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2.
J Biol Chem ; 300(3): 105739, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38342435

RESUMO

The p90 ribosomal S6 kinases (RSK) family of serine/threonine kinases comprises four isoforms (RSK1-4) that lie downstream of the ERK1/2 mitogen-activated protein kinase pathway. RSKs are implicated in fine tuning of cellular processes such as translation, transcription, proliferation, and motility. Previous work showed that pathogens such as Cardioviruses could hijack any of the four RSK isoforms to inhibit PKR activation or to disrupt cellular nucleocytoplasmic trafficking. In contrast, some reports suggest nonredundant functions for distinct RSK isoforms, whereas Coffin-Lowry syndrome has only been associated with mutations in the gene encoding RSK2. In this work, we used the analog-sensitive kinase strategy to ask whether the cellular substrates of distinct RSK isoforms differ. We compared the substrates of two of the most distant RSK isoforms: RSK1 and RSK4. We identified a series of potential substrates for both RSKs in cells and validated RanBP3, PDCD4, IRS2, and ZC3H11A as substrates of both RSK1 and RSK4, and SORBS2 as an RSK1 substrate. In addition, using mutagenesis and inhibitors, we confirmed analog-sensitive kinase data showing that endogenous RSKs phosphorylate TRIM33 at S1119. Our data thus identify a series of potential RSK substrates and suggest that the substrates of RSK1 and RSK4 largely overlap and that the specificity of the various RSK isoforms likely depends on their cell- or tissue-specific expression pattern.


Assuntos
Proteínas Quinases S6 Ribossômicas 90-kDa , Especificidade por Substrato , Humanos , Sistema de Sinalização das MAP Quinases , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/antagonistas & inibidores , Proteínas Quinases S6 Ribossômicas 90-kDa/química , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Reprodutibilidade dos Testes , Mutagênese
3.
Cell ; 141(4): 632-44, 2010 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-20434207

RESUMO

Extracellular signals regulate protein translation in many cell functions. A key advantage of control at the translational level is the opportunity to regulate protein synthesis within specific cellular subregions. However, little is known about mechanisms that may link extracellular cues to translation with spatial precision. Here, we show that a transmembrane receptor, DCC, forms a binding complex containing multiple translation components, including eukaryotic initiation factors, ribosomal large and small subunits, and monosomes. In neuronal axons and dendrites DCC colocalizes in particles with translation machinery, and newly synthesized protein. The extracellular ligand netrin promoted DCC-mediated translation and disassociation of translation components. The functional and physical association of a cell surface receptor with the translation machinery leads to a generalizable model for localization and extracellular regulation of protein synthesis, based on a transmembrane translation regulation complex.


Assuntos
Neurônios/metabolismo , Biossíntese de Proteínas , Receptores de Superfície Celular/metabolismo , Proteínas Ribossômicas/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Motivos de Aminoácidos , Animais , Axônios/metabolismo , Células Cultivadas , Embrião de Galinha , Dendritos/metabolismo , Hipocampo/citologia , Hipocampo/metabolismo , Humanos , Camundongos , Fatores de Crescimento Neural/metabolismo , Netrina-1 , Estrutura Terciária de Proteína , Ratos , Receptores de Superfície Celular/química , Ribossomos/metabolismo , Proteínas Supressoras de Tumor/química
4.
Mol Cell ; 67(3): 512-527.e4, 2017 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-28757207

RESUMO

Aberrant signaling by the mammalian target of rapamycin (mTOR) contributes to the devastating features of cancer cells. Thus, mTOR is a critical therapeutic target and catalytic inhibitors are being investigated as anti-cancer drugs. Although mTOR inhibitors initially block cell proliferation, cell viability and migration in some cancer cells are quickly restored. Despite sustained inhibition of mTORC1/2 signaling, Akt, a kinase regulating cell survival and migration, regains phosphorylation at its regulatory sites. Mechanistically, mTORC1/2 inhibition promotes reorganization of integrin/focal adhesion kinase-mediated adhesomes, induction of IGFR/IR-dependent PI3K activation, and Akt phosphorylation via an integrin/FAK/IGFR-dependent process. This resistance mechanism contributes to xenograft tumor cell growth, which is prevented with mTOR plus IGFR inhibitors, supporting this combination as a therapeutic approach for cancers.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias da Mama/tratamento farmacológico , Movimento Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Quinase 1 de Adesão Focal/metabolismo , Melanoma/tratamento farmacológico , Complexos Multiproteicos/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Receptores de Somatomedina/antagonistas & inibidores , Neoplasias Cutâneas/tratamento farmacológico , Serina-Treonina Quinases TOR/antagonistas & inibidores , Animais , Neoplasias da Mama/enzimologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Feminino , Quinase 1 de Adesão Focal/genética , Humanos , Integrina alfa2/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina , Alvo Mecanístico do Complexo 2 de Rapamicina , Melanoma/enzimologia , Melanoma/patologia , Camundongos Nus , Complexos Multiproteicos/metabolismo , Invasividade Neoplásica , Fosfatidilinositol 3-Quinase/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , Receptor IGF Tipo 1 , Receptores de Somatomedina/genética , Receptores de Somatomedina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Neoplasias Cutâneas/enzimologia , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Serina-Treonina Quinases TOR/metabolismo , Fatores de Tempo , Transfecção , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
5.
J Cell Sci ; 135(16)2022 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-35892282

RESUMO

Many animal cell shape changes are driven by gradients in the contractile tension of the actomyosin cortex, a thin cytoskeletal network supporting the plasma membrane. Elucidating cortical tension control is thus essential for understanding cell morphogenesis. Increasing evidence shows that alongside myosin activity, actin network organisation and composition are key to cortex tension regulation. However, owing to a poor understanding of how cortex composition changes when tension changes, which cortical components are important remains unclear. In this article, we compared cortices from cells with low and high cortex tensions. We purified cortex-enriched fractions from cells in interphase and mitosis, as mitosis is characterised by high cortical tension. Mass spectrometry analysis identified 922 proteins consistently represented in both interphase and mitotic cortices. Focusing on actin-related proteins narrowed down the list to 238 candidate regulators of the mitotic cortical tension increase. Among these candidates, we found that there is a role for septins in mitotic cell rounding control. Overall, our study provides a comprehensive dataset of candidate cortex regulators, paving the way for systematic investigations of the regulation of cell surface mechanics. This article has an associated First Person interview with the first author of the paper.


Assuntos
Actinas , Proteômica , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animais , Humanos , Interfase , Mitose
6.
PLoS Genet ; 17(6): e1009583, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34125833

RESUMO

Ribosome biogenesis lies at the nexus of various signaling pathways coordinating protein synthesis with cell growth and proliferation. This process is regulated by well-described transcriptional mechanisms, but a growing body of evidence indicates that other levels of regulation exist. Here we show that the Ras/mitogen-activated protein kinase (MAPK) pathway stimulates post-transcriptional stages of human ribosome synthesis. We identify RIOK2, a pre-40S particle assembly factor, as a new target of the MAPK-activated kinase RSK. RIOK2 phosphorylation by RSK stimulates cytoplasmic maturation of late pre-40S particles, which is required for optimal protein synthesis and cell proliferation. RIOK2 phosphorylation facilitates its release from pre-40S particles and its nuclear re-import, prior to completion of small ribosomal subunits. Our results bring a detailed mechanistic link between the Ras/MAPK pathway and the maturation of human pre-40S particles, which opens a hitherto poorly explored area of ribosome biogenesis.


Assuntos
Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Células HEK293 , Humanos , Mutação , Fosforilação , Transporte Proteico , Subunidades Ribossômicas Menores/metabolismo , Transdução de Sinais , Especificidade por Substrato , Transcrição Gênica
7.
Proc Natl Acad Sci U S A ; 117(33): 20139-20148, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32727899

RESUMO

Lung cancer causes more deaths annually than any other malignancy. A subset of non-small cell lung cancer (NSCLC) is driven by amplification and overexpression or activating mutation of the receptor tyrosine kinase (RTK) ERBB2 In some contexts, notably breast cancer, alternative splicing of ERBB2 causes skipping of exon 16, leading to the expression of an oncogenic ERBB2 isoform (ERBB2ΔEx16) that forms constitutively active homodimers. However, the broader implications of ERBB2 alternative splicing in human cancers have not been explored. Here, we have used genomic and transcriptomic analysis to identify elevated ERBB2ΔEx16 expression in a subset of NSCLC cases, as well as splicing site mutations facilitating exon 16 skipping and deletions of exon 16 in a subset of these lung tumors and in a number of other carcinomas. Supporting the potential of ERBB2ΔEx16 as a lung cancer driver, its expression transformed immortalized lung epithelial cells while a transgenic model featuring inducible ERBB2ΔEx16 specifically in the lung epithelium rapidly developed lung adenocarcinomas following transgene induction. Collectively, these observations indicate that ERBB2ΔEx16 is a lung cancer oncogene with potential clinical importance for a proportion of patients.


Assuntos
Carcinoma/genética , Predisposição Genética para Doença , Neoplasias Pulmonares/genética , Isoformas de Proteínas/genética , Receptor ErbB-2/metabolismo , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Masculino , Camundongos , Ratos , Receptor ErbB-2/genética , Microambiente Tumoral
8.
Mol Cell Proteomics ; 19(1): 50-64, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31678930

RESUMO

The RAS/mitogen-activated protein kinase (MAPK) signaling pathway regulates various biological functions, including cell survival, proliferation and migration. This pathway is frequently deregulated in cancer, including melanoma, which is the most aggressive form of skin cancer. RSK (p90 ribosomal S6 kinase) is a MAPK-activated protein kinase required for melanoma growth and proliferation, but relatively little is known about its function and the nature of its cellular partners. In this study, we used a proximity-based labeling approach to identify RSK proximity partners in cells. We identified many potential RSK-interacting proteins, including p120ctn (p120-catenin), which is an essential component of adherens junction (AJ). We found that RSK phosphorylates p120ctn on Ser320, which appears to be constitutively phosphorylated in melanoma cells. We also found that RSK inhibition increases melanoma cell-cell adhesion, suggesting that constitutive RAS/MAPK signaling negatively regulates AJ integrity. Together, our results indicate that RSK plays an important role in the regulation of melanoma cell-cell adhesion.


Assuntos
Cateninas/metabolismo , Adesão Celular/genética , Melanoma/metabolismo , Proteômica/métodos , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Cateninas/genética , Linhagem Celular Tumoral , Células HEK293 , Humanos , Sistema de Sinalização das MAP Quinases/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação , Interferência de RNA , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Transdução de Sinais/genética , delta Catenina
9.
Proc Natl Acad Sci U S A ; 116(8): 2967-2976, 2019 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-30728292

RESUMO

ERK is a key coordinator of the epithelial-to-mesenchymal transition (EMT) in that a variety of EMT-inducing factors activate signaling pathways that converge on ERK to regulate EMT transcription programs. However, the mechanisms by which ERK controls the EMT program are not well understood. Through an analysis of the global changes of gene expression mediated by ERK2, we identified the transcription factor FoxO1 as a potential mediator of ERK2-induced EMT, and thus we investigated the mechanism by which ERK2 regulates FoxO1. Additionally, our analysis revealed that ERK2 induced the expression of Dock10, a Rac1/Cdc42 GEF, during EMT. We demonstrate that the activation of the Rac1/JNK signaling axis downstream of Dock10 leads to an increase in FoxO1 expression and EMT. Taken together, our study uncovers mechanisms by which epithelial cells acquire less proliferative but more migratory mesenchymal properties and reveals potential therapeutic targets for cancers evolving into a metastatic disease state.


Assuntos
Transição Epitelial-Mesenquimal/genética , Proteína Forkhead Box O1/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Proteína Quinase 1 Ativada por Mitógeno/genética , Linhagem Celular Tumoral , Regulação da Expressão Gênica/genética , Humanos , Sistema de Sinalização das MAP Quinases/genética , Ativação Transcricional/genética , Proteínas rac1 de Ligação ao GTP/genética
10.
Genes Dev ; 28(4): 357-71, 2014 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-24532714

RESUMO

The mammalian target of rapamycin (mTOR) promotes cell growth and proliferation by promoting mRNA translation and increasing the protein synthetic capacity of the cell. Although mTOR globally promotes translation by regulating the mRNA 5' cap-binding protein eIF4E (eukaryotic initiation factor 4E), it also preferentially regulates the translation of certain classes of mRNA via unclear mechanisms. To help fill this gap in knowledge, we performed a quantitative proteomic screen to identify proteins that associate with the mRNA 5' cap in an mTOR-dependent manner. Using this approach, we identified many potential regulatory factors, including the putative RNA-binding protein LARP1 (La-related protein 1). Our results indicate that LARP1 associates with actively translating ribosomes via PABP and that LARP1 stimulates the translation of mRNAs containing a 5' terminal oligopyrimidine (TOP) motif, encoding for components of the translational machinery. We found that LARP1 associates with the mTOR complex 1 (mTORC1) and is required for global protein synthesis as well as cell growth and proliferation. Together, these data reveal important molecular mechanisms involved in TOP mRNA translation and implicate LARP1 as an important regulator of cell growth and proliferation.


Assuntos
Autoantígenos/metabolismo , Regulação da Expressão Gênica , Proteômica , Pirimidinas/metabolismo , RNA Mensageiro/genética , Ribonucleoproteínas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Autoantígenos/genética , Linhagem Celular , Linhagem Celular Tumoral , Células Cultivadas , Células HEK293 , Humanos , Camundongos , Proteínas de Ligação ao Cap de RNA/metabolismo , Ribonucleoproteínas/genética , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Antígeno SS-B
11.
Reproduction ; 161(2): 183-193, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33434153

RESUMO

Abolition of the LH-induced ERK1/2 pathway leads to dramatic changes in gene expression in granulosa cells, subsequently abrogating ovulation. Here we explored whether sustained ERK1/2 signaling beyond immediate-early hours of the LH surge is important for ovulation in mice. First, we examined the effect of inhibition of ERK1/2 activity at 4 h after hCG stimulation on ovulation in superovulated immature mice. Treatment with the ERK1/2 pathway inhibitor PD0325901 at 4 h post-hCG disrupted follicular rupture without altering cumulus expansion, oocyte meiotic maturation and luteinization. Profiling the expression pattern of genes of the RSK family of ERK1/2 signal mediators revealed that RSK3, but not other isoforms, was induced by hCG treatment. Further, RSK3-knockout mice were sub-fertile with reduced ovulation rate and smaller litter size compared to WT mice. Given that PD0325901 inhibits all mediators of ERK1/2 signaling, we chose to evaluate the gene expression underlying deficient follicular rupture in ERK1/2 inhibited mice. We found that inhibition of ERK1/2 signaling at 4 h post-hCG resulted in an imbalance in the expression of genes involved in extracellular matrix degradation and leukocyte infiltration necessary for follicular rupture. In conclusion, our data demonstrate that sustained ERK1/2 signaling during ovulation is not required for cumulus expansion, oocyte meiotic maturation and luteinization, but is required for follicular rupture.


Assuntos
Sistema de Sinalização das MAP Quinases , Ovulação , Animais , Feminino , Células da Granulosa/metabolismo , Luteinização , Camundongos , Camundongos Knockout
12.
RNA Biol ; 18(2): 207-217, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-32233986

RESUMO

The RNA-binding protein LARP1 has generated interest in recent years for its role in the mTOR signalling cascade and its regulation of terminal oligopyrimidine (TOP) mRNA translation. Paradoxically, some scientists have shown that LARP1 represses TOP translation while others that LARP1 activates it. Here, we present opinions from four leading scientists in the field to discuss these and other contradictory findings.


Assuntos
Autoantígenos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/metabolismo , Animais , Autoantígenos/química , Autoantígenos/genética , Sítios de Ligação , Proteínas de Transporte , Regulação da Expressão Gênica , Humanos , Família Multigênica , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , RNA/química , RNA/metabolismo , Clivagem do RNA , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Ribonucleoproteínas/química , Ribonucleoproteínas/genética , Transdução de Sinais , Especificidade por Substrato , Antígeno SS-B
13.
Am J Respir Crit Care Med ; 201(10): 1263-1276, 2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-31917615

RESUMO

Rationale: Vascular remodeling, including smooth muscle cell hypertrophy and proliferation, is the key pathological feature of pulmonary arterial hypertension (PAH). Prostaglandin I2 analogs (beraprost, iloprost, and treprostinil) are effective in the treatment of PAH. Of note, the clinically favorable effects of treprostinil in severe PAH may be attributable to concomitant activation of DP1 (D prostanoid receptor subtype 1).Objectives: To study the role of DP1 in the progression of PAH and its underlying mechanism.Methods: DP1 levels were examined in pulmonary arteries of patients and animals with PAH. Multiple genetic and pharmacologic approaches were used to investigate DP1-mediated signaling in PAH.Measurements and Main Results: DP1 expression was downregulated in hypoxia-treated pulmonary artery smooth muscle cells and in pulmonary arteries from rodent PAH models and patients with idiopathic PAH. DP1 deletion exacerbated pulmonary artery remodeling in hypoxia-induced PAH, whereas pharmacological activation or forced expression of the DP1 receptor had the opposite effect in different rodent models. DP1 deficiency promoted pulmonary artery smooth muscle cell hypertrophy and proliferation in response to hypoxia via induction of mTORC1 (mammalian target of rapamycin complex 1) activity. Rapamycin, an inhibitor of mTORC1, alleviated the hypoxia-induced exacerbation of PAH in DP1-knockout mice. DP1 activation facilitated raptor dissociation from mTORC1 and suppressed mTORC1 activity through PKA (protein kinase A)-dependent phosphorylation of raptor at Ser791. Moreover, treprostinil treatment blocked the progression of hypoxia-induced PAH in mice in part by targeting the DP1 receptor.Conclusions: DP1 activation attenuates hypoxia-induced pulmonary artery remodeling and PAH through PKA-mediated dissociation of raptor from mTORC1. These results suggest that the DP1 receptor may serve as a therapeutic target for the management of PAH.


Assuntos
Hipóxia/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Hipertensão Arterial Pulmonar/genética , Receptores Imunológicos/genética , Receptores de Prostaglandina/genética , Remodelação Vascular/genética , Animais , Anti-Hipertensivos/farmacologia , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Regulação para Baixo , Epoprostenol/análogos & derivados , Epoprostenol/farmacologia , Humanos , Hipertrofia , Imunossupressores/farmacologia , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Camundongos , Camundongos Knockout , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Hipertensão Arterial Pulmonar/tratamento farmacológico , Hipertensão Arterial Pulmonar/metabolismo , Artéria Pulmonar , RNA Mensageiro/metabolismo , Ratos , Sirolimo/farmacologia
14.
Genes Chromosomes Cancer ; 58(10): 723-730, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31102422

RESUMO

High hyperdiploidy (HD) is the most common cytogenetic subtype of childhood acute lymphoblastic leukemia (ALL), and a higher incidence of HD has been reported in ALL patients with congenital cancer syndromes. We assessed the frequency of predisposing germline mutations in 57 HD-ALL patients from the California Childhood Leukemia Study via targeted sequencing of cancer-relevant genes. Three out of 57 patients (5.3%) harbored confirmed germline mutations that were likely causal, in NBN, ETV6, and FLT3, with an additional six patients (10.5%) harboring putative predisposing mutations that were rare in unselected individuals (<0.01% allele frequency in the Exome Aggregation Consortium, ExAC) and predicted functional (scaled CADD score ≥ 20) in known or potential ALL predisposition genes (SH2B3, CREBBP, PMS2, MLL, ABL1, and MYH9). Three additional patients carried rare and predicted damaging germline mutations in GAB2, a known activator of the ERK/MAPK and PI3K/AKT pathways and binding partner of PTPN11-encoded SHP2. The frequency of rare and predicted functional germline GAB2 mutations was significantly higher in our patients (2.6%) than in ExAC (0.28%, P = 4.4 × 10-3 ), an observation that was replicated in ALL patients from the TARGET project (P = .034). We cloned patient GAB2 mutations and expressed mutant proteins in HEK293 cells and found that frameshift mutation P621fs led to reduced SHP2 binding and ERK1/2 phosphorylation but significantly increased AKT phosphorylation, suggesting possible RAS-independent leukemogenic effects. Our results support a significant contribution of rare, high penetrance germline mutations to HD-ALL etiology, and pinpoint GAB2 as a putative novel ALL predisposition gene.


Assuntos
Frequência do Gene , Mutação em Linhagem Germinativa , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Criança , Mutação da Fase de Leitura , Predisposição Genética para Doença , Células HEK293 , Humanos , Penetrância
15.
Semin Cancer Biol ; 48: 53-61, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-28476656

RESUMO

The 90kDa ribosomal S6 kinase (RSK) family is a group of Ser/Thr protein kinases (RSK1-4) that function downstream of the Ras/mitogen-activated protein kinase (MAPK) signalling pathway. RSK regulates many substrates involved in cell survival, growth, and proliferation, and as such, deregulated RSK activity has been associated with multiple cancer types. RSK expression and activity are dysregulated in several malignancies, including breast, prostate, and lung cancer, and available evidence suggests that RSK may be a promising cancer therapeutic target. Current limitations include the lack of RSK inhibitors with suitable pharmacokinetics and selectivity toward particular isoforms. This review briefly describes the current knowledge on RSK activation and function, with a particular emphasis on RSK-dependent mechanisms associated with tumorigenesis and pharmacological inhibition.


Assuntos
Neoplasias/patologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases S6 Ribossômicas/fisiologia , Animais , Adesão Celular , Ciclo Celular/fisiologia , Proliferação de Células , Ativação Enzimática , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Proteínas Quinases S6 Ribossômicas/antagonistas & inibidores
16.
Blood ; 129(25): 3344-3351, 2017 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-28408459

RESUMO

A small subset of human cord blood CD34+ cells express endothelial protein C receptor (EPCR/CD201/PROCR) when exposed to the hematopoietic stem cell (HSC) self-renewal agonist UM171. In this article, we show that EPCR-positive UM171-treated cells, as opposed to EPCR-negative cells, exhibit robust multilineage repopulation and serial reconstitution ability in immunocompromised mice. In contrast to other stem cell markers, such as CD38, EPCR expression is maintained when cells are introduced in culture, irrespective of UM171 treatment. Although engineered overexpression of EPCR fails to reproduce the effects of UM171 on HSC activity, its expression is required for the repopulating activity of human HSCs. Altogether, our results indicate that EPCR is a reliable and cell culture-compatible marker of UM171-expanded human cord blood HSCs.


Assuntos
Antígenos CD34/análise , Antígenos CD/análise , Sangue Fetal/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Indóis/farmacologia , Pirimidinas/farmacologia , Receptores de Superfície Celular/análise , Animais , Técnicas de Cultura de Células , Proliferação de Células/efeitos dos fármacos , Receptor de Proteína C Endotelial , Feminino , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Humanos , Camundongos Endogâmicos NOD , Camundongos SCID
17.
Proc Natl Acad Sci U S A ; 112(37): E5160-8, 2015 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-26324936

RESUMO

MAPKs are activated in response to G protein-coupled receptor (GPCR) stimulation and play essential roles in regulating cellular processes downstream of these receptors. However, very little is known about the reciprocal effect of MAPK activation on GPCRs. To investigate possible crosstalk between the MAPK and GPCRs, we assessed the effect of ERK1/2 on the activity of several GPCR family members. We found that ERK1/2 activation leads to a reduction in the steady-state cell-surface expression of many GPCRs because of their intracellular sequestration. This subcellular redistribution resulted in a global dampening of cell responsiveness, as illustrated by reduced ligand-mediated G-protein activation and second-messenger generation as well as blunted GPCR kinases and ß-arrestin recruitment. This ERK1/2-mediated regulatory process was observed for GPCRs that can interact with ß-arrestins, such as type-2 vasopressin, type-1 angiotensin, and CXC type-4 chemokine receptors, but not for the prostaglandin F receptor that cannot interact with ß-arrestin, implicating this scaffolding protein in the receptor's subcellular redistribution. Complementation experiments in mouse embryonic fibroblasts lacking ß-arrestins combined with in vitro kinase assays revealed that ß-arrestin-2 phosphorylation on Ser14 and Thr276 is essential for the ERK1/2-promoted GPCR sequestration. This previously unidentified regulatory mechanism was observed after constitutive activation as well as after receptor tyrosine kinase- or GPCR-mediated activation of ERK1/2, suggesting that it is a central node in the tonic regulation of cell responsiveness to GPCR stimulation, acting both as an effector and a negative regulator.


Assuntos
Arrestinas/metabolismo , Sistema de Sinalização das MAP Quinases , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , Membrana Celular/metabolismo , Citoplasma/metabolismo , Ativação Enzimática , Fibroblastos/metabolismo , Células HEK293 , Células HeLa , Humanos , Ligantes , Camundongos , Dados de Sequência Molecular , Peptídeos/química , Fosforilação , Ligação Proteica , Receptores de Prostaglandina/metabolismo , Homologia de Sequência de Aminoácidos , Transdução de Sinais , beta-Arrestina 2 , beta-Arrestinas
18.
Am J Hum Genet ; 94(6): 891-7, 2014 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-24814191

RESUMO

Epileptic encephalopathies are increasingly thought to be of genetic origin, although the exact etiology remains uncertain in many cases. We describe here three girls from two nonconsanguineous families affected by a clinical entity characterized by dysmorphic features, early-onset intractable epilepsy, intellectual disability, and cortical blindness. In individuals from each family, brain imaging also showed specific changes, including an abnormally marked pontobulbar sulcus and abnormal signals (T2 hyperintensities) and atrophy in the occipital lobe. Exome sequencing performed in the first family did not reveal any gene with rare homozygous variants shared by both affected siblings. It did, however, show one gene, DOCK7, with two rare heterozygous variants (c.2510delA [p.Asp837Alafs(∗)48] and c.3709C>T [p.Arg1237(∗)]) found in both affected sisters. Exome sequencing performed in the proband of the second family also showed the presence of two rare heterozygous variants (c.983C>G [p.Ser328(∗)] and c.6232G>T [p.Glu2078(∗)]) in DOCK7. Sanger sequencing confirmed that all three individuals are compound heterozygotes for these truncating mutations in DOCK7. These mutations have not been observed in public SNP databases and are predicted to abolish domains critical for DOCK7 function. DOCK7 codes for a Rac guanine nucleotide exchange factor that has been implicated in the genesis and polarization of newborn pyramidal neurons and in the morphological differentiation of GABAergic interneurons in the developing cortex. All together, these observations suggest that loss of DOCK7 function causes a syndromic form of epileptic encephalopathy by affecting multiple neuronal processes.


Assuntos
Cegueira Cortical/genética , Epilepsia/genética , Proteínas Ativadoras de GTPase/genética , Deficiência Intelectual/genética , Criança , Pré-Escolar , Epilepsias Mioclônicas/genética , Exoma , Feminino , Proteínas Ativadoras de GTPase/metabolismo , Genes Recessivos , Fatores de Troca do Nucleotídeo Guanina/genética , Heterozigoto , Homozigoto , Humanos , Lactente , Masculino , Mutação , Linhagem , Fenótipo , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNA , Espasmos Infantis/genética
20.
Proc Natl Acad Sci U S A ; 111(29): E2918-27, 2014 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-25002506

RESUMO

The Ras/MAPK signaling cascade regulates various biological functions, including cell growth and proliferation. As such, this pathway is frequently deregulated in several types of cancer, including most cases of melanoma. RSK (p90 ribosomal S6 kinase) is a MAPK-activated protein kinase required for melanoma growth and proliferation, but relatively little is known about its exact function and the nature of its substrates. Herein, we used a quantitative phosphoproteomics approach to define the signaling networks regulated by RSK in melanoma. To more accurately predict direct phosphorylation substrates, we defined the RSK consensus phosphorylation motif and found significant overlap with the binding consensus of 14-3-3 proteins. We thus characterized the phospho-dependent 14-3-3 interactome in melanoma cells and found that a large proportion of 14-3-3 binding proteins are also potential RSK substrates. Our results show that RSK phosphorylates the tumor suppressor PDCD4 (programmed cell death protein 4) on two serine residues (Ser76 and Ser457) that regulate its subcellular localization and interaction with 14-3-3 proteins. We found that 14-3-3 binding promotes PDCD4 degradation, suggesting an important role for RSK in the inactivation of PDCD4 in melanoma. In addition to this tumor suppressor, our results suggest the involvement of RSK in a vast array of unexplored biological functions with relevance in oncogenesis.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Fosfoproteínas/metabolismo , Proteômica/métodos , Proteínas de Ligação a RNA/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas 14-3-3/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Linhagem Celular , Núcleo Celular/metabolismo , Sequência Consenso , Humanos , Melanoma/metabolismo , Melanoma/patologia , Modelos Biológicos , Dados de Sequência Molecular , Biblioteca de Peptídeos , Fosforilação , Fosfosserina/metabolismo , Ligação Proteica , Transporte Proteico , Proteólise , Proteoma/metabolismo , Especificidade por Substrato
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