Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 852
Filtrar
1.
Cells ; 10(2)2021 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-33572403

RESUMO

Septins are GTP-binding proteins that form heteromeric filaments for proper cell growth and migration. Among the septins, septin7 (SEPT7) is an important component of all septin filaments. Here we show that protein kinase A (PKA) phosphorylates SEPT7 at Thr197, thus disrupting septin filament dynamics and ciliogenesis. The Thr197 residue of SEPT7, a PKA phosphorylating site, was conserved among different species. Treatment with cAMP or overexpression of PKA catalytic subunit (PKACA2) induced SEPT7 phosphorylation, followed by disruption of septin filament formation. Constitutive phosphorylation of SEPT7 at Thr197 reduced SEPT7‒SEPT7 interaction, but did not affect SEPT7‒SEPT6‒SEPT2 or SEPT4 interaction. Moreover, we noted that SEPT7 interacted with PKACA2 via its GTP-binding domain. Furthermore, PKA-mediated SEPT7 phosphorylation disrupted primary cilia formation. Thus, our data uncover the novel biological function of SEPT7 phosphorylation in septin filament polymerization and primary cilia formation.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Cílios/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Organogênese , Septinas/metabolismo , Sequência de Aminoácidos , Proteínas de Ciclo Celular/química , Sequência Conservada , Humanos , Fosforilação , Fosfotreonina/metabolismo , Ligação Proteica , Domínios Proteicos , Septinas/química , Especificidade da Espécie
2.
Plant Cell ; 33(5): 1790-1812, 2021 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-33630095

RESUMO

Calcium (Ca2+)/calmodulin (CaM)-dependent protein kinase (CCaMK) is an important positive regulator of abscisic acid (ABA) and abiotic stress signaling in plants and is believed to act upstream of mitogen-activated protein kinase (MAPK) in ABA signaling. However, it is unclear how CCaMK activates MAPK in ABA signaling. Here, we show that OsDMI3, a rice (Oryza sativa) CCaMK, directly interacts with and phosphorylates OsMKK1, a MAPK kinase (MKK) in rice, in vitro and in vivo. OsDMI3 was found to directly phosphorylate Thr-25 in the N-terminus of OsMKK1, and this Thr-25 phosphorylation is OsDMI3-specific in ABA signaling. The activation of OsMKK1 and its downstream kinase OsMPK1 is dependent on Thr-25 phosphorylation of OsMKK1 in ABA signaling. Moreover, ABA treatment induces phosphorylation in the activation loop of OsMKK1, and the two phosphorylations, in the N-terminus and in the activation loop, are independent. Further analyses revealed that OsDMI3-mediated phosphorylation of OsMKK1 positively regulates ABA responses in seed germination, root growth, and tolerance to both water stress and oxidative stress. Our results indicate that OsMKK1 is a direct target of OsDMI3, and OsDMI3-mediated phosphorylation of OsMKK1 plays an important role in activating the MAPK cascade and ABA signaling.


Assuntos
Ácido Abscísico/metabolismo , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Cálcio/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Oryza/enzimologia , Proteínas de Plantas/metabolismo , Ácido Abscísico/farmacologia , Quinases de Proteína Quinase Ativadas por Mitógeno/química , Modelos Biológicos , Oryza/efeitos dos fármacos , Oryza/fisiologia , Estresse Oxidativo/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Fosfotreonina/metabolismo , Ligação Proteica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Estresse Fisiológico/efeitos dos fármacos , Água
3.
Mol Cancer ; 20(1): 16, 2021 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-33461590

RESUMO

BACKGROUND: The IκB kinase (IKK) complex, comprising the two enzymes IKKα and IKKß, is the main activator of the inflammatory transcription factor NF-κB, which is constitutively active in many cancers. While several connections between NF-κB signaling and the oncogene c-Myc have been shown, functional links between the signaling molecules are still poorly studied. METHODS: Molecular interactions were shown by co-immunoprecipitation and FRET microscopy. Phosphorylation of c-Myc was shown by kinases assays and its activity by improved reporter gene systems. CRISPR/Cas9-mediated gene knockout and chemical inhibition were used to block IKK activity. The turnover of c-Myc variants was determined by degradation in presence of cycloheximide and by optical pulse-chase experiments.. Immunofluorescence of mouse prostate tissue and bioinformatics of human datasets were applied to correlate IKKα- and c-Myc levels. Cell proliferation was assessed by EdU incorporation and apoptosis by flow cytometry. RESULTS: We show that IKKα and IKKß bind to c-Myc and phosphorylate it at serines 67/71 within a sequence that is highly conserved. Knockout of IKKα decreased c-Myc-activity and increased its T58-phosphorylation, the target site for GSK3ß, triggering polyubiquitination and degradation. c-Myc-mutants mimicking IKK-mediated S67/S71-phosphorylation exhibited slower turnover, higher cell proliferation and lower apoptosis, while the opposite was observed for non-phosphorylatable A67/A71-mutants. A significant positive correlation of c-Myc and IKKα levels was noticed in the prostate epithelium of mice and in a variety of human cancers. CONCLUSIONS: Our data imply that IKKα phosphorylates c-Myc on serines-67/71, thereby stabilizing it, leading to increased transcriptional activity, higher proliferation and decreased apoptosis.


Assuntos
Quinase I-kappa B/metabolismo , Inflamação/enzimologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Sequência de Aminoácidos , Animais , Apoptose/genética , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Proliferação de Células , Células HEK293 , Humanos , Quinase I-kappa B/química , Inflamação/patologia , Masculino , Camundongos , Modelos Biológicos , Mutação/genética , Fosforilação , Fosfosserina/metabolismo , Fosfotreonina/metabolismo , Próstata/metabolismo , Ligação Proteica , Estabilidade Proteica , Transcrição Genética
4.
Nat Commun ; 12(1): 90, 2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33397943

RESUMO

Enterohemorrhagic Escherichia coli (EHEC) induces changes to the intestinal cell cytoskeleton and formation of attaching and effacing lesions, characterized by the effacement of microvilli and then formation of actin pedestals to which the bacteria are tightly attached. Here, we use a Caenorhabditis elegans model of EHEC infection to show that microvillar effacement is mediated by a signalling pathway including mitotic cyclin-dependent kinase 1 (CDK1) and diaphanous-related formin 1 (CYK1). Similar observations are also made using EHEC-infected human intestinal cells in vitro. Our results support the use of C. elegans as a host model for studying attaching and effacing lesions in vivo, and reveal that the CDK1-formin signal axis is necessary for EHEC-induced microvillar effacement.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Ciclo Celular/metabolismo , Escherichia coli Êntero-Hemorrágica/fisiologia , Interações Hospedeiro-Patógeno , Microvilosidades/microbiologia , Microvilosidades/patologia , Actinas/metabolismo , Animais , Células CACO-2 , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/microbiologia , Caenorhabditis elegans/ultraestrutura , Carboidratos Epimerases/metabolismo , Escherichia coli Êntero-Hemorrágica/patogenicidade , Forminas , Humanos , Intestinos/microbiologia , Microvilosidades/metabolismo , Fosforilação , Fosfotreonina/metabolismo , Virulência
5.
J Mol Neurosci ; 71(1): 89-100, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32557144

RESUMO

Hyperphosphorylated tau is the main component of neurofibrillary tangles and involved in the pathogenesis of Alzheimer's disease (AD). Increasing evidences suggest close associations between Porphyromonas gingivalis (P. gingivalis) and AD, but the relationship between P. gingivalis and tau hyperphosphorylation is still unclear. In this study, we investigated whether peripheral infection with P. gingivalis caused tau hyperphosphorylation by using wild Sprague-Dawley (SD) rats and HT-22 cells. The rats were injected with P. gingivalis suspension or phosphate-buffered saline 3 times per week. After 4 weeks or 12 weeks, the rats were sacrificed for analyzing systemic inflammation, neuroinflammation, and tau hyperphosphorylation. The results showed that the severity of phosphorylated tau at the AD-related sites Thr181 and Thr231 and the number of activated astrocytes were notably greater in the hippocampus of rats with P. gingivalis injection. And the levels of the inflammatory cytokines interleukin (IL)-1ß and IL-6 and tumor necrosis factor-α in serum and hippocampus were also increased in the rats with P. gingivalis injection. In addition, the activity of protein phosphatase 2A (PP2A) was significantly inhibited in the hippocampus of rats with P. gingivalis injection. In vitro, IL-1ß induced tau hyperphosphorylation by inhibiting the activity of PP2A in HT-22 cells and application of the PP2A promoter efficiently attenuated IL-1ß-induced tau hyperphosphorylation in HT-22 cells. These results indicated that P. gingivalis could induce tau hyperphosphorylation via, in part, attenuating the activity of PP2A through triggering systemic inflammation and neuroinflammation in wild-type SD rats.


Assuntos
Doença de Alzheimer/microbiologia , Infecções por Bacteroidaceae/metabolismo , Porphyromonas gingivalis/patogenicidade , Processamento de Proteína Pós-Traducional , Proteínas tau/metabolismo , Doença de Alzheimer/etiologia , Doença de Alzheimer/metabolismo , Animais , Astrócitos/metabolismo , Bacteriemia/metabolismo , Infecções por Bacteroidaceae/complicações , Infecções por Bacteroidaceae/microbiologia , Linhagem Celular , Citocinas/análise , Citocinas/sangue , Modelos Animais de Doenças , Ativação Enzimática , Hipocampo/citologia , Hipocampo/metabolismo , Hipocampo/patologia , Inflamação , Masculino , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Fosforilação , Fosfotreonina/metabolismo , Porphyromonas gingivalis/fisiologia , Proteína Fosfatase 2/antagonistas & inibidores , Proteína Fosfatase 2/genética , Ratos , Ratos Sprague-Dawley , Organismos Livres de Patógenos Específicos , Fator de Necrose Tumoral alfa/análise , Fator de Necrose Tumoral alfa/sangue
6.
Sci Rep ; 10(1): 21160, 2020 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-33273660

RESUMO

Mutations in genes that encode components of tuberous sclerosis complex 2 (TSC2) are associated with tuberous sclerosis complex disease. TSC2 interacts with tuberous sclerosis complex 1 to form a complex that negatively regulates cell growth and proliferation via the inactivation of mechanistic target of rapamycin complex 1. The activity of TSC2 is mainly regulated via posttranslational modifications such as phosphorylation. However, the control of TSC2 activity is not entirely achieved by phosphorylation. In this study, we show that TSC2 is methylated at R1457 and R1459 by protein arginine methyltransferase 1 (PRMT1). Methylation of these two residues can affect the phosphorylation status through protein kinase B (Akt) of TSC2 at T1462 and is essential for TSC2 stability. Taken together, these findings indicate that novel posttranslational modifications are important for the regulation of TSC2 stability through PRMT1-mediated methylation.


Assuntos
Arginina/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa/metabolismo , Células HEK293 , Células HeLa , Humanos , Metilação , Fosforilação , Fosfotreonina/metabolismo , Ligação Proteica , Estabilidade Proteica , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Repressoras/metabolismo
7.
Int J Mol Sci ; 21(23)2020 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-33256016

RESUMO

The strong association between diabetes mellitus type 2 and cancer is observed. The incidence of both diseases is increasing globally due to the interaction between them. Recent studies suggest that there is also an association between cancer incidence and anti-diabetic medications. An inhibitor of dipeptidyl-peptidase 4 (DPP-4), sitagliptin, is used in diabetes treatment. We examined the influence of sitagliptin alone or in combination with a cytostatic drug (paclitaxel) on the development of epithelial ovarian cancer cells and the process of metastasis. We examined migration, invasiveness, apoptosis, and metalloproteinases (MMPs) and their inhibitors' (TIMPs) production in two human ovarian cancer cell lines. Sitagliptin induced apoptosis by caspase 3/7 activation in paclitaxel-treated SKOV-3 and OVCAR-3 cells. Sitagliptin maintained paclitaxel influence on ERK and Akt signaling pathways. Sitagliptin additionally reduced migration and invasiveness of SKOV-3 cells. There were distinct differences of metalloproteinases production in sitagliptin-stimulated ovarian cancer cells in both cell lines, despite their identical histological classification. Only the SKOV-3 cell line expressed MMPs and TIMPs. SKOV-3 cells co-treated with sitagliptin and paclitaxel decreased concentrations of MMP-1, MMP-2, MMP-7, MMP-10, TIMP-1, TIMP-2. The obtained data showed that sitagliptin used with paclitaxel may be considered as a possibility of pharmacological modulation of intracellular transmission pathways to improve the response to chemotherapy.


Assuntos
Antineoplásicos/farmacologia , Neoplasias Ovarianas/patologia , Fosfato de Sitagliptina/farmacologia , Apoptose/efeitos dos fármacos , Caspases/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Dipeptidil Peptidase 4/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Inibidores de Metaloproteinases de Matriz/farmacologia , Metaloproteinases da Matriz/metabolismo , Invasividade Neoplásica , Neoplasias Ovarianas/enzimologia , Neoplasias Ovarianas/genética , Paclitaxel/farmacologia , Fosforilação/efeitos dos fármacos , Fosfotreonina/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
8.
Dev Cell ; 55(3): 367-380.e6, 2020 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-32891194

RESUMO

Plant stress responses involve dynamic growth regulation. Growth is restricted in harsh environmental conditions and is rapidly restored when conditions improve. Here, we identified BIN2, a glycogen synthase kinase 3 (GSK3)-like kinase, as a molecular switch in the transition to robust growth after salt stress in Arabidopsis thaliana. In the rapid recovery phase after salt stress, the calcium sensors SOS3 and SCaBP8 perceive a calcium signal and promote BIN2 localization to the plasma membrane to repress the salt stress response, and BIN2 inhibits SOS2 activity and enhances growth by releasing BZR1/BES1 transcriptional activity. The expression of stress- and brassinosteroid-responsive genes is coordinately regulated during this process. bin2-3bil1 and bin2-3bil2 mutants defective in BIN2 and its homologs BIL1 and BIL2, respectively, are hyposensitive to salt stress. Our study suggests that salt signaling modulates the subcellular localization and interactions of BIN2. By phosphorylating different substrates, BIN2 regulates the salt stress response and growth recovery.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Arabidopsis/crescimento & desenvolvimento , Quinase 3 da Glicogênio Sintase/metabolismo , Proteínas Quinases/metabolismo , Estresse Salino , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Homeostase , Modelos Biológicos , Fosforilação , Fosfotreonina/metabolismo , Ligação Proteica , Proteínas Quinases/genética
9.
Mol Cell ; 80(2): 296-310.e6, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-32979304

RESUMO

Necroptosis induction in vitro often requires caspase-8 (Casp8) inhibition by zVAD because pro-Casp8 cleaves RIP1 to disintegrate the necrosome. It has been unclear how the Casp8 blockade of necroptosis is eliminated naturally. Here, we show that pro-Casp8 within the necrosome can be inactivated by phosphorylation at Thr265 (pC8T265). pC8T265 occurs in vitro in various necroptotic cells and in the cecum of TNF-treated mice. p90 RSK is the kinase of pro-Casp8. It is activated by a mechanism that does not need ERK but PDK1, which is recruited to the RIP1-RIP3-MLKL-containing necrosome. Phosphorylation of pro-Casp8 at Thr265 can substitute for zVAD to permit necroptosis in vitro. pC8T265 mimic T265E knockin mice are embryonic lethal due to unconstrained necroptosis, and the pharmaceutical inhibition of RSK-mediated pC8T265 diminishes TNF-induced cecum damage and lethality in mice by halting necroptosis. Thus, phosphorylation of pro-Casp8 at Thr265 by RSK is an intrinsic mechanism for passing the Casp8 checkpoint of necroptosis.


Assuntos
Proteínas Quinases Dependentes de 3-Fosfoinositídeo/metabolismo , Caspase 8/metabolismo , Necroptose , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Transdução de Sinais , Animais , Ceco/lesões , Ceco/patologia , Linhagem Celular , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Camundongos Endogâmicos C57BL , Mutação/genética , Necroptose/efeitos dos fármacos , Especificidade de Órgãos , Fosforilação/efeitos dos fármacos , Fosfotreonina/metabolismo , Proteínas Quinases/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia
10.
Cell Rep ; 32(12): 108160, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32966793

RESUMO

The glyoxalase system is a highly conserved and ubiquitously expressed enzyme system, which is responsible for the detoxification of methylglyoxal (MG), a spontaneous by-product of energy metabolism. This study is able to show that a phosphorylation of threonine-107 (T107) in the (rate-limiting) Glyoxalase 1 (Glo1) protein, mediated by Ca2+/calmodulin-dependent kinase II delta (CamKIIδ), is associated with elevated catalytic efficiency of Glo1 (lower KM; higher Vmax). Additionally, we observe proteasomal degradation of non-phosphorylated Glo1 via ubiquitination does occur more rapidly as compared with native Glo1. The absence of CamKIIδ is associated with poor detoxification capacity and decreased protein content of Glo1 in a murine CamKIIδ knockout model. Therefore, phosphorylation of T107 in the Glo1 protein by CamKIIδ is a quick and precise mechanism regulating Glo1 activity, which is experimentally linked to an altered Glo1 status in cancer, diabetes, and during aging.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Lactoilglutationa Liase/metabolismo , Fosfotreonina/metabolismo , Proteômica , Envelhecimento/patologia , Animais , Linhagem Celular , Diabetes Mellitus/enzimologia , Diabetes Mellitus/patologia , Humanos , Inativação Metabólica , Cinética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neoplasias/enzimologia , Neoplasias/patologia , Fosforilação , Complexo de Endopeptidases do Proteassoma/metabolismo , Aldeído Pirúvico/metabolismo
11.
Mol Neurobiol ; 57(12): 5011-5025, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32820462

RESUMO

Alzheimer's disease (AD) is the most common neurodegenerative disease, but its underlying mechanism is still unclear and the identities of drugs for AD also lack. Tau acetylation has become potentially important post-translational modification of tau. Levels of tau acetylation are significantly enhanced in AD patients and transgenic mouse models of AD, but the underlying mechanism and roles of tau hyperacetylation in AD onset maintain elusive. In the current study, we found that tau acetylation is obviously enhanced and the activities of AMP-activated protein kinase (AMPK) and sirtuin1 (Sirt1) are significantly decreased in APP/PS1 and streptozotocin (STZ) mice and high glucose (HG)-treated cells. Moreover, we demonstrated that activation of AMPK reduces the level of tau acetylation and ameliorates memory impairment, and its mechanism is associated with activation of Sirt1. Taken together, AMPK might be a crucial upstream molecular to regulate acetylation of tau and become a new target for AD therapy in the future.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Transtornos da Memória/metabolismo , Sirtuína 1/metabolismo , Proteínas tau/metabolismo , Acetilação , Peptídeos beta-Amiloides/metabolismo , Animais , Regulação para Baixo , Células HEK293 , Hipocampo/metabolismo , Hipocampo/patologia , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fosforilação , Fosfotreonina/metabolismo , Presenilina-1/metabolismo , Estreptozocina , Regulação para Cima
12.
Mol Neurobiol ; 57(12): 5150-5166, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32860158

RESUMO

The signalling protein PKCγ is a major regulator of Purkinje cell development and synaptic function. We have shown previously that increased PKCγ activity impairs dendritic development of cerebellar Purkinje cells. Mutations in the protein kinase Cγ gene (PRKCG) cause spinocerebellar ataxia type 14 (SCA14). In a transgenic mouse model of SCA14 expressing the human S361G mutation, Purkinje cell dendritic development is impaired in cerebellar slice cultures similar to pharmacological activation of PKC. The mechanisms of PKCγ-driven inhibition of dendritic growth are still unclear. Using immunoprecipitation-coupled mass spectrometry analysis, we have identified collapsin response mediator protein 2 (CRMP2) as a protein interacting with constitutive active PKCγ(S361G) and confirmed the interaction with the Duolink™ proximity ligation assay. We show that in cerebellar slice cultures from PKCγ(S361G)-mice, phosphorylation of CRMP2 at the known PKC target site Thr555 is increased in Purkinje cells confirming phosphorylation of CRMP2 by PKCγ. miRNA-mediated CRMP2 knockdown decreased Purkinje cell dendritic outgrowth in dissociated cerebellar cultures as did the transfection of CRMP2 mutants with a modified Thr555 site. In contrast, dendritic development was normal after wild-type CRMP2 overexpression. In a novel knock-in mouse expressing only the phospho-defective T555A-mutant CRMP2, Purkinje cell dendritic development was reduced in dissociated cultures. This reduction could be rescued by transfecting wild-type CRMP2 but only partially by the phospho-mimetic T555D-mutant. Our findings establish CRMP2 as an important target of PKCγ phosphorylation in Purkinje cells mediating its control of dendritic development. Dynamic regulation of CRMP2 phosphorylation via PKCγ is required for its correct function.


Assuntos
Cerebelo/citologia , Dendritos/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteína Quinase C/metabolismo , Células de Purkinje/metabolismo , Animais , Sequência de Bases , Técnicas de Silenciamento de Genes , Camundongos Transgênicos , Modelos Biológicos , Fosforilação , Fosfotreonina/metabolismo , Ligação Proteica
13.
J Mol Cell Cardiol ; 148: 1-14, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32853649

RESUMO

RATIONALE: Among its many biological roles, fibroblast growth factor 2 (FGF2) protects the heart from dysfunction and damage associated with an ischemic attack. Our laboratory demonstrated that its protection against myocardial dysfunction occurs by the low molecular weight (LMW) isoform of FGF2, while the high molecular weight (HMW) isoforms are associated with a worsening in post-ischemic recovery of cardiac function. LMW FGF2-mediated cardioprotection is facilitated by activation of multiple kinases, including PKCalpha, PKCepsilon, and ERK, and inhibition of p38 and JNK. OBJECTIVE: Yet, the substrates of those kinases associated with LMW FGF2-induced cardioprotection against myocardial dysfunction remain to be elucidated. METHODS AND RESULTS: To identify substrates in LMW FGF2 improvement of post-ischemic cardiac function, mouse hearts expressing only LMW FGF2 were subjected to ischemia-reperfusion (I/R) injury and analyzed by a mass spectrometry (MS)-based quantitative phosphoproteomic strategy. MS analysis identified 50 phosphorylation sites from 7 sarcoendoplasmic reticulum (SR) proteins that were significantly altered in I/R-treated hearts only expressing LMW FGF2 compared to those hearts lacking FGF2. One of those phosphorylated SR proteins identified was phospholamban (PLB), which exhibited rapid, increased phosphorylation at Threonine-17 (Thr17) after I/R in hearts expressing only LMW FGF2; this was further validated using Selected Reaction Monitoring-based MS workflow. To demonstrate a mechanistic role of phospho-Thr17 PLB in LMW FGF2-mediated cardioprotection, hearts only expressing LMW FGF2 and those expressing only LMW FGF2 with a mutant PLB lacking phosphorylatable Thr17 (Thr17Ala PLB) were subjected to I/R. Hearts only expressing LMW FGF2 showed significantly improved recovery of cardiac function following I/R (p < 0.05), and this functional improvement was significantly abrogated in hearts expressing LMW FGF2 and Thr17Ala PLB (p < 0.05). CONCLUSION: The findings indicate that LMW FGF2 modulates intracellular calcium handling/cycling via regulatory changes in SR proteins essential for recovery from I/R injury, and thereby protects the heart from post-ischemic cardiac dysfunction.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Cardiotônicos/farmacologia , Fator 2 de Crescimento de Fibroblastos/metabolismo , Isquemia Miocárdica/prevenção & controle , Isquemia Miocárdica/fisiopatologia , Fosfoproteínas/metabolismo , Fosfotreonina/metabolismo , Proteômica , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Camundongos , Modelos Biológicos , Peso Molecular , Fosforilação , Proteína Quinase C-alfa/metabolismo , Retículo Sarcoplasmático/metabolismo
14.
Cell Death Dis ; 11(6): 410, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32483123

RESUMO

Tumor initiating cells (TIC) of lung cancer are mainly induced by stress-related plasticity. Calcium/Calmodulin dependent protein kinase II alpha (CAMK2A) is a key calcium signaling molecule activated by exogenous and endogenous stimuli with effects on multiple cell functions but little is known about its role on TIC. In human lung adenocarcinomas (AD), CAMK2A was aberrantly activated in a proportion of cases and was an independent risk factor predicting shorter survivals. Functionally, CAMK2A enhanced TIC phenotypes in vitro and in vivo. CAMK2A regulated SOX2 expression by reducing H3K27me3 and EZH2 occupancy at SOX2 regulatory regions, leading to its epigenetic de-repression with functional consequences. Further, CAMK2A caused kinase-dependent phosphorylation of EZH2 at T487 with suppression of EZH2 activity. Together, the data demonstrated the CAMK2A-EZH2-SOX2 axis on TIC regulation. This study provided phenotypic and mechanistic evidence for the TIC supportive role of CAMK2A, implicating a novel predictive and therapeutic target for lung cancer.


Assuntos
Adenocarcinoma de Pulmão/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/genética , Células-Tronco Neoplásicas/patologia , Fatores de Transcrição SOXB1/genética , Regulação para Cima/genética , Adenocarcinoma de Pulmão/patologia , Animais , Apoptose/genética , Carcinogênese/genética , Carcinogênese/patologia , Linhagem Celular Tumoral , Autorrenovação Celular/genética , Humanos , Neoplasias Pulmonares/patologia , Camundongos SCID , Terapia de Alvo Molecular , Células-Tronco Neoplásicas/metabolismo , Fosforilação , Fosfotreonina/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Fatores de Transcrição SOXB1/metabolismo , Análise de Sobrevida
15.
Cell Cycle ; 19(14): 1817-1832, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32573322

RESUMO

Polo-like kinase 1 (Plk1) is a cell cycle kinase essential for mitosis progression, but also important for checkpoint recovery and adaptation in response to DNA damage and replication stress. However, although Plk1 is expressed in S phase, little is known about its function during unperturbed DNA replication. Using Xenopus laevis egg extracts, mimicking early embryonic replication, we demonstrate that Plk1 is simultaneously recruited to chromatin with pre-replication proteins where it accumulates throughout S phase. Further, we found that chromatin-bound Plk1 is phosphorylated on its activating site T201, which appears to be sensitive to dephosphorylation by protein phosphatase 2A. Extracts immunodepleted of Plk1 showed a decrease in DNA replication, rescued by wild type recombinant Plk1. Inversely, modest Plk1 overexpression accelerated DNA replication. Plk1 depletion led to an increase in Chk1 phosphorylation and to a decrease in Cdk2 activity, which strongly suggests that Plk1 could inhibit the ATR/Chk1-dependent intra-S phase checkpoint during normal S phase. In addition, we observed that phosphorylated Plk1 levels are high during the rapid, early cell cycles of Xenopus development but decrease after the mid-blastula transition when the cell cycle and the replication program slow down along with more active checkpoints. These data shed new light on the role of Plk1 as a positive regulating factor for DNA replication in early, rapidly dividing embryos.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Replicação do DNA , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Xenopus laevis/metabolismo , Animais , Blástula/metabolismo , Cromatina/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Complexos Multiproteicos/metabolismo , Fosforilação , Fosfosserina/metabolismo , Fosfotreonina/metabolismo , Proteína Fosfatase 2/metabolismo , Fase S , Estresse Fisiológico
16.
Mol Oncol ; 14(6): 1268-1281, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32306542

RESUMO

Cross-linking of the B-cell receptor (BCR) induces transcriptional activation of immediate early genes (IEGs) including EGR1 and DUSP2 in chronic lymphocytic leukaemia (CLL). Here, we have shown that this transcriptional activation correlated with histone H3 threonine 6 and 11 phosphorylation. Both transcription and histone post-translational modifications are repressed by ibrutinib, a small molecule inhibitor used in CLL treatment. Moreover, we have identified the death-associated protein kinase 3 (DAPK3), as the kinase mediating these histone phosphorylation marks in response to activation of the BCR signalling pathway with this kinase being recruited to RNA polymerase II in an anti-IgM-dependent manner. DAPK inhibition mimics ibrutinib-induced repression of both IEG mRNA and histone H3 phosphorylation and has anti-proliferative effect comparable to ibrutinib in CLL in vitro. DAPK inhibitor does not repress transcription itself but impacts on mRNA processing and has a broader anti-tumour effect than ibrutinib, by repressing both anti-IgM- and CD40L-dependent activation.


Assuntos
Proteínas Quinases Associadas com Morte Celular/genética , Genes Precoces , Leucemia Linfocítica Crônica de Células B/genética , Processamento Pós-Transcricional do RNA/genética , Ligante de CD40/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Proteínas Quinases Associadas com Morte Celular/antagonistas & inibidores , Proteínas Quinases Associadas com Morte Celular/metabolismo , Loci Gênicos , Histonas/metabolismo , Humanos , Imunoglobulina M/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Fosforilação/efeitos dos fármacos , Fosfotreonina/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo
17.
Nat Commun ; 11(1): 1819, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32286328

RESUMO

The BRCA2 tumor suppressor protein is involved in the maintenance of genome integrity through its role in homologous recombination. In mitosis, BRCA2 is phosphorylated by Polo-like kinase 1 (PLK1). Here we describe how this phosphorylation contributes to the control of mitosis. We identify a conserved phosphorylation site at T207 of BRCA2 that constitutes a bona fide docking site for PLK1 and is phosphorylated in mitotic cells. We show that BRCA2 bound to PLK1 forms a complex with the phosphatase PP2A and phosphorylated-BUBR1. Reducing BRCA2 binding to PLK1, as observed in BRCA2 breast cancer variants S206C and T207A, alters the tetrameric complex resulting in unstable kinetochore-microtubule interactions, misaligned chromosomes, faulty chromosome segregation and aneuploidy. We thus reveal a role of BRCA2 in the alignment of chromosomes, distinct from its DNA repair function, with important consequences on chromosome stability. These findings may explain in part the aneuploidy observed in BRCA2-mutated tumors.


Assuntos
Proteína BRCA2/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromossomos Humanos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Aneuploidia , Neoplasias da Mama/genética , Segregação de Cromossomos , Feminino , Variação Genética , Células HeLa , Recombinação Homóloga , Humanos , Cinética , Cinetocoros , Mitose , Simulação de Acoplamento Molecular , Fosforilação , Fosfosserina/metabolismo , Fosfotreonina/metabolismo , Ligação Proteica , Proteína Fosfatase 2/metabolismo
18.
J Exp Med ; 217(6)2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32271880

RESUMO

Hyperactivation of YAP has been commonly associated with tumorigenesis, and emerging evidence hints at multilayered Hippo-independent regulations of YAP. In this study, we identified a new MST4-YAP axis, which acts as a noncanonical Hippo signaling pathway that limits stress-induced YAP activation. MST4 kinase directly phosphorylated YAP at Thr83 to block its binding with importin α, therefore leading to YAP cytoplasmic retention and inactivation. Due to a consequential interplay between MST4-mediated YAP phospho-Thr83 signaling and the classical YAP phospho-Ser127 signaling, the phosphorylation level of YAP at Thr83 was correlated to that at Ser127. Mutation of T83E mimicking MST4-mediated alternative signaling restrained the activity of both wild-type YAP and its S127A mutant mimicking loss of classical Hippo signal. Depletion of MST4 in mice promoted gastric tumorigenesis with diminished Thr83 phosphorylation and hyperactivation of YAP. Moreover, loss of MST4-YAP signaling was associated with poor prognosis of human gastric cancer. Collectively, our study uncovered a noncanonical MST4-YAP signaling axis essential for suppressing gastric tumorigenesis.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Carcinogênese/metabolismo , Carcinogênese/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Neoplasias Gástricas/enzimologia , Neoplasias Gástricas/patologia , Fatores de Transcrição/metabolismo , Transporte Ativo do Núcleo Celular , Proteínas Adaptadoras de Transdução de Sinal/química , Sequência de Aminoácidos , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Proliferação de Células , Meios de Cultura Livres de Soro , Feminino , Células HEK293 , Humanos , Masculino , Pessoa de Meia-Idade , Fosforilação , Fosfotreonina/metabolismo , Ligação Proteica , Proteínas Serina-Treonina Quinases/deficiência , Estresse Fisiológico , Fatores de Transcrição/química , Resultado do Tratamento , Proteínas Supressoras de Tumor/metabolismo
19.
Mol Biol Cell ; 31(10): 1032-1046, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32129703

RESUMO

Primary cilia are organelles necessary for proper implementation of developmental and homeostasis processes. To initiate their assembly, coordinated actions of multiple proteins are needed. Tau tubulin kinase 2 (TTBK2) is a key player in the cilium assembly pathway, controlling the final step of cilia initiation. The function of TTBK2 in ciliogenesis is critically dependent on its kinase activity; however, the precise mechanism of TTBK2 action has so far not been fully understood due to the very limited information about its relevant substrates. In this study, we demonstrate that CEP83, CEP89, CCDC92, Rabin8, and DVL3 are substrates of TTBK2 kinase activity. Further, we characterize a set of phosphosites of those substrates and CEP164 induced by TTBK2 in vitro and in vivo. Intriguingly, we further show that identified TTBK2 phosphosites and consensus sequence delineated from those are distinct from motifs previously assigned to TTBK2. Finally, we show that TTBK2 is also required for efficient phosphorylation of many S/T sites in CEP164 and provide evidence that TTBK2-induced phosphorylations of CEP164 modulate its function, which in turn seems relevant for the process of cilia formation. In summary, our work provides important insight into the substrates-TTBK2 kinase relationship and suggests that phosphorylation of substrates on multiple sites by TTBK2 is probably involved in the control of ciliogenesis in human cells.


Assuntos
Cílios/metabolismo , Complexos Multiproteicos/metabolismo , Organogênese , Proteínas Serina-Treonina Quinases/metabolismo , Motivos de Aminoácidos , Caseína Quinase I/metabolismo , Células HEK293 , Humanos , Fosforilação , Fosfosserina/metabolismo , Fosfotreonina/metabolismo , Proteínas Serina-Treonina Quinases/química , Especificidade por Substrato
20.
Nat Commun ; 11(1): 1567, 2020 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-32218435

RESUMO

Voltage-gated K+ channels function in macromolecular complexes with accessory subunits to regulate brain function. Here, we describe a peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1)-dependent mechanism that regulates the association of the A-type K+ channel subunit Kv4.2 with its auxiliary subunit dipeptidyl peptidase 6 (DPP6), and thereby modulates neuronal excitability and cognitive flexibility. We show that activity-induced Kv4.2 phosphorylation triggers Pin1 binding to, and isomerization of, Kv4.2 at the pThr607-Pro motif, leading to the dissociation of the Kv4.2-DPP6 complex. We generated a novel mouse line harboring a knock-in Thr607 to Ala (Kv4.2TA) mutation that abolished dynamic Pin1 binding to Kv4.2. CA1 pyramidal neurons of the hippocampus from these mice exhibited altered Kv4.2-DPP6 interaction, increased A-type K+ current, and reduced neuronal excitability. Behaviorally, Kv4.2TA mice displayed normal initial learning but improved reversal learning in both Morris water maze and lever press paradigms. These findings reveal a Pin1-mediated mechanism regulating reversal learning and provide potential targets for the treatment of neuropsychiatric disorders characterized by cognitive inflexibility.


Assuntos
Cognição , Peptidilprolil Isomerase de Interação com NIMA/metabolismo , Canais de Potássio Shal/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Células HEK293 , Humanos , Imidazóis/farmacologia , Ativação do Canal Iônico/efeitos dos fármacos , Isomerismo , Aprendizagem , Camundongos , Modelos Biológicos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fosforilação/efeitos dos fármacos , Fosfotreonina/metabolismo , Ligação Proteica , Células Piramidais/efeitos dos fármacos , Células Piramidais/metabolismo , Piridinas/farmacologia , Convulsões/metabolismo , Convulsões/patologia , Canais de Potássio Shal/química , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...