliferación Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Células HeLa , Humanos , Células K562 , Modelos Moleculares , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Unión Proteica , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteómica/métodos , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Transducción de Señal/efectos de los fármacos , Relación Estructura-Actividad , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Triazoles/químicaRESUMEN
mRNA processing, transport, translation, and ultimately degradation involve a series of dedicated protein complexes that often assemble into large membraneless structures such as stress granules (SGs) and processing bodies (PBs). Here, systematic in vivo proximity-dependent biotinylation (BioID) analysis of 119 human proteins associated with different aspects of mRNA biology uncovers 7424 unique proximity interactions with 1,792 proteins. Classical bait-prey analysis reveals connections of hundreds of proteins to distinct mRNA-associated processes or complexes, including the splicing and transcriptional elongation machineries (protein phosphatase 4) and the CCR4-NOT deadenylase complex (CEP85, RNF219, and KIAA0355). Analysis of correlated patterns between endogenous preys uncovers the spatial organization of RNA regulatory structures and enables the definition of 144 core components of SGs and PBs. We report preexisting contacts between most core SG proteins under normal growth conditions and demonstrate that several core SG proteins (UBAP2L, CSDE1, and PRRC2C) are critical for the formation of microscopically visible SGs.
Asunto(s)
Citoplasma/ultraestructura , Gránulos Citoplasmáticos/metabolismo , ARN Mensajero/metabolismo , Proteínas Portadoras/metabolismo , Citoplasma/metabolismo , Proteínas de Unión al ADN/metabolismo , Humanos , Espacio Intracelular , Proteínas/metabolismo , ARN/metabolismo , Proteínas de Unión al ARN/metabolismo , Estrés FisiológicoRESUMEN
The Hippo tumor suppressor pathway regulates organ size and tissue homoeostasis in response to diverse signaling inputs. The core of the pathway consists of a short kinase cascade: MST1 and MST2 phosphorylate and activate LATS1 and LATS2, which in turn phosphorylate and inactivate key transcriptional coactivators, YAP1 and TAZ (gene WWTR1). The MOB1 adapter protein regulates both phosphorylation reactions firstly by concurrently binding to the upstream MST and downstream LATS kinases to enable the trans phosphorylation reaction, and secondly by allosterically activating the catalytic function of LATS1 and LATS2 to directly stimulate phosphorylation of YAP and TAZ. Studies of yeast Mob1 and human MOB1 revealed that the ability to recognize phosphopeptide sequences in their interactors, Nud1 and MST2 respectively, was critical to their roles in regulating the Mitotic Exit Network in yeast and the Hippo pathway in metazoans. However, the underlying rules of phosphopeptide recognition by human MOB1, the implications of binding specificity for Hippo pathway signaling, and the generality of phosphopeptide binding function to other human MOB family members remained elusive.Employing proteomics, peptide arrays and biochemical analyses, we systematically examine the phosphopeptide binding specificity of MOB1 and find it to be highly complementary to the substrate phosphorylation specificity of MST1 and MST2. We demonstrate that autophosphorylation of MST1 and MST2 on several threonine residues provides multiple MOB1 binding sites with varying binding affinities which in turn contribute to a redundancy of MST1-MOB1 protein interactions in cells. The crystal structures of MOB1A in complex with two favored phosphopeptide sites in MST1 allow for a full description of the MOB1A phosphopeptide-binding consensus. Lastly, we show that the phosphopeptide binding properties of MOB1A are conserved in all but one of the seven MOB family members in humans, thus providing a starting point for uncovering their elusive cellular functions.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular , Fosfopéptidos/metabolismo , Fosforilación , Unión Proteica , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Proteínas Recombinantes/metabolismo , Serina-Treonina Quinasa 3 , Transducción de SeñalRESUMEN
Reversible phosphorylation is a fundamental regulatory mechanism, intricately coordinated by kinases and phosphatases, two classes of enzymes widely disrupted in human disease. To better understand the functions of the relatively understudied phosphatases, we have used complementary affinity purification and proximity-based interaction proteomics approaches to generate a physical interactome for 140 human proteins harboring phosphatase catalytic domains. We identified 1,335 high-confidence interactions (1,104 previously unreported), implicating these phosphatases in the regulation of a variety of cellular processes. Systematic phenotypic profiling of phosphatase catalytic and regulatory subunits revealed that phosphatases from every evolutionary family impinge on mitosis. Using clues from the interactome, we have uncovered unsuspected roles for DUSP19 in mitotic exit, CDC14A in regulating microtubule integrity, PTPRF in mitotic retraction fiber integrity, and DUSP23 in centriole duplication. The functional phosphatase interactome further provides a rich resource for ascribing functions for this important class of enzymes.
Asunto(s)
Mitosis , Monoéster Fosfórico Hidrolasas/metabolismo , Mapas de Interacción de Proteínas , Evolución Biológica , Centriolos/metabolismo , Fosfatasas de Especificidad Dual/metabolismo , Células HeLa , Humanos , Fenotipo , Unión Proteica , Proteínas Serina-Treonina Quinasas/metabolismo , Subunidades de Proteína/metabolismo , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/metabolismo , Reproducibilidad de los ResultadosRESUMEN
Affinity purification coupled with mass spectrometry (AP-MS) is a powerful technique for the identification and quantification of physical interactions. AP-MS requires careful experimental design, appropriate control selection and quantitative workflows to successfully identify bona fide interactors amongst a large background of contaminants. We previously introduced ProHits, a Laboratory Information Management System for interaction proteomics, which tracks all samples in a mass spectrometry facility, initiates database searches and provides visualization tools for spectral counting-based AP-MS approaches. More recently, we implemented Significance Analysis of INTeractome (SAINT) within ProHits to provide scoring of interactions based on spectral counts. Here, we provide an update to ProHits to support Data Independent Acquisition (DIA) with identification software (DIA-Umpire and MSPLIT-DIA), quantification tools (through DIA-Umpire, or externally via targeted extraction), and assessment of quantitative enrichment (through mapDIA) and scoring of interactions (through SAINT-intensity). With additional improvements, notably support of the iProphet pipeline, facilitated deposition into ProteomeXchange repositories and enhanced export and viewing functions, ProHits 4.0 offers a comprehensive suite of tools to facilitate affinity proteomics studies. SIGNIFICANCE: It remains challenging to score, annotate and analyze proteomics data in a transparent manner. ProHits was previously introduced as a LIMS to enable storing, tracking and analysis of standard AP-MS data. In this revised version, we expand ProHits to include integration with a number of identification and quantification tools based on Data-Independent Acquisition (DIA). ProHits 4.0 also facilitates data deposition into public repositories, and the transfer of data to new visualization tools.
Asunto(s)
Bases de Datos de Proteínas , Proteómica/métodos , Programas Informáticos , Cromatografía de Afinidad/métodos , Espectrometría de Masas/métodos , Péptidos/análisis , Péptidos/metabolismo , Mapeo de Interacción de Proteínas , Proteínas/análisis , Proteínas/metabolismoRESUMEN
SAINT (Significance Analysis of INTeractome) is a probabilistic method for scoring bait-prey interactions against negative controls in affinity purification - mass spectrometry (AP-MS) experiments. Our published SAINT algorithms use spectral counts or protein intensities as the input for calculating the probability of true interaction, which enables objective selection of high-confidence interactions with false discovery control. With the advent of new protein quantification methods such as Data Independent Acquisition (DIA), we redeveloped the scoring method to utilize the reproducibility information embedded in the peptide or fragment intensity data as a key scoring criterion, bypassing protein intensity summarization required in the previous SAINT workflow. The new software package, SAINTq, addresses key issues in the interaction scoring based on intensity data, including treatment of missing values and selection of peptides and fragments for scoring each prey protein. We applied SAINTq to two independent DIA AP-MS data sets profiling the interactome of MEPCE and EIF4A2 and that of 14-3-3ß, and benchmarked the performance in terms of recovering previously reported literature interactions in the iRefIndex database. In both data sets, the SAINTq analysis using the fragment-level intensity data led to the most sensitive detection of literature interactions at the same level of specificity. This analysis outperforms the analysis using protein intensity data summed from fragment intensity data that is equivalent to the model in SAINTexpress.
Asunto(s)
Cromatografía de Afinidad/métodos , Espectrometría de Masas/métodos , Péptidos/análisis , Biología Computacional , Unión ProteicaRESUMEN
The mechanisms governing apical membrane assembly during biological tube development are poorly understood. Here, we show that extension of the C. elegans excretory canal requires cerebral cavernous malformation 3 (CCM-3), independent of the CCM1 orthologue KRI-1. Loss of ccm-3 causes canal truncations and aggregations of canaliculular vesicles, which form ectopic lumen (cysts). We show that CCM-3 localizes to the apical membrane, and in cooperation with GCK-1 and STRIPAK, promotes CDC-42 signalling, Golgi stability and endocytic recycling. We propose that endocytic recycling is mediated through the CDC-42-binding kinase MRCK-1, which interacts physically with CCM-3-STRIPAK. We further show canal membrane integrity to be dependent on the exocyst complex and the actin cytoskeleton. This work reveals novel in vivo roles of CCM-3·STRIPAK in regulating tube extension and membrane integrity through small GTPase signalling and vesicle dynamics, which may help explain the severity of CCM3 mutations in patients.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiología , Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al GTP/metabolismo , Discapacidad Intelectual/metabolismo , Micrognatismo/metabolismo , Morfogénesis/fisiología , Costillas/anomalías , Transducción de Señal/fisiología , Vesículas Transportadoras/fisiología , Animales , Caenorhabditis elegans/metabolismo , Aparato de Golgi/metabolismo , Intestinos/crecimiento & desarrollo , Microscopía Electrónica de Transmisión , Microscopía de Interferencia , Interferencia de ARN , Costillas/metabolismoRESUMEN
Quantitative interaction proteomics data can be a challenge to efficiently analyze and subsequently present to an audience in a simple and easy to understand format that still conveys sufficient levels of information. Here we present freely accessible and open-source web tools for displaying multiple parameters from quantitative protein-protein interaction data sets in a visually intuitive format. Given a set of "bait" proteins with detected "prey" interactions, dot plots can be generated to display absolute spectral counts for the preys, relative spectral counts between baits and confidence levels for the interactions (e.g. as determined by SAINTexpress). Additional tools are available for displaying fold change results between numerous baits with their associated confidence level (e.g. resulting from intensity measurements) and pairwise bait analyses displaying spectral counts, confidence score and fold change differences in a scatter plot format. These tools make it easy for the user to identify important interaction changes, interpret their data, and present this information to others in an intuitive way.
Asunto(s)
Mapeo de Interacción de Proteínas , Programas Informáticos , Bases de Datos de Proteínas , Internet , ProteómicaRESUMEN
Mapping protein-protein interactions for chromatin-associated proteins remains challenging. Here we explore the use of BioID, a proximity biotinylation approach in which a mutated biotin ligase (BirA*) is fused to a bait of interest, allowing for the local activation of biotin and subsequent biotinylation of proteins in the bait vicinity. BioID allowed for successful interactome mapping of core histones and members of the mediator complex. We explored the background signal produced by the BioID approach and found that using distinct types of controls increased the stringency of our statistical analysis with SAINTexpress. A direct comparison of BioID with our AP-MS protocol optimized for chromatin-associated protein complexes revealed that the approaches identified few shared interaction partners and enriched for distinct biological processes; yet, both approaches permitted the recovery of biologically meaningful interactions. While no clear bias could be observed for either technique toward protein complexes of particular functions, BioID allowed for the purification of proteins of lower cellular abundance. Finally, we were able to identify a strong association of MED4 with the centrosome by BioID and validated this finding by immunofluorescence. In summary, BioID complements AP-MS for the study of chromatin-associated protein complexes. BIOLOGICAL SIGNIFICANCE: This manuscript describes the application of BioID, a proximity biotinylation approach, to chromatin-associated proteins, namely core histones and members of the mediator complex. We observed that BioID was successful at identifying known interaction partners for the baits tested, but also allowed novel putative interaction partners to be identified. By performing a detailed comparison of BioID versus a standard method for interactome mapping (affinity purification coupled to mass spectrometry, AP-MS), we show that the approaches were complementary, allowing for purification of different interaction partners. These interaction partners were different in the biological processes they are associated with, but also in their abundance. BioID represents a significant technical development in the field of chromatin research by expanding the search space for interactome mapping beyond what is possible with AP-MS. This article is part of a Special Issue entitled: Protein dynamics in health and disease. Guest Editors: Pierre Thibault and Anne-Claude Gingras.
Asunto(s)
Ligasas de Carbono-Nitrógeno , Cromatina , Proteínas de Escherichia coli , Escherichia coli , Complejo Mediador , Proteínas Represoras , Ligasas de Carbono-Nitrógeno/genética , Ligasas de Carbono-Nitrógeno/metabolismo , Cromatina/genética , Cromatina/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Humanos , Complejo Mediador/genética , Complejo Mediador/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismoRESUMEN
Post-translational modifications of proteins regulate diverse cellular functions, with mounting evidence suggesting that hierarchical cross-talk between distinct modifications may fine-tune cellular responses. For example, in apoptosis, caspases promote cell death via cleavage of key structural and enzymatic proteins that in some instances is inhibited by phosphorylation near the scissile bond. In this study, we systematically investigated how protein phosphorylation affects susceptibility to caspase cleavage using an N-terminomic strategy, namely, a modified terminal amino isotopic labeling of substrates (TAILS) workflow, to identify proteins for which caspase-catalyzed cleavage is modulated by phosphatase treatment. We validated the effects of phosphorylation on three of the identified proteins and found that Yap1 and Golgin-160 exhibit decreased cleavage when phosphorylated, whereas cleavage of MST3 was promoted by phosphorylation. Furthermore, using synthetic peptides we systematically examined the influence of phosphoserine throughout the entirety of caspase-3, -7, and -8 recognition motifs and observed a general inhibitory effect of phosphorylation even at residues considered outside the classical consensus motif. Overall, our work demonstrates a role for phosphorylation in controlling caspase-mediated cleavage and shows that N-terminomic strategies can be tailored to study cross-talk between phosphorylation and proteolysis.
Asunto(s)
Caspasas/química , Caspasas/metabolismo , Péptidos/metabolismo , Proteómica/métodos , Células HeLa , Humanos , Marcaje Isotópico , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Fosfoproteínas/metabolismo , Fosforilación , Procesamiento Proteico-Postraduccional , Proteínas Serina-Treonina Quinasas/metabolismo , ProteolisisRESUMEN
The Hippo pathway regulates organ size and tissue homeostasis in response to multiple stimuli, including cell density and mechanotransduction. Pharmacological inhibition of phosphatases can also stimulate Hippo signaling in cell culture. We defined the Hippo protein-protein interaction network with and without inhibition of serine and threonine phosphatases by okadaic acid. We identified 749 protein interactions, including 599 previously unrecognized interactions, and demonstrated that several interactions with serine and threonine phosphatases were phosphorylation-dependent. Mutation of the T-loop of MST2 (mammalian STE20-like protein kinase 2), which prevented autophosphorylation, disrupted its association with STRIPAK (striatin-interacting phosphatase and kinase complex). Deletion of the amino-terminal forkhead-associated domain of SLMAP (sarcolemmal membrane-associated protein), a component of the STRIPAK complex, prevented its association with MST1 and MST2. Phosphatase inhibition produced temporally distinct changes in proteins that interacted with MOB1A and MOB1B (Mps one binder kinase activator-like 1A and 1B) and promoted interactions with upstream Hippo pathway proteins, such as MST1 and MST2, and with the trimeric protein phosphatase 6 complex (PP6). Mutation of three basic amino acids that are part of a phospho-serine- and phospho-threonine-binding domain in human MOB1B prevented its interaction with MST1 and PP6 in cells treated with okadaic acid. Collectively, our results indicated that changes in phosphorylation orchestrate interactions between kinases and phosphatases in Hippo signaling, providing a putative mechanism for pathway regulation.
Asunto(s)
Monoéster Fosfórico Hidrolasas/metabolismo , Mapas de Interacción de Proteínas , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Sitios de Unión , Western Blotting , Análisis por Conglomerados , Células HEK293 , Células HeLa , Vía de Señalización Hippo , Humanos , Péptidos y Proteínas de Señalización Intracelular , Luciferasas/genética , Luciferasas/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Mutación , Fosfoproteínas Fosfatasas/genética , Fosfoproteínas Fosfatasas/metabolismo , Monoéster Fosfórico Hidrolasas/clasificación , Monoéster Fosfórico Hidrolasas/genética , Fosforilación , Unión Proteica , Mapeo de Interacción de Proteínas/métodos , Proteínas Serina-Treonina Quinasas/clasificación , Proteínas Serina-Treonina Quinasas/genética , Serina-Treonina Quinasa 3RESUMEN
Protein kinases are the second largest human protein family, but in terms of research interest, both basic and applied, they are surely the most popular. Over the past decade, many techniques and approaches for studying the kinome have been described and the pace of development is ever increasing. Presently, a molecular biologist can approach the kinome from many different angles: what kinases are active during a specific cell state of interest or become activated in response to a specific stimulus? What are the effects of controlling the activation status of an individual kinase? What substrates are targeted by a particular kinase, either in general or under particular conditions? And what kinase is responsible for targeting a specific phosphorylation site of interest? These are some of the more commonly asked questions during any kinase-centric research project and different strategies have been devised for answering such queries. In this review, we outline the most promising of these approaches, particularly those with a capacity for high-throughput studies. This article is part of a Special Issue entitled: From protein structures to clinical applications.
Asunto(s)
Proteínas Quinasas/metabolismo , Proteoma/metabolismo , Proteómica/métodos , Animales , Humanos , Proteínas Quinasas/química , Proteoma/químicaRESUMEN
The TrkA receptor tyrosine kinase induces death in medulloblastoma cells via an interaction with the cerebral cavernous malformation 2 (CCM2) protein. We used affinity proteomics to identify the germinal center kinase class III (GCKIII) kinases STK24 and STK25 as novel CCM2 interactors. Down-modulation of STK25, but not STK24, rescued medulloblastoma cells from NGF-induced TrkA-dependent cell death, suggesting that STK25 is part of the death-signaling pathway initiated by TrkA and CCM2. CCM2 can be phosphorylated by STK25, and the kinase activity of STK25 is required for death signaling. Finally, STK25 expression in tumors is correlated with positive prognosis in neuroblastoma patients. These findings delineate a death-signaling pathway downstream of neurotrophic receptor tyrosine kinases that may provide targets for therapeutic intervention in pediatric tumors of neural origin.