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1.
Nature ; 574(7776): 103-107, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31511700

RESUMO

The sequencing of ancient DNA has enabled the reconstruction of speciation, migration and admixture events for extinct taxa1. However, the irreversible post-mortem degradation2 of ancient DNA has so far limited its recovery-outside permafrost areas-to specimens that are not older than approximately 0.5 million years (Myr)3. By contrast, tandem mass spectrometry has enabled the sequencing of approximately 1.5-Myr-old collagen type I4, and suggested the presence of protein residues in fossils of the Cretaceous period5-although with limited phylogenetic use6. In the absence of molecular evidence, the speciation of several extinct species of the Early and Middle Pleistocene epoch remains contentious. Here we address the phylogenetic relationships of the Eurasian Rhinocerotidae of the Pleistocene epoch7-9, using the proteome of dental enamel from a Stephanorhinus tooth that is approximately 1.77-Myr old, recovered from the archaeological site of Dmanisi (South Caucasus, Georgia)10. Molecular phylogenetic analyses place this Stephanorhinus as a sister group to the clade formed by the woolly rhinoceros (Coelodonta antiquitatis) and Merck's rhinoceros (Stephanorhinus kirchbergensis). We show that Coelodonta evolved from an early Stephanorhinus lineage, and that this latter genus includes at least two distinct evolutionary lines. The genus Stephanorhinus is therefore currently paraphyletic, and its systematic revision is needed. We demonstrate that sequencing the proteome of Early Pleistocene dental enamel overcomes the limitations of phylogenetic inference based on ancient collagen or DNA. Our approach also provides additional information about the sex and taxonomic assignment of other specimens from Dmanisi. Our findings reveal that proteomic investigation of ancient dental enamel-which is the hardest tissue in vertebrates11, and is highly abundant in the fossil record-can push the reconstruction of molecular evolution further back into the Early Pleistocene epoch, beyond the currently known limits of ancient DNA preservation.


Assuntos
DNA Antigo/análise , Esmalte Dentário/metabolismo , Fósseis , Perissodáctilos/classificação , Perissodáctilos/genética , Filogenia , Proteoma/genética , Proteômica , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Teorema de Bayes , História Antiga , Humanos , Masculino , Perissodáctilos/metabolismo , Fosforilação/genética , Proteoma/análise
2.
Nat Commun ; 10(1): 3090, 2019 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-31300647

RESUMO

The role of brain somatic mutations in Alzheimer's disease (AD) is not well understood. Here, we perform deep whole-exome sequencing (average read depth 584×) in 111 postmortem hippocampal formation and matched blood samples from 52 patients with AD and 11 individuals not affected by AD. The number of somatic single nucleotide variations (SNVs) in AD brain specimens increases significantly with aging, and the rate of mutation accumulation in the brain is 4.8-fold slower than that in AD blood. The putatively pathogenic brain somatic mutations identified in 26.9% (14 of 52) of AD individuals are enriched in PI3K-AKT, MAPK, and AMPK pathway genes known to contribute to hyperphosphorylation of tau. We show that a pathogenic brain somatic mutation in PIN1 leads to a loss-of-function mutation. In vitro mimicking of haploinsufficiency of PIN1 aberrantly increases tau phosphorylation and aggregation. This study provides new insights into the genetic architecture underlying the pathogenesis of AD.


Assuntos
Doença de Alzheimer/genética , Peptidilprolil Isomerase de Interação com NIMA/genética , Agregação Patológica de Proteínas/genética , Proteínas tau/metabolismo , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/genética , Doença de Alzheimer/patologia , Animais , Linhagem Celular Tumoral , Feminino , Técnicas de Silenciamento de Genes , Haploinsuficiência , Hipocampo/citologia , Hipocampo/patologia , Humanos , Mutação com Perda de Função , Masculino , Camundongos , Pessoa de Meia-Idade , Taxa de Mutação , Peptidilprolil Isomerase de Interação com NIMA/metabolismo , Neurônios , Fosforilação/genética , Polimorfismo de Nucleotídeo Único , Agregação Patológica de Proteínas/patologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sequenciamento Completo do Exoma
3.
Cancer Invest ; 37(4-5): 199-208, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31181967

RESUMO

Prostate cancer (PCa) is the most common malignant tumor for men. But the mechanism is unclear. EIF3C was shown to be overexpressed in PCa tissues and cell lines. EIF3C overexpression was correlated to age and tumor stage in PCa patients and indicated poor survival. The proliferation, migration, and invasiveness of PC3 cells were all inhibited after EIF3C knockdown. Additionally, the phosphorylation level of PI3K and Akt was downregulated while total NF-κB and Myc decreased after EIF3C knockdown. But the expression of IκB increased reversely. Therefore, EIF3C at least partially regulates the activity of PI3K/Akt/NF-κB signaling pathway in PC3 cells.


Assuntos
Carcinogênese/genética , Fator de Iniciação 3 em Eucariotos/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias da Próstata/genética , Animais , Carcinogênese/patologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Regulação para Baixo , Fator de Iniciação 3 em Eucariotos/genética , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/genética , Prognóstico , Neoplasias da Próstata/mortalidade , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proto-Oncogenes , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/genética , Taxa de Sobrevida , Ensaios Antitumorais Modelo de Xenoenxerto
4.
PLoS Genet ; 15(6): e1008206, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31194741

RESUMO

The septation initiation network (SIN), composed of a conserved SepH (Cdc7p) kinase cascade, plays an essential role in fungal cytokinesis/septation and conidiation for asexual reproduction, while the mitogen-activated protein kinase (MAPK) pathway depends on successive signaling cascade phosphorylation to sense and respond to stress and environmental factors. In this study, a SepH suppressor-PomA in the filamentous fungus A. nidulans is identified as a negative regulator of septation and conidiation such that the pomA mutant is able to cure defects of sepH1 in septation and conidiation and overexpression of pomA remarkably suppresses septation. Under the normal cultural condition, SepH positively regulates the phosphorylation of MAPK-HogA, while PomA reversely affects this process. In the absence of PbsB (MAPKK, a putative upstream member of HogA), PomA and SepH are unable to affect the phosphorylation level of HogA. Under the osmostress condition, the induced phosphorylated HogA is capable of bypassing the requirement of SepH, a key player for early events during cytokinesis but not for MobA/SidB, the last one in the core SIN protein kinase cascade, indicating the osmotic stimuli-induced septation is capable of bypassing requirement of SepH but unable to bypass the whole SIN requirement. Findings demonstrate that crosstalk exists between the SIN and MAPK pathways. PomA and SepH indirectly regulate HogA phosphorylation through affecting HogA-P upstream kinases.


Assuntos
Aspergillus nidulans/genética , Proteínas Fúngicas/genética , Proteínas Quinases Ativadas por Mitógeno/genética , Reprodução Assexuada/genética , Aspergillus nidulans/crescimento & desenvolvimento , Proteínas de Ciclo Celular/genética , Citocinese/genética , Mutação/genética , Proteínas Nucleares/genética , Pressão Osmótica , Fosforilação/genética , Polimorfismo de Nucleotídeo Único/genética , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , Schizosaccharomyces/genética , Schizosaccharomyces/crescimento & desenvolvimento , Proteínas de Schizosaccharomyces pombe/genética , Transdução de Sinais/genética
5.
Mol Carcinog ; 58(9): 1691-1700, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31237025

RESUMO

Non-melanoma skin cancer frequently results from chronic exposure to ultraviolet (UV) irradiation. UV-induced DNA damage activates cell cycle arrest checkpoints through degradation of the cyclin-dependent kinase activators, the cell division cycle 25 (CDC25) phosphatases. We previously reported increased CDC25A in nonmelanoma skin cancer, but CDC25B and CDC25C had not been previously examined. Consequently, we hypothesized that increased expression of CDC25B and CDC25C increases tumor cell proliferation and skin tumor growth. We found that CDC25B and CDC25C were increased in mouse and human skin cancers. CDC25B was primarily cytoplasmic in skin and skin tumors and was significantly increased in the squamous cell carcinoma (SCC), while CDC25C was mostly nuclear in the skin, with an increased cytoplasmic signal in the premalignant and malignant tumors. Surprisingly, forced expression of CDC25B or CDC25C in cultured SCC cells did not affect proliferation, but instead suppressed apoptosis, while CDC25C silencing increased apoptosis without impacting proliferation. Targeting CDC25C to the nucleus via mutation of its nuclear export sequence, however, increased proliferation in SCC cells. Overexpression of CDC25C in the nuclear compartment did not hinder the ability of CDC25C to suppress apoptosis, neither did mutation of sites necessary for its interaction with 14-3-3 proteins. Analysis of apoptotic signaling pathways revealed that CDC25C increased activating phosphorylation of Akt on Ser473 , increased inhibitory phosphorylation of proapoptotic BAD on Ser136 , and increased the survival protein Survivin. Silencing of CDC25C significantly reduced Survivin levels. Taken together, these data suggest that increased expression of CDC25B or CDC25C are mechanisms by which skin cancers evade apoptotic cell death.


Assuntos
Morte Celular/genética , Neoplasias Cutâneas/genética , Fosfatases cdc25/genética , Animais , Apoptose/genética , Carcinoma de Células Escamosas/genética , Ciclo Celular/genética , Linhagem Celular Tumoral , Núcleo Celular/genética , Proliferação de Células/genética , Citoplasma/genética , Dano ao DNA/genética , Humanos , Camundongos , Camundongos Transgênicos/genética , Fosforilação/genética , Transdução de Sinais/genética
6.
RNA ; 25(9): 1192-1201, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31239298

RESUMO

Protein kinase RNA-activated (PKR) is an interferon-inducible kinase that is potently activated by long double-stranded RNA (dsRNA). In a previous study, we found that snoRNAs exhibit increased association with PKR in response to metabolic stress. While it was unclear if snoRNAs also activated PKR in cells, activation in vitro was observed. snoRNAs do not exhibit the double-stranded character typically required for activation of PKR, but some studies suggest such RNAs can activate PKR if triphosphorylated at the 5' terminus, or if they are able to form intermolecular dimers. To interrogate the mechanism of PKR activation by snoRNAs in vitro we focused on SNORD113. Using multiple methods for defining the 5'-phosphorylation state, we find that activation of PKR by SNORD113 does not require a 5'-triphosphate. Gel purification from a native gel followed by analysis using analytical ultracentrifugation showed that dimerization was also not responsible for activation. We isolated distinct conformers of SNORD113 from a native polyacrylamide gel and tracked the activating species to dsRNA formed from antisense RNA synthesized during in vitro transcription with T7 RNA polymerase. Similar studies with additional snoRNAs and small RNAs showed the generality of our results. Our studies suggest that a 5' triphosphate is not an activating ligand for PKR, and emphasize the insidious nature of antisense contamination.


Assuntos
Ativação Enzimática/genética , Polifosfatos/metabolismo , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Dimerização , Humanos , Ligantes , Fosforilação/genética , Ligação Proteica/genética , RNA de Cadeia Dupla/genética , RNA Nucleolar Pequeno/genética , Transcrição Genética/genética , Ultracentrifugação/métodos , Proteínas Virais/metabolismo
7.
Plant Cell Physiol ; 60(8): 1804-1810, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31119298

RESUMO

While ligand-induced autophosphorylation of receptor-like kinases (RLKs) is known to be critical for triggering the downstream responses, biochemical mechanism by which each phosphorylation site contributes to the initiation of corresponding signaling cascades is only poorly understood, except the involvement of some phosphorylation sites in the regulation of catalytic activity of these RLKs. In this article, we first confirmed that the phosphorylation of S493 of AtCERK1 is involved in the regulation of chitin-induced defense responses by the complementation of an atcerk1 mutant with AtCERK1(S493A) cDNA. In vitro kinase assay with the heterologously expressed kinase domain of AtCERK1, GST-AtCERK1cyt, showed that the S493A mutation did not affect the autophosphorylation of AtCERK1 itself but diminished the transphosphorylation of downstream signaling components, PBL27 and PUB4. On the other hand, a phosphomimetic mutant, GST-AtCERK1(S493D)cyt, transphosphorylated these substrates as similar to the wild type AtCERK1. These results suggested that the phosphorylation of S493 does not contribute to the regulation of catalytic activity but plays an important role for the transphosphorylation of the downstream signaling components, thus contributing to the initiation of chitin signaling. To our knowledge, it is a novel finding that a specific phosphorylation site contributes to the regulation of transphosphorylation activity of RLKs. Further studies on the structural basis by which S493 phosphorylation contributes to the regulation of transphosphorylation would contribute to the understanding how the ligand-induced autophosphorylation of RLKs properly regulates the downstream signaling.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Arabidopsis/fisiologia , Quitina/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Fosforilação/genética , Fosforilação/fisiologia , Imunidade Vegetal/genética , Imunidade Vegetal/fisiologia , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
8.
Biochim Biophys Acta Proteins Proteom ; 1867(7-8): 710-721, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31096047

RESUMO

Members of the casein kinase 1 (CK1) family are involved in regulation of crucial cellular pathways including chromosomal segregation, DNA repair, and apoptosis. Therefore, the activity of CK1 isoforms needs to be tightly regulated in order to avoid pathogenesis of proliferative diseases. Regulation of cellular CK1 activity is mainly mediated by (auto-) phosphorylation within its C-terminal regulatory domain. Cellular kinases, among them protein kinase A (PKA), checkpoint kinase 1 (Chk1), protein kinase C α (PKCα), and cyclin-dependent kinases (CDKs) have already been identified to C-terminally phosphorylate CK1δ, thereby modulating its kinase activity. In the present study we analyzed the CK1δ kinase domain for phosphorylation sites targeted by PKCα. Several phosphorylation sites were identified in vitro by initially using GST-CK1δ wild type and phosphorylation-site mutant protein fragments originating from the CK1δ kinase domain. Residues S53, T176, and S181 could finally be confirmed as targets for PKCα. Determination of kinetic parameters of full-length wild type and mutant GST-CK1δ-mediated substrate phosphorylation revealed that integrity of residue T176 is crucial for maintaining CK1δ kinase activity. Functional biochemical and cell culture-based analysis discovered that site-specific phosphorylation of CK1δ by PKCα contributes to fine-tuning of CK1δ kinase activity. In summary, our work for the first time demonstrates the effects of PKCα-mediated site-specific phosphorylation in the CK1δ kinase domain and enhances our knowledge about the regulation of the disease-associated CK1 kinase family.


Assuntos
Proteína Quinase C-alfa/metabolismo , Proteína Quinase C-delta/metabolismo , Substituição de Aminoácidos , Células HEK293 , Humanos , Mutação de Sentido Incorreto , Fosforilação/genética , Domínios Proteicos , Proteína Quinase C-alfa/genética , Proteína Quinase C-delta/genética
9.
Mol Carcinog ; 58(9): 1701-1710, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31124185

RESUMO

The general transcription factor E2F1 reportedly functions in a protumorigenic manner in several cancer models. We show that the genetic context of cancer cells influence E2F1's role to impede the protumorigenic role. Thirty to fifty percent of melanoma patients carry mutant BRAF with about 90% of mutant BRAF melanomas being V600E mutation. Tissue microarrays from melanoma patients were used to establish an association between E2F1 and BRAFV600E . We show for the first time that low E2F1 levels in BRAFV600E melanomas are associated with lymph node metastasis. Genetic manipulation of E2F1 in BRAFV600E and BRAFwt cells were used to determine its role in malignant melanoma progression by examining effects on migration and invasion. E2F1-mediated negative regulation of myosin light chain kinase (MYLK) increased migration and invasion in BRAFV600E cells by phosphorylating myosin light chain and increased stress fiber formation. We show that E2F1 inhibits extracellular signal-regulated kinase (ERK) activation in BRAFV600E cells and provide evidence for a negative feedback loop between E2F1 and ERK in these cells. This study shows for the first time that E2F1 has a cancer protective role in oncogenic BRAF-activated melanoma cells and that loss of E2F1 can allow disease progression through a novel mechanism of E2F1-mediated MYLK regulation. This study has implications for oncogenic BRAF-activated tumors and resistance to targeted oncogenic BRAF therapy.


Assuntos
Movimento Celular/genética , Fator de Transcrição E2F1/genética , Melanoma/genética , Melanoma/patologia , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Metástase Linfática/genética , Metástase Linfática/patologia , Sistema de Sinalização das MAP Quinases/genética , Mutação/genética , Fosforilação/genética , Proteínas Proto-Oncogênicas B-raf/genética
10.
Mol Cell ; 74(5): 1086-1102.e5, 2019 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-31101498

RESUMO

Kinase and phosphatase overexpression drives tumorigenesis and drug resistance. We previously developed a mass-cytometry-based single-cell proteomics approach that enables quantitative assessment of overexpression effects on cell signaling. Here, we applied this approach in a human kinome- and phosphatome-wide study to assess how 649 individually overexpressed proteins modulated cancer-related signaling in HEK293T cells in an abundance-dependent manner. Based on these data, we expanded the functional classification of human kinases and phosphatases and showed that the overexpression effects include non-catalytic roles. We detected 208 previously unreported signaling relationships. The signaling dynamics analysis indicated that the overexpression of ERK-specific phosphatases sustains proliferative signaling. This suggests a phosphatase-driven mechanism of cancer progression. Moreover, our analysis revealed a drug-resistant mechanism through which overexpression of tyrosine kinases, including SRC, FES, YES1, and BLK, induced MEK-independent ERK activation in melanoma A375 cells. These proteins could predict drug sensitivity to BRAF-MEK concurrent inhibition in cells carrying BRAF mutations.


Assuntos
Carcinogênese/genética , Melanoma/genética , Monoéster Fosfórico Hidrolases/genética , Fosfotransferases/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proliferação de Células/genética , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Melanoma/enzimologia , Melanoma/patologia , Mutação , Fosforilação/genética , Inibidores de Proteínas Quinases/farmacologia , Proteômica , Transdução de Sinais/efeitos dos fármacos
11.
Mol Plant Microbe Interact ; 32(9): 1229-1242, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31012804

RESUMO

The plasma membrane (PM) is at the interface of plant-pathogen interactions and, thus, many bacterial type-III effector (T3E) proteins target membrane-associated processes to interfere with immunity. The Pseudomonas syringae T3E HopZ1a is a host cell PM-localized effector protein that has several immunity-associated host targets but also activates effector-triggered immunity in resistant backgrounds. Although HopZ1a has been shown to interfere with early defense signaling at the PM, no dedicated PM-associated HopZ1a target protein has been identified until now. Here, we show that HopZ1a interacts with the PM-associated remorin protein NbREM4 from Nicotiana benthamiana in several independent assays. NbREM4 relocalizes to membrane nanodomains after treatment with the bacterial elicitor flg22 and transient overexpression of NbREM4 in N. benthamiana induces the expression of a subset of defense-related genes. We can further show that NbREM4 interacts with the immune-related receptor-like cytoplasmic kinase avrPphB-susceptible 1 (PBS1) and is phosphorylated by PBS1 on several residues in vitro. Thus, we conclude that NbREM4 is associated with early defense signaling at the PM. The possible relevance of the HopZ1a-NbREM4 interaction for HopZ1a virulence and avirulence functions is discussed.Copyright © 2019 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.


Assuntos
Proteínas de Bactérias , Proteínas de Transporte , Interações Hospedeiro-Patógeno , Fosfoproteínas , Proteínas de Plantas , Proteínas Serina-Treonina Quinases , Pseudomonas syringae , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Fosfoproteínas/metabolismo , Fosforilação/genética , Proteínas de Plantas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Pseudomonas syringae/metabolismo , Tabaco/enzimologia , Tabaco/microbiologia
12.
Oncol Rep ; 41(6): 3292-3304, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31002345

RESUMO

The ubiquitin­specific protease 9X (USP9X) is a conserved deubiquitinase that has been investigated in several types of human cancer. However, the clinical significance and the biological roles of USP9X in prostate cancer remain unexplored. In the present study, an investigation into the expression and clinical significance of USP9X in prostate cancer revealed that USP9X expression was downregulated in prostate cancer tissues compared with that in healthy tissues. In addition, decreased USP9X expression was associated with a higher Gleason score and local invasion. Depletion of USP9X in prostate cancer LNCaP and PC­3 cells by small interfering RNA promoted cell invasion and migration. Furthermore, USP9X depletion upregulated matrix metalloproteinase 9 (MMP9) and the phosphorylation of dynamin­related protein 1 (DRP1). Notably, a significant increase in phosphorylated extracellular signal­regulated kinase (ERK), an upstream activator of MMP9 and DRP1, was observed. To investigate whether ERK activation was able to increase MMP9 protein levels and induce DRP1 phosphorylation, an ERK inhibitor was used, demonstrating that ERK­mediated MMP9 production and change in mitochondrial function was critical for the biological function of USP9X in prostate cancer cells. In conclusion, the present study demonstrated that USP9X is downregulated in prostate cancer and functions as an inhibitor of tumor cell invasion, possibly through the regulation of the ERK signaling pathway.


Assuntos
GTP Fosfo-Hidrolases/genética , Metaloproteinase 9 da Matriz/genética , Proteínas Associadas aos Microtúbulos/genética , Proteínas Mitocondriais/genética , Neoplasias da Próstata/genética , Ubiquitina Tiolesterase/genética , Idoso , Movimento Celular/genética , Proliferação de Células/genética , Enzimas Desubiquitinantes/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Sistema de Sinalização das MAP Quinases/genética , Masculino , Pessoa de Meia-Idade , Dinâmica Mitocondrial/genética , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Fosforilação/genética , Neoplasias da Próstata/patologia
13.
Mol Cell ; 74(4): 758-770.e4, 2019 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-30982746

RESUMO

The cyclin-dependent kinases Cdk4 and Cdk6 form complexes with D-type cyclins to drive cell proliferation. A well-known target of cyclin D-Cdk4,6 is the retinoblastoma protein Rb, which inhibits cell-cycle progression until its inactivation by phosphorylation. However, the role of Rb phosphorylation by cyclin D-Cdk4,6 in cell-cycle progression is unclear because Rb can be phosphorylated by other cyclin-Cdks, and cyclin D-Cdk4,6 has other targets involved in cell division. Here, we show that cyclin D-Cdk4,6 docks one side of an alpha-helix in the Rb C terminus, which is not recognized by cyclins E, A, and B. This helix-based docking mechanism is shared by the p107 and p130 Rb-family members across metazoans. Mutation of the Rb C-terminal helix prevents its phosphorylation, promotes G1 arrest, and enhances Rb's tumor suppressive function. Our work conclusively demonstrates that the cyclin D-Rb interaction drives cell division and expands the diversity of known cyclin-based protein docking mechanisms.


Assuntos
Proliferação de Células/genética , Ciclina D/genética , Mapas de Interação de Proteínas/genética , Proteína do Retinoblastoma/genética , Ciclo Celular/genética , Proteína Substrato Associada a Crk/genética , Ciclina D/química , Quinase 4 Dependente de Ciclina/química , Quinase 4 Dependente de Ciclina/genética , Quinase 6 Dependente de Ciclina/química , Quinase 6 Dependente de Ciclina/genética , Ciclinas/genética , Fase G1/genética , Humanos , Simulação de Acoplamento Molecular , Fosforilação/genética , Ligação Proteica/genética , Conformação Proteica em alfa-Hélice/genética , Proteína do Retinoblastoma/química , Proteína p107 Retinoblastoma-Like/genética , Fase S/genética
14.
Mol Cell ; 74(4): 742-757.e8, 2019 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-30979586

RESUMO

Disturbances in autophagy and stress granule dynamics have been implicated as potential mechanisms underlying inclusion body myopathy (IBM) and related disorders. Yet the roles of core autophagy proteins in IBM and stress granule dynamics remain poorly characterized. Here, we demonstrate that disrupted expression of the core autophagy proteins ULK1 and ULK2 in mice causes a vacuolar myopathy with ubiquitin and TDP-43-positive inclusions; this myopathy is similar to that caused by VCP/p97 mutations, the most common cause of familial IBM. Mechanistically, we show that ULK1/2 localize to stress granules and phosphorylate VCP, thereby increasing VCP's activity and ability to disassemble stress granules. These data suggest that VCP dysregulation and defective stress granule disassembly contribute to IBM-like disease in Ulk1/2-deficient mice. In addition, stress granule disassembly is accelerated by an ULK1/2 agonist, suggesting ULK1/2 as targets for exploiting the higher-order regulation of stress granules for therapeutic intervention of IBM and related disorders.


Assuntos
Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Doenças por Armazenamento dos Lisossomos/genética , Doenças Musculares/genética , Proteínas Serina-Treonina Quinases/genética , Proteína com Valosina/genética , Adenosina Trifosfatases/genética , Animais , Autofagia/genética , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Humanos , Corpos de Inclusão/genética , Corpos de Inclusão/patologia , Doenças por Armazenamento dos Lisossomos/metabolismo , Doenças por Armazenamento dos Lisossomos/patologia , Camundongos , Doenças Musculares/metabolismo , Doenças Musculares/patologia , Fosforilação/genética , Estresse Fisiológico/genética , Ubiquitina/genética
15.
J Biol Chem ; 294(15): 5935-5944, 2019 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-30819803

RESUMO

X-linked inhibitor of apoptosis protein (XIAP) suppresses apoptosis and plays key roles in the development, growth, migration, and invasion of cancer cells. Therefore, XIAP has recently attracted much attention as a potential antineoplastic therapeutic target, requiring elucidation of the molecular mechanisms underlying its biological activities. Here, using shRNA-mediated gene silencing, immunoblotting, quantitative RT-PCR, anchorage-independent growth assay, and invasive assay, we found that XIAP's RING domain, but not its BIR domain, is crucial for XIAP-mediated up-regulation of c-Myc protein expression in human bladder cancer (BC) cells. Mechanistically, we observed that the RING domain stabilizes c-Myc by inhibiting its phosphorylation at Thr-58 and that this inhibition is due to activated ERK1/2-mediated phosphorylation of glycogen synthase kinase-3ß (GSK-3ß) at Ser-9. Functional studies further revealed that c-Myc protein promotes anchorage-independent growth and invasion stimulated by the XIAP RING domain in human BC cells. Collectively, the findings in our study uncover that the RING domain of XIAP supports c-Myc protein stability, providing insight into the molecular mechanism and role of c-Myc overexpression in cancer progression. Our observations support the notion of targeting XIAP's RING domain and c-Myc in cancer therapy.


Assuntos
Regulação Neoplásica da Expressão Gênica , Proteínas Proto-Oncogênicas c-myc/biossíntese , Neoplasias da Bexiga Urinária/metabolismo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Linhagem Celular Tumoral , Glicogênio Sintase Quinase 3 beta/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Invasividade Neoplásica/genética , Fosforilação/genética , Domínios Proteicos , Estabilidade Proteica , Proteínas Proto-Oncogênicas c-myc/genética , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/patologia , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genética
16.
PLoS Biol ; 17(3): e2006540, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30897078

RESUMO

Specificity within protein kinase signaling cascades is determined by direct and indirect interactions between kinases and their substrates. While the impact of localization and recruitment on kinase-substrate targeting can be readily assessed, evaluating the relative importance of direct phosphorylation site interactions remains challenging. In this study, we examine the STE20 family of protein serine-threonine kinases to investigate basic mechanisms of substrate targeting. We used peptide arrays to define the phosphorylation site specificity for the majority of STE20 kinases and categorized them into four distinct groups. Using structure-guided mutagenesis, we identified key specificity-determining residues within the kinase catalytic cleft, including an unappreciated role for the kinase ß3-αC loop region in controlling specificity. Exchanging key residues between the STE20 kinases p21-activated kinase 4 (PAK4) and Mammalian sterile 20 kinase 4 (MST4) largely interconverted their phosphorylation site preferences. In cells, a reprogrammed PAK4 mutant, engineered to recognize MST substrates, failed to phosphorylate PAK4 substrates or to mediate remodeling of the actin cytoskeleton. In contrast, this mutant could rescue signaling through the Hippo pathway in cells lacking multiple MST kinases. These observations formally demonstrate the importance of catalytic site specificity for directing protein kinase signal transduction pathways. Our findings further suggest that phosphorylation site specificity is both necessary and sufficient to mediate distinct signaling outputs of STE20 kinases and imply broad applicability to other kinase signaling systems.


Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/fisiologia , Quinases Ativadas por p21/metabolismo , Catálise , Linhagem Celular , Humanos , Mutagênese/genética , Mutagênese/fisiologia , Fosforilação/genética , Fosforilação/fisiologia , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais/genética , Quinases Ativadas por p21/genética
17.
Nucleic Acids Res ; 47(9): 4663-4683, 2019 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-30916345

RESUMO

Cleavage factor I mammalian (CFIm) complex, composed of cleavage and polyadenylation specificity factor 5 (CPSF5) and serine/arginine-like protein CPSF6, regulates alternative polyadenylation (APA). Loss of CFIm function results in proximal polyadenylation site usage, shortening mRNA 3' untranslated regions (UTRs). Although CPSF6 plays additional roles in human disease, its nuclear translocation mechanism remains unresolved. Two ß-karyopherins, transportin (TNPO) 1 and TNPO3, can bind CPSF6 in vitro, and we demonstrate here that while the TNPO1 binding site is dispensable for CPSF6 nuclear import, the arginine/serine (RS)-like domain (RSLD) that mediates TNPO3 binding is critical. The crystal structure of the RSLD-TNPO3 complex revealed potential CPSF6 interaction residues, which were confirmed to mediate TNPO3 binding and CPSF6 nuclear import. Both binding and nuclear import were independent of RSLD phosphorylation, though a hyperphosphorylated mimetic mutant failed to bind TNPO3 and mislocalized to the cell cytoplasm. Although hypophosphorylated CPSF6 largely supported normal polyadenylation site usage, a significant number of mRNAs harbored unnaturally extended 3' UTRs, similar to what is observed when other APA regulators, such as CFIIm component proteins, are depleted. Our results clarify the mechanism of CPSF6 nuclear import and highlight differential roles for RSLD phosphorylation in nuclear translocation versus regulation of APA.


Assuntos
Poliadenilação/genética , Conformação Proteica , Proteínas de Ligação a RNA/química , beta Carioferinas/química , Transporte Ativo do Núcleo Celular/genética , Cristalografia por Raios X , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Humanos , Fosforilação/genética , Ligação Proteica/genética , Proteínas Serina-Treonina Quinases/química , Proteínas Tirosina Quinases/química , RNA Mensageiro , Proteínas de Ligação a RNA/genética , beta Carioferinas/genética , Fatores de Poliadenilação e Clivagem de mRNA/química , Fatores de Poliadenilação e Clivagem de mRNA/genética
18.
PLoS Biol ; 17(3): e3000175, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30840614

RESUMO

G protein-coupled receptors (GPCRs) play an important role in physiology and disease and represent the most productive drug targets. Orphan GPCRs, with their endogenous ligands unknown, were considered a source of drug targets and consequently attract great interest to identify their endogenous cognate ligands for deorphanization. However, a contrary view to the ubiquitous existence of endogenous ligands for every GPCR is that there might be a significant overlooked fraction of orphan GPCRs that function constitutively in a ligand-independent manner only. Here, we investigated the evolution of the bombesin receptor-ligand family in vertebrates in which one member-bombesin receptor subtype-3 (BRS3)-is a potential orphan GPCR. With analysis of 17 vertebrate BRS3 structures and 10 vertebrate BRS3 functional data, our results demonstrated that nonplacental vertebrate BRS3 still connects to the original ligands-neuromedin B (NMB) and gastrin-releasing peptide (GRP)-because of adaptive evolution, with significantly changed protein structure, especially in three altered key residues (Q127R, P205S, and R294H) originally involved in ligand binding/activation, whereas the placental mammalian BRS3 lost the binding affinity to NMB/GRP and constitutively activates Gs/Gq/G12 signaling in a ligand-independent manner. Moreover, the N terminus of placental mammalian BRS3 underwent positive selection, exhibiting significant structural differences compared to nonplacental vertebrate BRS3, and this domain plays an important role in constitutive activity of placental mammalian BRS3. In conclusion, constitutively active BRS3 is a genuinely orphan GPCR in placental mammals, including human. To our knowledge, this study identified the first example that might represent a new group of genuinely orphan GPCRs that will never be deorphanized by the discovery of a natural ligand and provided new perspectives in addition to the current ligand-driven GPCR deorphanization.


Assuntos
Receptores da Bombesina/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Animais , Peptídeo Liberador de Gastrina/genética , Peptídeo Liberador de Gastrina/metabolismo , Células HEK293 , Humanos , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Mutação/genética , Neurocinina B/análogos & derivados , Neurocinina B/genética , Neurocinina B/metabolismo , Fosforilação/genética , Fosforilação/fisiologia , Filogenia , Receptores da Bombesina/genética , Receptores Acoplados a Proteínas-G/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia
19.
Mol Carcinog ; 58(7): 1168-1180, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30834575

RESUMO

Ras-association domain family (RASSF) proteins exert distinct cellular functions. The expression of RASSF10 in non-small cell lung cancer and its underlying mechanism have not been reported. Herein, we explored the roles of RASSF10 in lung cancer cells and potential molecular mechanisms. We found low RASSF10 expression in lung cancer specimens, which was associated with low differentiation, advanced pTNM stage, positive lymph node metastasis, and poor prognosis in patients. Furthermore, RASSF10 overexpression inhibited the proliferation and invasion of lung cancer cells, which was the result of Wnt signaling suppression. However, we found that RASSF10 had no influence on Hippo signaling, while RASSF10 bound to LRP6 via the coiled-coil domains and reduced p-LRP6 level, eventually prohibiting ß-catenin nuclear translocation. However, deleting the coiled-coil domains ablated this function. These findings expound the interaction between RASSF10 and LRP6 and uncover a potential link between N-terminal RASSFs and the Wnt pathway.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/patologia , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Neoplasias Pulmonares/patologia , Proteínas Supressoras de Tumor/genética , Via de Sinalização Wnt/genética , Células A549 , Transporte Ativo do Núcleo Celular/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Linhagem Celular Tumoral , Proliferação de Células/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/mortalidade , Metástase Linfática/patologia , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica/genética , Fosforilação/genética , Prognóstico , Ligação Proteica/genética , Proteínas Serina-Treonina Quinases/metabolismo , beta Catenina/metabolismo
20.
Nat Commun ; 10(1): 1242, 2019 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-30886146

RESUMO

The ataxia-telangiectasia mutated (ATM) kinase, an upstream kinase of the DNA damage response (DDR), is rapidly activated following DNA damage, and phosphorylates its downstream targets to launch DDR signaling. However, the mechanism of ATM activation is still not completely understood. Here we report that UFM1 specific ligase 1 (UFL1), an ufmylation E3 ligase, is important for ATM activation. UFL1 is recruited to double strand breaks by the MRE11/RAD50/NBS1 complex, and monoufmylates histone H4 following DNA damage. Monoufmylated histone H4 is important for Suv39h1 and Tip60 recruitment. Furthermore, ATM phosphorylates UFL1 at serine 462, enhancing UFL1 E3 ligase activity and promoting ATM activation in a positive feedback loop. These findings reveal that ufmylation of histone H4 by UFL1 is an important step for amplification of ATM activation and maintenance of genomic integrity.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Reparo do DNA , Histonas/metabolismo , Processamento de Proteína Pós-Traducional/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células HEK293 , Humanos , Lisina Acetiltransferase 5/metabolismo , Proteína Homóloga a MRE11/metabolismo , Metiltransferases/metabolismo , Proteínas Nucleares/metabolismo , Fosforilação/genética , RNA Interferente Pequeno/metabolismo , Proteínas Repressoras/metabolismo , Serina/metabolismo , Ubiquitina-Proteína Ligases/genética
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