RESUMO
Previous studies of the protein kinase, ERK2, using NMR and hydrogen-exchange measurements have shown changes in dynamics accompanying its activation by phosphorylation. However, knowledge about the conformational motions involved is incomplete. Here, we examined ERK2 using long conventional molecular dynamics (MD) simulations starting from crystal structures of phosphorylated (2P) and unphosphorylated (0P) forms. Individual trajectories were run for (5 to 25) µs, totaling 727 µs. The results show unexpected flexibility of the A-loop, with multiple long-lived (>5 µs) conformational states in both 2P- and 0P-ERK2. Differential contact network and principal component analyses reveal coupling between the A-loop fold and active site dynamics, with evidence for conformational selection in the kinase core of 2P-ERK2 but not 0P-ERK2. Simulations of 2P-ERK2 show A-loop states corresponding to restrained dynamics within the N-lobe, including regions around catalytic residues. One A-loop conformer forms lasting interactions with the L16 segment, leading to reduced RMSF and greater compaction in the active site. By contrast, simulations of 0P-ERK2 reveal excursions of A-loop residues away from the C-lobe, leading to greater active site mobility. Thus, the A-loop in ERK2 switches between distinct conformations that reflect coupling with the active site, possibly via the L16 segment. Crystal packing interactions suggest that lattice contacts with the A-loop may restrain its structural variation in X-ray structures of ERK2. The novel conformational states identified by MD expand our understanding of ERK2 regulation, by linking the activated state of the kinase to reduced dynamics and greater compaction surrounding the catalytic site.
Assuntos
Domínio AAA , Domínio Catalítico , Proteína Quinase 1 Ativada por Mitógeno , Simulação de Dinâmica Molecular , Fosforilação , Proteína Quinase 1 Ativada por Mitógeno/química , Ativação Enzimática , Cristalografia por Raios XRESUMO
Eukaryotic protein kinases (EPKs) adopt an active conformation following phosphorylation of a particular activation loop residue. Most EPKs spontaneously autophosphorylate this residue. While structure-function relationships of the active conformation are essentially understood, those of the "prone-to-autophosphorylate" conformation are unclear. Here, we propose that a site within the αC-helix of EPKs, occupied by Arg in the mitogen-activated protein kinase (MAPK) Erk1/2 (Arg84/65), impacts spontaneous autophosphorylation. MAPKs lack spontaneous autoactivation, but we found that converting Arg84/65 of Erk1/2 to various residues enables spontaneous autophosphorylation. Furthermore, Erk1 molecules mutated in Arg84 are oncogenic. Arg84/65 thus obstructs the adoption of the "prone-to-autophosphorylate" conformation. All MAPKs harbor an Arg that is equivalent to Arg84/65 of Erks, whereas Arg is rarely found at the equivalent position in other EPKs. We observed that Arg84/65 of Erk1/2 interacts with the DFG motif, suggesting that autophosphorylation may be inhibited by the Arg84/65-DFG interactions. Erk1/2s mutated in Arg84/65 autophosphorylate not only the TEY motif, known as critical for catalysis, but also on Thr207/188. Our MS/MS analysis revealed that a large proportion of the Erk2R65H population is phosphorylated on Thr188 or on Tyr185 + Thr188, and a small fraction is phosphorylated on the TEY motif. No molecules phosphorylated on Thr183 + Thr188 were detected. Thus, phosphorylation of Thr183 and Thr188 is mutually exclusive suggesting that not only TEY-phosphorylated molecules are active but perhaps also those phosphorylated on Tyr185 + Thr188. The effect of mutating Arg84/65 may mimic a physiological scenario in which allosteric effectors cause Erk1/2 activation by autophosphorylation.
Assuntos
Arginina , Proteína Quinase 1 Ativada por Mitógeno , Proteína Quinase 3 Ativada por Mitógeno , Fosforilação , Arginina/metabolismo , Humanos , Animais , Camundongos , Linhagem Celular , Células HEK293 , Ativação Enzimática/genética , Mutação , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteína Quinase 1 Ativada por Mitógeno/química , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/química , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Estrutura Terciária de Proteína , Modelos Moleculares , Cristalização , Sequência de AminoácidosRESUMO
The combination of ultrahigh-throughput screening and sequencing informs on function and intragenic epistasis within combinatorial protein mutant libraries. Establishing a droplet-based, in vitro compartmentalised approach for robust expression and screening of protein kinase cascades (>107 variants/day) allowed us to dissect the intrinsic molecular features of the MKK-ERK signalling pathway, without interference from endogenous cellular components. In a six-residue combinatorial library of the MKK1 docking domain, we identified 29,563 sequence permutations that allow MKK1 to efficiently phosphorylate and activate its downstream target kinase ERK2. A flexibly placed hydrophobic sequence motif emerges which is defined by higher order epistatic interactions between six residues, suggesting synergy that enables high connectivity in the sequence landscape. Through positive epistasis, MKK1 maintains function during mutagenesis, establishing the importance of co-dependent residues in mammalian protein kinase-substrate interactions, and creating a scenario for the evolution of diverse human signalling networks.
Assuntos
Epistasia Genética , Proteínas Quinases Ativadas por Mitógeno/química , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfatos/metabolismo , Catálise , Humanos , MAP Quinase Quinase 1/química , MAP Quinase Quinase 1/metabolismo , Sistema de Sinalização das MAP Quinases , Proteína Quinase 1 Ativada por Mitógeno/química , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Simulação de Acoplamento Molecular , Fosforilação , Domínios Proteicos , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais , Especificidade por SubstratoRESUMO
Phosphoprotein enriched in astrocytes, 15 kDa (PEA-15) is a death-effector domain (DED) containing protein involved in regulating mitogen-activated protein kinase and apoptosis pathways. In this molecular dynamics study, we examined how phosphorylation of the PEA-15 C-terminal tail residues, Ser-104 and Ser-116, allosterically mediates conformational changes of the DED and alters the binding specificity from extracellular-regulated kinase (ERK) to Fas-associated death domain (FADD) protein. We delineated that the binding interfaces between the unphosphorylated PEA-15 and ERK2 and between the doubly phosphorylated PEA-15 and FADD are similarly composed of a scaffold that includes both the DED and the C-terminal tail residues of PEA-15. While the unphosphorylated serine residues do not directly interact with ERK2, the phosphorylated Ser-116 engages in strong electrostatic interactions with arginine residues on FADD DED. Upon PEA-15 binding, FADD repositions its death domain (DD) relative to the DED, an essential conformational change to allow the death-inducing signaling complex (DISC) assembly.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Proteína de Domínio de Morte Associada a Fas/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteínas Reguladoras de Apoptose/química , Proteína de Domínio de Morte Associada a Fas/química , Humanos , Proteína Quinase 1 Ativada por Mitógeno/química , Simulação de Dinâmica Molecular , Complexos Multiproteicos , Fosforilação , Ligação Proteica , Conformação Proteica , Serina , Eletricidade Estática , Relação Estrutura-AtividadeRESUMO
The Kaposi's sarcoma associated herpesvirus protein ORF45 binds the extracellular signal-regulated kinase (ERK) and the p90 Ribosomal S6 kinase (RSK). ORF45 was shown to be a kinase activator in cells but a kinase inhibitor in vitro, and its effects on the ERK-RSK complex are unknown. Here, we demonstrate that ORF45 binds ERK and RSK using optimized linear binding motifs. The crystal structure of the ORF45-ERK2 complex shows how kinase docking motifs recognize the activated form of ERK. The crystal structure of the ORF45-RSK2 complex reveals an AGC kinase docking system, for which we provide evidence that it is functional in the host. We find that ORF45 manipulates ERK-RSK signaling by favoring the formation of a complex, in which activated kinases are better protected from phosphatases and docking motif-independent RSK substrate phosphorylation is selectively up-regulated. As such, our data suggest that ORF45 interferes with the natural design of kinase docking systems in the host.
Assuntos
Cristalografia por Raios X/métodos , Herpesvirus Humano 8/metabolismo , Proteínas Imediatamente Precoces/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/química , Proteínas Quinases S6 Ribossômicas 90-kDa/química , Sarcoma de Kaposi/metabolismo , Linhagem Celular , Biologia Computacional , Herpesvirus Humano 8/química , Herpesvirus Humano 8/isolamento & purificação , Humanos , Proteínas Imediatamente Precoces/química , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Fosforilação , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Sarcoma de Kaposi/patologia , Sarcoma de Kaposi/virologia , Transdução de SinaisRESUMO
Arrestin binding to active phosphorylated G protein-coupled receptors terminates G protein coupling and initiates another wave of signaling. Among the effectors that bind directly to receptor-associated arrestins are extracellular signal-regulated kinases 1/2 (ERK1/2), which promote cellular proliferation and survival. Arrestins may also engage ERK1/2 in isolation in a pre- or post-signaling complex that is likely in equilibrium with the full signal initiation complex. Molecular details of these binary complexes remain unknown. Here, we investigate the molecular mechanisms whereby arrestin-2 and arrestin-3 (a.k.a. ß-arrestin1 and ß-arrestin2, respectively) engage ERK1/2 in pairwise interactions. We find that purified arrestin-3 binds ERK2 more avidly than arrestin-2. A combination of biophysical techniques and peptide array analysis demonstrates that the molecular basis in this difference of binding strength is that the two non-visual arrestins bind ERK2 via different parts of the molecule. We propose a structural model of the ERK2-arrestin-3 complex in solution using size-exclusion chromatography coupled to small angle X-ray scattering (SEC-SAXS). This binary complex exhibits conformational heterogeneity. We speculate that this drives the equilibrium either toward the full signaling complex with receptor-bound arrestin at the membrane or toward full dissociation in the cytoplasm. As ERK1/2 regulates cell migration, proliferation, and survival, understanding complexes that relate to its activation could be exploited to control cell fate.
Assuntos
Proteína Quinase 1 Ativada por Mitógeno , beta-Arrestina 1 , beta-Arrestina 2 , Proteína Quinase 1 Ativada por Mitógeno/química , Ligação Proteica , Espalhamento a Baixo Ângulo , Difração de Raios X , beta-Arrestina 1/química , beta-Arrestina 2/químicaRESUMO
Aberrant activation of the mitogen-activated protein kinase pathway frequently drives tumor growth, and the ERK1/2 kinases are positioned at a key node in this pathway, making them important targets for therapeutic intervention. Recently, a number of ERK1/2 inhibitors have been advanced to investigational clinical trials in patients with activating mutations in B-Raf proto-oncogene or Ras. Here, we describe the discovery of the clinical candidate ASTX029 (15) through structure-guided optimization of our previously published isoindolinone lead (7). The medicinal chemistry campaign focused on addressing CYP3A4-mediated metabolism and maintaining favorable physicochemical properties. These efforts led to the identification of ASTX029, which showed the desired pharmacological profile combining ERK1/2 inhibition with suppression of phospho-ERK1/2 (pERK) levels, and in addition, it possesses suitable preclinical pharmacokinetic properties predictive of once daily dosing in humans. ASTX029 is currently in a phase I-II clinical trial in patients with advanced solid tumors.
Assuntos
Antineoplásicos/uso terapêutico , Indóis/uso terapêutico , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Pirimidinas/uso terapêutico , Animais , Antineoplásicos/síntese química , Antineoplásicos/metabolismo , Antineoplásicos/farmacocinética , Cristalografia por Raios X , Cães , Humanos , Indóis/síntese química , Indóis/metabolismo , Indóis/farmacocinética , Masculino , Camundongos Endogâmicos BALB C , Proteína Quinase 1 Ativada por Mitógeno/química , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Estrutura Molecular , Fosforilação/efeitos dos fármacos , Ligação Proteica , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacocinética , Proto-Oncogene Mas , Pirimidinas/síntese química , Pirimidinas/metabolismo , Pirimidinas/farmacocinética , Ratos Sprague-Dawley , Ratos Wistar , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Nasopharyngeal carcinoma (NPC) originates in the nasopharynx epithelium. Although concurrent chemoradiation therapy followed by chemotherapy is considered as an effective treatment, there is substantial drug resistance in locally advanced NPC patients. One major contributor to the chemoresistance includes aberrant expression of cell adhesion molecules, such as integrin α and ß subunits, giving rise to cell adhesion-mediated drug resistance. Thus, the aim of this study was to investigate the effect of integrin α5 on the development of intrinsic cisplatin resistance in NPC and the associated underlying mechanisms using in vitro three-dimensional (3D) spheroid models, as well as induced cisplatin-resistant NPC (NPCcisR). We demonstrated that established 3D highly- (5-8F) and lowly- (6-10B) metastatic NPC spheroids overexpressed integrin α5 and aggravated their resistance to cisplatin. Besides, enhanced integrin α5 resulted in substantially reduced growth, corresponding to G0/G1 and G2/M cell cycle arrest. In addition, 5-8FcisR and 6-10BcisR cells in 3D forms synergistically strengthened endurance of their spheroids to cisplatin treatment as observed by increased resistance index (RI) and decreased apoptosis. Mechanistically, the aberrantly expressed integrin α5 decreased drug susceptibility in NPC spheroids by inactivating ERK and inhibition of caspase-3 inducing apoptosis. Furthermore, the effect of integrin α5 inducing intrinsic resistance was verified via treatment with ATN-161, a peptide inhibitor for integrin α5ß1. The results showed dramatic reduction in integrin α5 expression, reversal of ERK phosphorylation and caspase-3 cleavage, together with elevated cisplatin sensitivity, indicating regulation of innate drug resistance via integrin α5. Taken together, our findings suggest that integrin α5 could act as a promising target to enhance the chemotherapeutic sensitivity in NPC.
Assuntos
Apoptose , Caspase 3/química , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos , Integrina alfa5/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/química , Carcinoma Nasofaríngeo/patologia , Esferoides Celulares/patologia , Antineoplásicos/farmacologia , Caspase 3/genética , Caspase 3/metabolismo , Técnicas de Cultura de Células , Pontos de Checagem do Ciclo Celular , Humanos , Integrina alfa5/genética , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Carcinoma Nasofaríngeo/tratamento farmacológico , Carcinoma Nasofaríngeo/metabolismo , Neoplasias Nasofaríngeas/tratamento farmacológico , Neoplasias Nasofaríngeas/metabolismo , Neoplasias Nasofaríngeas/secundário , Fosforilação , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismoRESUMO
We develop an approach by which reliable estimates of the transfer entropy can be obtained from the variance-covariance matrix of atomic fluctuations, which converges quickly and retains sensitivity to the full chemical profile of the biomolecular system. We validate our method on ERK2, a well-studied kinase involved in the MAPK signaling cascade for which considerable computational, experimental, and mutation data are available. We present the results of transfer entropy analysis on data obtained from molecular dynamics simulations of wild-type active and inactive ERK2, along with mutants Q103A, I84A, L73P, and G83A. We show that our method is systematically consistent within the context of other approaches for calculating transfer entropy, and we provide a method for interpreting networks of interconnected residues in the protein from a perspective of allosteric coupling. We introduce new insights about possible allosteric activity of the extreme N-terminal region of the kinase, and we describe evidence that suggests that activation may occur by different paths or routes in different mutants. Our results highlight systematic advantages and disadvantages of each method for calculating transfer entropy and show the important role of transfer entropy analysis for understanding allosteric behavior in biomolecular systems.
Assuntos
Entropia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Regulação Alostérica , Substituição de Aminoácidos , Proteína Quinase 1 Ativada por Mitógeno/química , Simulação de Dinâmica Molecular , Conformação ProteicaRESUMO
BACKGROUND: A novel type of noncoding RNA, circRNA has been reported to participate in the occurrence and development of diseases through many mechanisms. The MAPK pathway is a common signal transduction pathway involved in cell proliferation, inflammation and apoptosis and plays a particularly important role in cancers. However, the role of circRNAs related to the MAPK pathway in gastric cancer has not been explored. METHODS: A bioinformatics analysis was performed to profile and identify the circRNAs involved in the MAPK pathway in gastric cancer. The tumor-suppressive role of circMAPK1 was confirmed both in vitro and in vivo. Mass spectrometry, Western blot and immunofluorescence staining assays were used to validate the existence and expression of MAPK1-109aa. The molecular mechanism of circMAPK1 was investigated by mass spectrometry and immunoprecipitation analyses. RESULTS: In this study, we identified that circMAPK1 (hsa_circ_0004872) was downregulated in gastric cancer tissues compared with adjacent normal tissues. Importantly, lower circMAPK1 expression predicted poor survival in GC patients. CircMAPK1 inhibited the proliferation and invasion of gastric cancer cells in vitro and in vivo. Next, we found that circMAPK1 encoded a novel protein with 109 amino acids in length. Through a series of functional experiments, we confirmed that circMAPK1 exerted a tumor-suppressing effect via the encoded protein MAPK1-109aa. Mechanistically, the tumor suppressor MAPK1-109aa inhibited the phosphorylation of MAPK1 by competitively binding to MEK1, thereby suppressing the activation of MAPK1 and its downstream factors in MAPK pathway. CONCLUSIONS: Our study revealed that circMAPK1 inhibits the malignant biological behavior of gastric cancer cells through its encoded protein MAPK1-109aa. More importantly, circMAPK1 is a favorable predictor for gastric cancer patients and may provide a new therapeutic target in the treatment of gastric cancer.
Assuntos
Biomarcadores Tumorais , Sistema de Sinalização das MAP Quinases , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , RNA Circular/genética , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Adulto , Idoso , Sequência de Aminoácidos , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Suscetibilidade a Doenças , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Proteína Quinase 1 Ativada por Mitógeno/química , Metástase Neoplásica , Estadiamento de Neoplasias , Fosforilação , Neoplasias Gástricas/patologia , Carga TumoralRESUMO
Constitutively active extracellular signal-regulated kinase (ERK) 1/2 signaling promotes cancer cell proliferation and survival. We previously described a class of compounds containing a 1,1-dioxido-2,5-dihydrothiophen-3-yl 4-benzenesulfonate scaffold that targeted ERK2 substrate docking sites and selectively inhibited ERK1/2-dependent functions, including activator protein-1-mediated transcription and growth of cancer cells containing active ERK1/2 due to mutations in Ras G-proteins or BRAF, Proto-oncogene B-RAF (Rapidly Acclerated Fibrosarcoma) kinase. The current study identified chemical features required for biologic activity and global effects on gene and protein levels in A375 melanoma cells containing mutant BRAF (V600E). Saturation transfer difference-NMR and mass spectrometry analyses revealed interactions between a lead compound (SF-3-030) and ERK2, including the formation of a covalent adduct on cysteine 252 that is located near the docking site for ERK/FXF (DEF) motif for substrate recruitment. Cells treated with SF-3-030 showed rapid changes in immediate early gene levels, including DEF motif-containing ERK1/2 substrates in the Fos family. Analysis of transcriptome and proteome changes showed that the SF-3-030 effects overlapped with ATP-competitive or catalytic site inhibitors of MAPK/ERK Kinase 1/2 (MEK1/2) or ERK1/2. Like other ERK1/2 pathway inhibitors, SF-3-030 induced reactive oxygen species (ROS) and genes associated with oxidative stress, including nuclear factor erythroid 2-related factor 2 (NRF2). Whereas the addition of the ROS inhibitor N-acetyl cysteine reversed SF-3-030-induced ROS and inhibition of A375 cell proliferation, the addition of NRF2 inhibitors has little effect on cell proliferation. These studies provide mechanistic information on a novel chemical scaffold that selectively regulates ERK1/2-targeted transcription factors and inhibits the proliferation of A375 melanoma cells through a ROS-dependent mechanism. SIGNIFICANCE STATEMENT: Constitutive activation of the extracellular signal-regulated kinase (ERK1/2) pathway drives the proliferation and survival of many cancer cell types. Given the diversity of cellular functions regulated by ERK1/2, the current studies have examined the mechanism of a novel chemical scaffold that targets ERK2 near a substrate binding site and inhibits select ERK functions. Using transcriptomic and proteomic analyses, we provide a mechanistic basis for how this class of compounds inhibits melanoma cells containing mutated BRAF and active ERK1/2.
Assuntos
Antineoplásicos/química , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Melanoma/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Estresse Oxidativo , Antineoplásicos/farmacologia , Domínio Catalítico , Proliferação de Células/efeitos dos fármacos , Células HeLa , Humanos , Células Jurkat , Proteína Quinase 1 Ativada por Mitógeno/química , Ligação Proteica , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas B-raf/genéticaRESUMO
CD40-activated CD40L reverse signaling is a major physiological regulator of axon and dendrite growth from developing hippocampal pyramidal neurons. Here we have studied how CD40L-mediated reverse signaling promotes the growth of these processes. Cultures of hippocampal pyramidal neurons were established from Cd40-/- mouse embryos to eliminate endogenous CD40/CD40L signaling, and CD40L reverse signaling was stimulated by a CD40-Fc chimera. CD40L reverse signaling increased phosphorylation and hence activation of proteins in the PKC, ERK, and JNK signaling pathways. Pharmacological activators and inhibitors of these pathways revealed that whereas activation of JNK inhibited growth, activation of PKC and ERK1/ERK2 enhanced growth. Experiments using combinations of pharmacological reagents revealed that these signaling pathways regulate growth by functioning as an interconnected and interdependent network rather than acting in a simple linear sequence. Immunoprecipitation studies suggested that stimulation of CD40L reverse signaling generated a receptor complex comprising CD40L, PKCß, and the Syk tyrosine kinase. Our studies have begun to elucidate the molecular network and interactions that promote axon and dendrite growth from developing hippocampal neurons following activation of CD40L reverse signaling.
Assuntos
Axônios/metabolismo , Ligante de CD40/metabolismo , Dendritos/fisiologia , Transdução de Sinais , Animais , Butadienos/farmacologia , Antígenos CD40/deficiência , Antígenos CD40/genética , Células Cultivadas , Dendritos/efeitos dos fármacos , Hipocampo/citologia , Hipocampo/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/química , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/química , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/química , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Nitrilas/farmacologia , Fosforilação , Domínios e Motivos de Interação entre Proteínas , Proteína Quinase C/metabolismo , Transdução de Sinais/efeitos dos fármacos , Quinase Syk/metabolismoRESUMO
The adhesion G-protein-coupled receptor (GPCR) latrophilin 3 (ADGRL3) has been associated with increased risk of attention deficit hyperactivity disorder (ADHD) and substance use in human genetic studies. Knockdown in multiple species leads to hyperlocomotion and altered dopamine signaling. Thus, ADGRL3 is a potential target for treatment of neuropsychiatric disorders that involve dopamine dysfunction, but its basic signaling properties are poorly understood. Identification of adhesion GPCR signaling partners has been limited by a lack of tools to acutely activate these receptors in living cells. Here, we design a novel acute activation strategy to characterize ADGRL3 signaling by engineering a receptor construct in which we could trigger acute activation enzymatically. Using this assay, we found that ADGRL3 signals through G12/G13 and Gq, with G12/13 the most robustly activated. Gα12/13 is a new player in ADGRL3 biology, opening up unexplored roles for ADGRL3 in the brain. Our methodological advancements should be broadly useful in adhesion GPCR research.
Assuntos
Fator 6 Ativador da Transcrição/metabolismo , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/metabolismo , Peptídeos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Peptídeos/metabolismo , Fator 6 Ativador da Transcrição/agonistas , Fator 6 Ativador da Transcrição/química , Fator 6 Ativador da Transcrição/genética , Animais , Arrestina/química , Arrestina/genética , Arrestina/metabolismo , Sistemas CRISPR-Cas , Engenharia Celular , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/química , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/química , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Expressão Gênica , Células HEK293 , Humanos , Cinética , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/química , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/química , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Peptídeos/química , Peptídeos/farmacologia , Ligação Proteica , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Receptores de Peptídeos/química , Receptores de Peptídeos/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de SinaisRESUMO
The activation loop segment in protein kinases is a common site for regulatory phosphorylation. In extracellular signal-regulated kinase 2 (ERK2), dual phosphorylation and conformational rearrangement of the activation loop accompany enzyme activation. X-ray structures show the active conformation to be stabilized by multiple ion pair interactions between phosphorylated threonine and tyrosine residues in the loop and six arginine residues in the kinase core. Despite the extensive salt bridge network, nuclear magnetic resonance Carr-Purcell-Meiboom-Gill relaxation dispersion experiments show that the phosphorylated activation loop is conformationally mobile on a microsecond to millisecond time scale. The dynamics of the loop match those of previously reported global exchange within the kinase core region and surrounding the catalytic site that have been found to facilitate productive nucleotide binding. Mutations in the core region that alter these global motions also alter the dynamics of the activation loop. Conversely, mutations in the activation loop perturb the global exchange within the kinase core. Together, these findings provide evidence for coupling between motions in the activation loop and those surrounding the catalytic site in the active state of the kinase. Thus, the activation loop segment in dual-phosphorylated ERK2 is not held statically in the active X-ray conformation but instead undergoes exchange between conformers separated by a small energetic barrier, serving as part of a dynamic allosteric network controlling nucleotide binding and catalytic function.
Assuntos
Proteína Quinase 1 Ativada por Mitógeno/química , Animais , Cristalografia por Raios X , Ativação Enzimática , Modelos Moleculares , Movimento (Física) , Ressonância Magnética Nuclear Biomolecular , Fosforilação , Conformação Proteica , RatosRESUMO
Mitogen-activated protein kinases (MAPKs) control essential eukaryotic signaling pathways. While much has been learned about MAPK activation, much less is known about substrate recruitment and specificity. MAPK substrates may be other kinases that are crucial to promote a further diversification of the signaling outcomes. Here, we used a variety of molecular and cellular tools to investigate the recruitment of two substrate kinases, RSK1 and MK2, to three MAPKs (ERK2, p38α, and ERK5). Unexpectedly, we identified that kinase heterodimers form structurally and functionally distinct complexes depending on the activation state of the MAPK. These may be incompatible with downstream signaling, but naturally they may also form structures that are compatible with the phosphorylation of the downstream kinase at the activation loop, or alternatively at other allosteric sites. Furthermore, we show that small-molecule inhibitors may affect the quaternary arrangement of kinase heterodimers and thus influence downstream signaling in a specific manner.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/química , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Ativação Enzimática , Células HEK293 , Humanos , Espectroscopia de Ressonância Magnética , Proteína Quinase 1 Ativada por Mitógeno/química , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 14 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 14 Ativada por Mitógeno/química , Proteína Quinase 14 Ativada por Mitógeno/genética , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Proteína Quinase 7 Ativada por Mitógeno/química , Proteína Quinase 7 Ativada por Mitógeno/genética , Proteína Quinase 7 Ativada por Mitógeno/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Fosforilação , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Multimerização Proteica , Estrutura Quaternária de Proteína , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
Although intrinsically disordered proteins and intrinsically disordered regions (IDRs) in folded proteins are not able to form stable structures, it is known that they play critically important roles in various biological processes. However, despite multiple studies, the molecular mechanisms of their functions remain not fully understood. In this work, we theoretically investigate the role of IDRs in acceleration of protein-protein association processes. Our hypothesis is that, in protein pairs with several independent binding sites, the association process goes faster if some of these binding sites are located on IDRs or connected by IDRs. To test this idea, we employed analytical modeling, numerical calculations, and Brownian dynamics computer simulations to calculate protein-protein association reaction rates for the ERK2-EtsΔ138 system, belonging to the RAS-RAF-MEK-ERK signaling pathway in living cells. It is found that putting a binding site on IDR accelerates the association process by a factor of 3 to 4. Possible molecular explanations for these observations are presented, and other systems that might use this mechanism are also mentioned.
Assuntos
Proteínas Intrinsicamente Desordenadas/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Sítios de Ligação , Humanos , Proteínas Intrinsicamente Desordenadas/química , Proteína Quinase 1 Ativada por Mitógeno/química , Simulação de Dinâmica Molecular , Ligação Proteica , Domínios Proteicos , Estrutura Terciária de Proteína , Transdução de SinaisRESUMO
Protein kinases (PKs) are allosteric enzymes that play an essential role in signal transduction by regulating a variety of key cellular processes. Most PKs suffer conformational rearrangements upon phosphorylation that strongly enhance the catalytic activity. Generally, it involves the movement of the phosphorylated loop toward the active site and the rotation of the whole C-terminal lobe. However, not all kinases undergo such a large configurational change: The MAPK extracellular signal-regulated protein kinases ERK1 and ERK2 achieve a 50â¯000 fold increase in kinase activity with only a small motion of the C-terminal region. In the present work, we used a combination of molecular simulation tools to characterize the conformational landscape of ERK2 in the active (phosphorylated) and inactive (unphosphorylated) states in solution in agreement with NMR experiments. We show that the chemical reaction barrier is strongly dependent on ATP conformation and that the "active" low-barrier configuration is subtly regulated by phosphorylation, which stabilizes a key salt bridge between the conserved Lys52 and Glu69 belonging to helix-C and promotes binding of a second Mg ion. Our study highlights that the on-off switch embedded in the kinase fold can be regulated by small, medium, and large conformational changes.
Assuntos
Proteína Quinase 1 Ativada por Mitógeno/química , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Sequência Conservada , Dissulfetos/química , Ativação Enzimática , Simulação de Dinâmica Molecular , Fosforilação , Conformação ProteicaRESUMO
Sepsis-associated encephalopathy (SAE) is a potentially irreversible acute cognitive dysfunction with unclear mechanism. Striatal-enriched protein tyrosine phosphatase (STEP) is a brain-specific phosphatase which normally opposes synaptic strengthening by regulating key signaling molecules involved in synaptic plasticity and neuronal function. Thus, we hypothesized that abnormal STEP signaling pathway was involved in sepsis-induced cognitive impairment evoked by lipopolysaccharides (LPS) injection. The levels of STEP, phosphorylation of GluN2B (pGluN2B), the kinases extracellular signal-regulated kinase 1/2 (pERK), cAMP-response element binding protein (CREB), synaptophysin, brain derived neurotrophic factor (BDNF), and post-synaptic density protein 95 (PSD95) in the hippocampus, prefrontal cortex, and striatum were determined at the indicated time points. In the present study, we found that STEP levels were significantly increased in the hippocampus, prefrontal cortex, and striatum following LPS injection, which might resulted from the disruption of the ubiquitin-proteasome system. Notably, a STEP inhibitor TC-2153 treatment alleviated sepsis-induced memory impairment by increasing phosphorylation of GluN2B and ERK1/2, CREB/BDNF, and PSD95. In summary, our results support the key role of STEP in sepsis-induced memory impairment in a mouse model of SAE, whereas inhibition of STEP may provide a novel therapeutic approach for this disorder and possible other neurodegenerative diseases.
Assuntos
Transtornos da Memória/fisiopatologia , Proteínas Tirosina Fosfatases não Receptoras/metabolismo , Encefalopatia Associada a Sepse/fisiopatologia , Transdução de Sinais/fisiologia , Animais , Benzotiepinas/farmacologia , Fator Neurotrófico Derivado do Encéfalo/química , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Corpo Estriado/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/química , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteína 4 Homóloga a Disks-Large/química , Proteína 4 Homóloga a Disks-Large/metabolismo , Hipocampo/metabolismo , Lipopolissacarídeos , Masculino , Memória/efeitos dos fármacos , Memória/fisiologia , Transtornos da Memória/induzido quimicamente , Camundongos Endogâmicos C57BL , Proteína Quinase 1 Ativada por Mitógeno/química , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/química , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação/efeitos dos fármacos , Córtex Pré-Frontal/metabolismo , Proteínas Tirosina Fosfatases não Receptoras/antagonistas & inibidores , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/metabolismo , Encefalopatia Associada a Sepse/induzido quimicamente , Transdução de Sinais/efeitos dos fármacosRESUMO
Extracellular signal-regulated kinases (ERK1/2) are mitogen-activated protein kinases (MAPKs) that play a pro-tumorigenic role in numerous cancers. ERK1/2 possess two protein-docking sites that are distinct from the active site: the D-recruitment site (DRS) and the F-recruitment site. These docking sites facilitate substrate recognition, intracellular localization, signaling specificity, and protein complex assembly. Targeting these sites on ERK in a therapeutic context may overcome many problems associated with traditional ATP-competitive inhibitors. Here, we identified a new class of inhibitors that target the ERK DRS by screening a synthetic combinatorial library of more than 30 million compounds. The screen detects the competitive displacement of a fluorescent peptide from the DRS of ERK2. The top molecular scaffold from the screen was optimized for structure-activity relationship by positional scanning of different functional groups. This resulted in 10 compounds with similar binding affinities and a shared core structure consisting of a tertiary amine hub with three functionalized cyclic guanidino branches. Compound 2507-1 inhibited ERK2 from phosphorylating a DRS-targeting substrate and prevented the phosphorylation of ERK2 by a constitutively active MEK1 (MAPK/ERK kinase 1) mutant. Interaction between an analogue, 2507-8, and the ERK2 DRS was confirmed by nuclear magnetic resonance and X-ray crystallography. 2507-8 forms critical interactions at the common docking domain residue Asp319 via an arginine-like moiety that is shared by all 10 hits, suggesting a common binding mode. The structural and biochemical insights reported here provide the basis for developing new ERK inhibitors that are not ATP-competitive but instead function by disrupting critical protein-protein interactions.
Assuntos
Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Ativação Enzimática , Guanidina/farmacologia , Humanos , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/química , Ressonância Magnética Nuclear Biomolecular , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Especificidade por SubstratoRESUMO
Aberrant activation of the extracellular signalregulated kinases (ERKs)/ribosomal S6 kinase 2 (RSK2) signaling pathway is frequently determined in various human tumor types, including liver cancer, and has been considered as a promising target for cancer chemoprevention and therapy. In the present study, using computeraided virtual screening and molecular docking, isobavachalcone (IBC), a natural chalcone compound, was identified to be an ATPcompetitive inhibitor targeting ERK1/2 and RSK2. Cell Counting Kit8, EdU incorporation and colony formation assays were used to detect the effects of IBC on cell viability and proliferation, and the results demonstrated that IBC effectively inhibited the proliferation of liver cancer HepG2 and Hep3B cells, whereas it had no notable cytotoxic effect on immortal liver L02 cells. Flow cytometric analysis and western blotting further revealed that IBC caused significant levels of apoptosis on liver cancer cells via the caspasedependent mitochondria pathway. The computer prediction was confirmed with pulldown and in vitro kinase assays, in which IBC directly bound with ERK1/2 and RSK2, and dosedependently blocked RSK2 kinase activity in liver cancer cells. Treatment of HepG2 or Hep3B cells with IBC significantly attenuated epidermal growth factorinduced phosphorylation of RSK2 and resulted in the reduced activation of its downstream substrates including cAMP response elementbinding protein, activating transcription factor 1, histone H3 and activating protein1. Enforced RSK2 expression in L02 cells could increase the effect of IBC on suppressing cell growth. Conversely, knockdown of RSK2 reduced the inhibitory effect of IBC on HepG2 cell proliferation. Overall, the present data indicated that ERKs/RSK2 signaling serves a pivotal role in IBCinduced suppression of liver cancer cells and that IBC may be a potential therapeutic candidate for human cancer with elevated ERKs/RSK2 activity.