RESUMO
The recruitment of signaling proteins into activated receptor tyrosine kinases (RTKs) to produce rapid, high-fidelity downstream response is exposed to the ambiguity of random diffusion to the target site. Liquid-liquid phase separation (LLPS) overcomes this by providing elevated, localized concentrations of the required proteins while impeding competitor ligands. Here, we show a subset of phosphorylation-dependent RTK-mediated LLPS states. We then investigate the formation of phase-separated droplets comprising a ternary complex including the RTK, (FGFR2); the phosphatase, SHP2; and the phospholipase, PLCγ1, which assembles in response to receptor phosphorylation. SHP2 and activated PLCγ1 interact through their tandem SH2 domains via a previously undescribed interface. The complex of FGFR2 and SHP2 combines kinase and phosphatase activities to control the phosphorylation state of the assembly while providing a scaffold for active PLCγ1 to facilitate access to its plasma membrane substrate. Thus, LLPS modulates RTK signaling, with potential consequences for therapeutic intervention.
Assuntos
Proteína Tirosina Fosfatase não Receptora Tipo 11 , Transdução de Sinais , Fosforilação , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Tirosina/metabolismo , Domínios de Homologia de srcRESUMO
The probability of a given receptor tyrosine kinase (RTK) triggering a defined cellular outcome is low because of the promiscuous nature of signalling, the randomness of molecular diffusion through the cell, and the ongoing nonfunctional submembrane signalling activity or noise. Signal transduction is therefore a 'numbers game', where enough cell surface receptors and effector proteins must initially be engaged to guarantee formation of a functional signalling complex against a background of redundant events. The presence of intracellular liquid-liquid phase separation (LLPS) at the plasma membrane provides a mechanism through which the probabilistic nature of signalling can be weighted in favour of the required, discrete cellular outcome and mutual exclusivity in signal initiation.
Assuntos
Receptores Proteína Tirosina Quinases , Transdução de Sinais , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais/fisiologia , Probabilidade , Sistemas de Liberação de MedicamentosRESUMO
Receptor tyrosine kinase (RTK) overexpression is linked to the development and progression of multiple cancers. RTKs are classically considered to initiate cytoplasmic signalling pathways via ligand-induced tyrosine phosphorylation, however recent evidence points to a second tier of signalling contingent on interactions mediated by the proline-rich motif (PRM) regions of non-activated RTKs. The presence of PRMs on the C-termini of >40 % of all RTKs and the abundance of PRM-binding proteins encoded by the human genome suggests that there is likely to be a large number of previously unexplored interactions which add to the RTK intracellular interactome. Here, we explore the RTK PRM interactome and its potential significance using affinity purification mass spectrometry and in silico enrichment analyses. Peptides comprising PRM-containing C-terminal tail regions of EGFR, FGFR2 and HER2 were used as bait to affinity purify bound proteins from different cancer cell line lysates. 490 unique interactors were identified, amongst which proteins with metabolic, homeostatic and migratory functions were overrepresented. This suggests that PRMs from RTKs may sustain a diverse interactome in cancer cells. Since RTK overexpression is common in cancer, RTK PRM-derived signalling may be an important, but as yet underexplored, contributor to negative cancer outcomes including resistance to kinase inhibitors.
RESUMO
Receptor tyrosine kinase (RTK)-mediated signal transduction is fundamental to cell function and drives important cellular outcomes which, when dysregulated, can lead to malignant tumour growth and metastasis. The initiation of signals from plasma membrane-bound RTKs is subjected to multiple regulatory mechanisms that control downstream effector protein recruitment and function. The high propensity of RTKs to condense via liquid-liquid phase separation (LLPS) into membraneless organelles with downstream effector proteins provides a further fundamental mechanism for signal regulation. Herein we highlight how this phenomenon contributes to cancer signalling and consider the potential impact of LLPS on outcomes for cancer patients.
RESUMO
Protein interactions with the microRNA (miRNA)-mediated gene silencing protein Argonaute 2 (AGO2) control miRNA expression. miRNA biogenesis starts with the production of precursor transcripts and culminates with the loading of mature miRNA onto AGO2 by DICER1. Here we reveal an additional component to the regulatory mechanism for miRNA biogenesis involving the adaptor protein, growth factor receptor-bound protein 2 (GRB2). The N-terminal SH3 domain of GRB2 is recruited to the PAZ domain of AGO2 forming a ternary complex containing GRB2, AGO2 and DICER1. Using small-RNA sequencing we identified two groups of miRNAs which are regulated by the binding of GRB2. First, mature and precursor transcripts of mir-17~92 and mir-221 miRNAs are enhanced. Second, mature, but not precursor, let-7 family miRNAs are diminished suggesting that GRB2 directly affects loading of these miRNAs. Notably, the resulting loss of let-7 augments expression of oncogenic targets such as RAS. Thus, a new role for GRB2 is established with implications for cancer pathogenesis through regulation of miRNA biogenesis and oncogene expression.
Assuntos
MicroRNAs , MicroRNAs/metabolismo , Inativação Gênica , Sequência de Bases , Proteína Adaptadora GRB2/genética , Proteína Adaptadora GRB2/metabolismo , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismoRESUMO
Receptor tyrosine kinases (RTKs) are typically activated through a precise sequence of intracellular phosphorylation events starting with a tyrosine residue on the activation loop (A-loop) of the kinase domain (KD). From this point the mono-phosphorylated enzyme is active, but subject to stringent regulatory mechanisms which can vary dramatically across the different RTKs. In the absence of extracellular stimulation, fibroblast growth factor receptor 2 (FGFR2) exists in the mono-phosphorylated state in which catalytic activity is regulated to allow rapid response upon ligand binding, whilst restricting ligand-independent activation. Failure of this regulation is responsible for pathologic outcomes including cancer. Here we reveal the molecular mechanistic detail of KD control based on combinatorial interactions of the juxtamembrane (JM) and the C-terminal tail (CT) regions of the receptor. JM stabilizes the asymmetric dimeric KD required for substrate phosphorylation, whilst CT binding opposes dimerization, and down-regulates activity. Direct binding between JM and CT delays the recruitment of downstream effector proteins adding a further control step as the receptor proceeds to full activation. Our findings underscore the diversity in mechanisms of RTK oligomerisation and activation.
Assuntos
Receptor Tipo 2 de Fator de Crescimento de Fibroblastos , Tirosina , Fosforilação , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/química , Ligantes , Membrana CelularRESUMO
Background: Methylation of carbon-5 of cytosines (m 5C) is a conserved post-transcriptional nucleotide modification of RNA with widespread distribution across organisms. It can be further modified to yield 5-hydroxymethylcytidine (hm 5C), 5-formylcytidine (f 5C), 2´-O-methyl-5-hydroxymethylcytidine (hm 5Cm) and 2´-O-methyl-5-formylcytidine (f 5Cm). How m 5C, and specially its derivates, contribute to biology mechanistically is poorly understood. We recently showed that m 5C is required for Caenorhabditis elegans development and fertility under heat stress. m 5C has been shown to participate in mRNA transport and maintain mRNA stability through its recognition by the reader proteins ALYREF and YBX1, respectively. Hence, identifying readers for RNA modifications can enhance our understanding in the biological roles of these modifications. Methods: To contribute to the understanding of how m 5C and its oxidative derivatives mediate their functions, we developed RNA baits bearing modified cytosines in diverse structural contexts to pulldown potential readers in C. elegans. Potential readers were identified using mass spectrometry. The interaction of two of the putative readers with m 5C was validated using immunoblotting. Results: Our mass spectrometry analyses revealed unique binding proteins for each of the modifications. In silico analysis for phenotype enrichments suggested that hm 5Cm unique readers are enriched in proteins involved in RNA processing, while readers for m 5C, hm 5C and f 5C are involved in germline processes. We validated our dataset by demonstrating that the nematode ALYREF homologues ALY-1 and ALY-2 preferentially bind m 5C in vitro. Finally, sequence alignment analysis showed that several of the putative m 5C readers contain the conserved RNA recognition motif (RRM), including ALY-1 and ALY-2. Conclusions: The dataset presented here serves as an important scientific resource that will support the discovery of new functions of m 5C and its derivatives. Furthermore, we demonstrate that ALY-1 and ALY-2 bind to m 5C in C. elegans.
RESUMO
The regulation of phosphatase activity is fundamental to the control of intracellular signalling and in particular the tyrosine kinase-mediated mitogen-activated protein kinase (MAPK) pathway. Shp2 is a ubiquitously expressed protein tyrosine phosphatase and its kinase-induced hyperactivity is associated with many cancer types. In non-stimulated cells we find that binding of the adaptor protein Grb2, in its monomeric state, initiates Shp2 activity independent of phosphatase phosphorylation. Grb2 forms a bidentate interaction with both the N-terminal SH2 and the catalytic domains of Shp2, releasing the phosphatase from its auto-inhibited conformation. Grb2 typically exists as a dimer in the cytoplasm. However, its monomeric state prevails under basal conditions when it is expressed at low concentration, or when it is constitutively phosphorylated on a specific tyrosine residue (Y160). Thus, Grb2 can activate Shp2 and downstream signal transduction, in the absence of extracellular growth factor stimulation or kinase-activating mutations, in response to defined cellular conditions. Therefore, direct binding of Grb2 activates Shp2 phosphatase in the absence of receptor tyrosine kinase up-regulation.
Assuntos
Proteína Adaptadora GRB2/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Transdução de Sinais , Ativação Enzimática , Proteína Adaptadora GRB2/metabolismo , Fosforilação , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismoRESUMO
Membraneless organelles are sites for RNA biology including small non-coding RNA (ncRNA) mediated gene silencing. How small ncRNAs utilise phase separated environments for their function is unclear. We investigated how the PIWI-interacting RNA (piRNA) pathway engages with the membraneless organelle P granule in Caenorhabditis elegans. Proteomic analysis of the PIWI protein PRG-1 reveals an interaction with the constitutive P granule protein DEPS-1. DEPS-1 is not required for piRNA biogenesis but piRNA-dependent silencing: deps-1 mutants fail to produce the secondary endo-siRNAs required for the silencing of piRNA targets. We identify a motif on DEPS-1 which mediates a direct interaction with PRG-1. DEPS-1 and PRG-1 form intertwining clusters to build elongated condensates in vivo which are dependent on the Piwi-interacting motif of DEPS-1. Additionally, we identify EDG-1 as an interactor of DEPS-1 and PRG-1. Our study reveals how specific protein-protein interactions drive the spatial organisation and piRNA-dependent silencing within membraneless organelles.
Assuntos
Proteínas Argonautas/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Inativação Gênica , RNA Interferente Pequeno/metabolismo , Animais , Proteínas Argonautas/genética , Sítios de Ligação , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Grânulos Citoplasmáticos/metabolismo , Células Germinativas/metabolismo , Mutação , Ligação Proteica , Proteômica , Interferência de RNA , RNA de Cadeia Dupla/metabolismo , RNA Interferente Pequeno/genéticaRESUMO
Receptor tyrosine kinase (RTK)-mediated hyperactivation of the MAPK/Erk pathway is responsible for a large number of pathogenic outcomes including many cancers. Considerable effort has been directed at targeting this pathway with varying degrees of long term therapeutic success. Under non-stimulated conditions Erk is bound to the adaptor protein Shc preventing aberrant signalling by sequestering Erk from activation by Mek. Activated RTK recruits Shc, via its phosphotyrosine binding (PTB) domain (ShcPTB), precipitating the release of Erk to engage in a signalling response. Here we describe a novel approach to inhibition of MAP kinase signal transduction through attempting to preserve the Shc-Erk complex under conditions of activated receptor. A library of existing drug molecules was computationally screened for hits that would bind to the ShcPTB and block its interaction with the RTKs EGFR and ErbB2. The primary hit from the screen was indomethacin, a non-steroidal anti-inflammatory drug. Validation of this molecule in vitro and in cellular efficacy studies in cancer cells provides proof of principle of the approach to pathway down-regulation and a potential optimizable lead compound.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Antineoplásicos/farmacologia , Reposicionamento de Medicamentos , Indometacina/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Proteínas Adaptadoras da Sinalização Shc/antagonistas & inibidores , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/metabolismo , Antineoplásicos/química , Antineoplásicos/metabolismo , Movimento Celular/efeitos dos fármacos , Receptores ErbB/química , Receptores ErbB/metabolismo , Células HeLa , Humanos , Indometacina/química , Indometacina/metabolismo , Células MCF-7 , Simulação de Acoplamento Molecular , Invasividade Neoplásica , Neoplasias/enzimologia , Neoplasias/patologia , Fosforilação , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Adaptadoras da Sinalização Shc/química , Proteínas Adaptadoras da Sinalização Shc/metabolismo , Relação Estrutura-AtividadeRESUMO
Signaling pathways and small RNAs direct diverse cellular events, but few examples are known of defined signaling pathways directly regulating small RNA biogenesis. We show that ERK phosphorylates Dicer on two conserved residues in its RNase IIIb and double-stranded RNA (dsRNA)-binding domains and that phosphorylation of these residues is necessary and sufficient to trigger Dicer's nuclear translocation in worms, mice, and human cells. Phosphorylation of Dicer on either site inhibits Dicer function in the female germline and dampens small RNA repertoire. Our data demonstrate that ERK phosphorylates and inhibits Dicer during meiosis I for oogenesis to proceed normally in Caenorhabditis elegans and that this inhibition is released before fertilization for embryogenesis to proceed normally. The conserved Dicer residues, their phosphorylation by ERK, and the consequences of the resulting modifications implicate an ERK-Dicer nexus as a fundamental component of the oocyte-to-embryo transition and an underlying mechanism coupling extracellular cues to small RNA production.
Assuntos
Caenorhabditis elegans/enzimologia , Sistema de Sinalização das MAP Quinases/fisiologia , Oócitos/metabolismo , RNA de Cadeia Dupla/metabolismo , Ribonuclease III/metabolismo , Animais , Sequência de Bases/fisiologia , Caenorhabditis elegans/embriologia , Camundongos , Oogênese/fisiologia , FosforilaçãoRESUMO
Control mechanisms that prevent aberrant signaling are necessary to maintain cellular homeostasis. We describe a new mechanism by which the adaptor protein Shc directly binds the MAP kinase Erk, thus preventing its activation in the absence of extracellular stimuli. The Shc-Erk complex restricts Erk nuclear translocation, restraining Erk-dependent transcription of genes, including those responsible for oncogenic growth. The complex forms through unique binding sites on both the Shc PTB domain and the N-terminal lobe of Erk. Upon receptor tyrosine kinase stimulation, a conformational change within Shc-induced through interaction with the phosphorylated receptor-releases Erk, allowing it to fulfill its role in signaling. Thus, in addition to its established role in promoting MAP kinase signaling in stimulated cells, Shc negatively regulates Erk activation in the absence of growth factors and thus could be considered a tumor suppressor in human cells.
Assuntos
Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Mapeamento de Interação de Proteínas , Proteínas Adaptadoras da Sinalização Shc/metabolismo , Transdução de Sinais , Sítios de Ligação , Linhagem Celular , Humanos , Ligação Proteica , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de SrcRESUMO
The adaptor protein Grb2 is recruited to intracellular early signalling complexes of many receptor tyrosine kinases and plays an important role transducing signals leading to MAP kinase activation. To date the SH2 domain of Grb2 has been shown to mediate receptor interactions with phosphorylated tyrosine residues sited directly on the receptor or on auxiliary docking proteins. Here we report that FGFR2 recruits Grb2 through its C-terminal SH3 domain. The binding site of this domain was mapped to the proline-rich C-terminus of the receptor. Deletion of the last 10 amino acids of FGFR2 abrogates interaction with Grb2. Synthetic peptides based on the C-terminus of FGFR2 bind to full length Grb2 with low micromolar affinity. The function of this novel mode of Grb2 binding provides resistance to site-specific Shp2-mediated receptor dephosphorylation.