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1.
Nat Commun ; 14(1): 7791, 2023 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-38057326

RESUMO

Oncogenic lesions in pancreatic ductal adenocarcinoma (PDAC) hijack the epigenetic machinery in stromal components to establish a desmoplastic and therapeutic resistant tumor microenvironment (TME). Here we identify Class I histone deacetylases (HDACs) as key epigenetic factors facilitating the induction of pro-desmoplastic and pro-tumorigenic transcriptional programs in pancreatic stromal fibroblasts. Mechanistically, HDAC-mediated changes in chromatin architecture enable the activation of pro-desmoplastic programs directed by serum response factor (SRF) and forkhead box M1 (FOXM1). HDACs also coordinate fibroblast pro-inflammatory programs inducing leukemia inhibitory factor (LIF) expression, supporting paracrine pro-tumorigenic crosstalk. HDAC depletion in cancer-associated fibroblasts (CAFs) and treatment with the HDAC inhibitor entinostat (Ent) in PDAC mouse models reduce stromal activation and curb tumor progression. Notably, HDAC inhibition (HDACi) enriches a lipogenic fibroblast subpopulation, a potential precursor for myofibroblasts in the PDAC stroma. Overall, our study reveals the stromal targeting potential of HDACi, highlighting the utility of this epigenetic modulating approach in PDAC therapeutics.


Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Animais , Camundongos , Linhagem Celular Tumoral , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Pâncreas/metabolismo , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Fibroblastos/metabolismo , Carcinogênese/patologia , Microambiente Tumoral
2.
Nat Commun ; 14(1): 5195, 2023 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-37673892

RESUMO

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy in need of new therapeutic options. Using unbiased analyses of super-enhancers (SEs) as sentinels of core genes involved in cell-specific function, here we uncover a druggable SE-mediated RNA-binding protein (RBP) cascade that supports PDAC growth through enhanced mRNA translation. This cascade is driven by a SE associated with the RBP heterogeneous nuclear ribonucleoprotein F, which stabilizes protein arginine methyltransferase 1 (PRMT1) to, in turn, control the translational mediator ubiquitin-associated protein 2-like. All three of these genes and the regulatory SE are essential for PDAC growth and coordinately regulated by the Myc oncogene. In line with this, modulation of the RBP network by PRMT1 inhibition reveals a unique vulnerability in Myc-high PDAC patient organoids and markedly reduces tumor growth in male mice. Our study highlights a functional link between epigenetic regulation and mRNA translation and identifies components that comprise unexpected therapeutic targets for PDAC.


Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Masculino , Animais , Camundongos , RNA , Epigênese Genética , Sequências Reguladoras de Ácido Nucleico , Neoplasias Pancreáticas/genética , Carcinoma Ductal Pancreático/genética , Metiltransferases , Proteínas de Ligação a RNA/genética
3.
bioRxiv ; 2023 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-37745372

RESUMO

Oncogenic lesions in pancreatic ductal adenocarcinoma (PDAC) hijack the epigenetic machinery in stromal components to establish a desmoplastic and therapeutic resistant tumor microenvironment (TME). Here we identify Class I histone deacetylases (HDACs) as key epigenetic factors facilitating the induction of pro-desmoplastic and pro-tumorigenic transcriptional programs in pancreatic stromal fibroblasts. Mechanistically, HDAC-mediated changes in chromatin architecture enable the activation of pro-desmoplastic programs directed by serum response factor (SRF) and forkhead box M1 (FOXM1). HDACs also coordinate fibroblast pro-inflammatory programs inducing leukemia inhibitory factor (LIF) expression, supporting paracrine pro-tumorigenic crosstalk. HDAC depletion in cancer-associated fibroblasts (CAFs) and treatment with the HDAC inhibitor entinostat (Ent) in PDAC mouse models reduce stromal activation and curb tumor progression. Notably, HDAC inhibition (HDACi) enriches a lipogenic fibroblast subpopulation, a potential precursor for myofibroblasts in the PDAC stroma. Overall, our study reveals the stromal targeting potential of HDACi, highlighting the utility of this epigenetic modulating approach in PDAC therapeutics.

5.
J Biol Chem ; 296: 100445, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33617877

RESUMO

Within the AGC kinase superfamily, gene fusions resulting from chromosomal rearrangements have been most frequently described for protein kinase C (PKC), with gene fragments encoding either the C-terminal catalytic domain or the N-terminal regulatory moiety fused to other genes. Kinase fusions that eliminate regulatory domains are typically gain of function and often oncogenic. However, several quality control pathways prevent accumulation of aberrant PKC, suggesting that PKC fusions may paradoxically be loss of function. To explore this topic, we used biochemical, cellular, and genome editing approaches to investigate the function of fusions that retain the portion of the gene encoding either the catalytic domain or regulatory domain of PKC. Overexpression studies revealed that PKC catalytic domain fusions were constitutively active but vulnerable to degradation. Genome editing of endogenous genes to generate a cancer-associated PKC fusion resulted in cells with detectable levels of fusion transcript but no detectable protein. Hence, PKC catalytic domain fusions are paradoxically loss of function as a result of their instability, preventing appreciable accumulation of protein in cells. Overexpression of a PKC regulatory domain fusion suppressed both basal and agonist-induced endogenous PKC activity, acting in a dominant-negative manner by competing for diacylglycerol. For both catalytic and regulatory domain fusions, the PKC component of the fusion proteins mediated the effects of the full-length fusions on the parameters examined, suggesting that the partner protein is dispensable in these contexts. Taken together, our findings reveal that PKC gene fusions are distinct from oncogenic fusions and present a mechanism by which loss of PKC function occurs in cancer.


Assuntos
Neoplasias/metabolismo , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Animais , Sítios de Ligação , Células COS , Domínio Catalítico , Linhagem Celular Tumoral , Chlorocebus aethiops , Diglicerídeos/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Humanos , Mutação com Perda de Função/genética , Fosforilação , Domínios Proteicos , Proteína Quinase C-alfa/genética , Proteína Quinase C-alfa/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
6.
Oncogenesis ; 9(11): 100, 2020 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-33168807

RESUMO

The tumor microenvironment in pancreatic ductal adenocarcinoma (PDAC) is highly heterogeneous, fibrotic, and hypovascular, marked by extensive desmoplasia and maintained by the tumor cells, cancer-associated fibroblasts (CAFs) and other stromal cells. There is an urgent need to identify and develop treatment strategies that not only target the tumor cells but can also modulate the stromal cells. A growing number of studies implicate the role of regulatory DNA elements called super-enhancers (SE) in maintaining cell-type-specific gene expression networks in both normal and cancer cells. Using chromatin activation marks, we first mapped SE networks in pancreatic CAFs and epithelial tumor cells and found them to have distinct SE profiles. Next, we explored the role of triptolide (TPL), a natural compound with antitumor activity, in the context of modulating cell-type-specific SE signatures in PDAC. We found that TPL, cytotoxic to both pancreatic tumor cells and CAFs, disrupted SEs in a manner that resulted in the downregulation of SE-associated genes (e.g., BRD4, MYC, RNA Pol II, and Collagen 1) in both cell types at mRNA and protein levels. Our observations suggest that TPL acts as a SE interactive agent and may elicit its antitumor activity through SE disruption to re-program cellular cross talk and signaling in PDAC. Based on our findings, epigenetic reprogramming of transcriptional regulation using SE modulating compounds such as TPL may provide means for effective treatment options for pancreatic cancer patients.

7.
Cancer Res ; 80(20): 4324-4334, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-32928922

RESUMO

Adenosquamous cancer of the pancreas (ASCP) is a subtype of pancreatic cancer that has a worse prognosis and greater metastatic potential than the more common pancreatic ductal adenocarcinoma (PDAC) subtype. To distinguish the genomic landscape of ASCP and identify actionable targets for this lethal cancer, we applied DNA content flow cytometry to a series of 15 tumor samples including five patient-derived xenografts (PDX). We interrogated purified sorted tumor fractions from these samples with whole-genome copy-number variant (CNV), whole-exome sequencing, and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) analyses. These identified a variety of somatic genomic lesions targeting chromatin regulators in ASCP genomes that were superimposed on well-characterized genomic lesions including mutations in TP53 (87%) and KRAS (73%), amplification of MYC (47%), and homozygous deletion of CDKN2A (40%) that are common in PDACs. Furthermore, a comparison of ATAC-seq profiles of three ASCP and three PDAC genomes using flow-sorted PDX models identified genes with accessible chromatin unique to the ASCP genomes, including the lysine methyltransferase SMYD2 and the pancreatic cancer stem cell regulator RORC in all three ASCPs, and a FGFR1-ERLIN2 fusion associated with focal CNVs in both genes in a single ASCP. Finally, we demonstrate significant activity of a pan FGFR inhibitor against organoids derived from the FGFR1-ERLIN2 fusion-positive ASCP PDX model. Our results suggest that the genomic and epigenomic landscape of ASCP provide new strategies for targeting this aggressive subtype of pancreatic cancer. SIGNIFICANCE: These data provide a unique description of the ASCP genomic and epigenomic landscape and identify candidate therapeutic targets for this dismal cancer.


Assuntos
Carcinoma Adenoescamoso/genética , Cromatina/genética , Epigenoma , Mutação , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogênicas p21(ras) , Carcinoma Adenoescamoso/tratamento farmacológico , Carcinoma Adenoescamoso/patologia , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Cromatina/metabolismo , Humanos , Organoides , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Análise de Célula Única , Proteína Smad4/genética , Sequenciamento do Exoma
8.
Nature ; 569(7754): 131-135, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30996350

RESUMO

Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis largely owing to inefficient diagnosis and tenacious drug resistance. Activation of pancreatic stellate cells (PSCs) and consequent development of dense stroma are prominent features accounting for this aggressive biology1,2. The reciprocal interplay between PSCs and pancreatic cancer cells (PCCs) not only enhances tumour progression and metastasis but also sustains their own activation, facilitating a vicious cycle to exacerbate tumorigenesis and drug resistance3-7. Furthermore, PSC activation occurs very early during PDAC tumorigenesis8-10, and activated PSCs comprise a substantial fraction of the tumour mass, providing a rich source of readily detectable factors. Therefore, we hypothesized that the communication between PSCs and PCCs could be an exploitable target to develop effective strategies for PDAC therapy and diagnosis. Here, starting with a systematic proteomic investigation of secreted disease mediators and underlying molecular mechanisms, we reveal that leukaemia inhibitory factor (LIF) is a key paracrine factor from activated PSCs acting on cancer cells. Both pharmacologic LIF blockade and genetic Lifr deletion markedly slow tumour progression and augment the efficacy of chemotherapy to prolong survival of PDAC mouse models, mainly by modulating cancer cell differentiation and epithelial-mesenchymal transition status. Moreover, in both mouse models and human PDAC, aberrant production of LIF in the pancreas is restricted to pathological conditions and correlates with PDAC pathogenesis, and changes in the levels of circulating LIF correlate well with tumour response to therapy. Collectively, these findings reveal a function of LIF in PDAC tumorigenesis, and suggest its translational potential as an attractive therapeutic target and circulating marker. Our studies underscore how a better understanding of cell-cell communication within the tumour microenvironment can suggest novel strategies for cancer therapy.


Assuntos
Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/patologia , Fator Inibidor de Leucemia/metabolismo , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/patologia , Comunicação Parácrina , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Carcinogênese/genética , Carcinoma Ductal Pancreático/diagnóstico , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Linhagem Celular Tumoral , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos , Transição Epitelial-Mesenquimal , Feminino , Humanos , Fator Inibidor de Leucemia/antagonistas & inibidores , Fator Inibidor de Leucemia/sangue , Masculino , Espectrometria de Massas , Camundongos , Neoplasias Pancreáticas/diagnóstico , Comunicação Parácrina/efeitos dos fármacos , Receptores de OSM-LIF/deficiência , Receptores de OSM-LIF/genética , Receptores de OSM-LIF/metabolismo , Microambiente Tumoral
9.
Clin Sci (Lond) ; 130(17): 1499-510, 2016 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-27433023

RESUMO

Protein phosphorylation is a highly-regulated and reversible process that is precisely controlled by the actions of protein kinases and protein phosphatases. Factors that tip the balance of protein phosphorylation lead to changes in a wide range of cellular responses, including cell proliferation, differentiation and survival. The protein kinase C (PKC) family of serine/threonine kinases sits at nodal points in many signal transduction pathways; PKC enzymes have been the focus of considerable attention since they contribute to both normal physiological responses as well as maladaptive pathological responses that drive a wide range of clinical disorders. This review provides a background on the mechanisms that regulate individual PKC isoenzymes followed by a discussion of recent insights into their role in the pathogenesis of diseases such as cancer. We then provide an overview on the role of individual PKC isoenzymes in the regulation of cardiac contractility and pathophysiological growth responses, with a focus on the PKC-dependent mechanisms that regulate pump function and/or contribute to the pathogenesis of heart failure.


Assuntos
Coração/fisiologia , Miocárdio/enzimologia , Proteína Quinase C/metabolismo , Animais , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Fosforilação , Proteína Quinase C/genética
10.
Sci Signal ; 9(427): ra47, 2016 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-27165780

RESUMO

Alzheimer's disease (AD) is a progressive dementia disorder characterized by synaptic degeneration and amyloid-ß (Aß) accumulation in the brain. Through whole-genome sequencing of 1345 individuals from 410 families with late-onset AD (LOAD), we identified three highly penetrant variants in PRKCA, the gene that encodes protein kinase Cα (PKCα), in five of the families. All three variants linked with LOAD displayed increased catalytic activity relative to wild-type PKCα as assessed in live-cell imaging experiments using a genetically encoded PKC activity reporter. Deleting PRKCA in mice or adding PKC antagonists to mouse hippocampal slices infected with a virus expressing the Aß precursor CT100 revealed that PKCα was required for the reduced synaptic activity caused by Aß. In PRKCA(-/-) neurons expressing CT100, introduction of PKCα, but not PKCα lacking a PDZ interaction moiety, rescued synaptic depression, suggesting that a scaffolding interaction bringing PKCα to the synapse is required for its mediation of the effects of Aß. Thus, enhanced PKCα activity may contribute to AD, possibly by mediating the actions of Aß on synapses. In contrast, reduced PKCα activity is implicated in cancer. Hence, these findings reinforce the importance of maintaining a careful balance in the activity of this enzyme.


Assuntos
Doença de Alzheimer/genética , Mutação , Proteína Quinase C-alfa/genética , Sinapses/patologia , Animais , Células COS , Chlorocebus aethiops , Saúde da Família , Genoma , Genoma Humano , Hipocampo/metabolismo , Humanos , Camundongos , Neoplasias/metabolismo , Doenças Neurodegenerativas/metabolismo , Domínios Proteicos
11.
Cell Rep ; 12(8): 1252-60, 2015 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-26279568

RESUMO

The signaling output of protein kinase C (PKC) is exquisitely controlled, with its disruption resulting in pathophysiologies. Identifying the structural basis for autoinhibition is central to developing effective therapies for cancer, where PKC activity needs to be enhanced, or neurodegenerative diseases, where PKC activity should be inhibited. Here, we reinterpret a previously reported crystal structure of PKCßII and use docking and functional analysis to propose an alternative structure that is consistent with previous literature on PKC regulation. Mutagenesis of predicted contact residues establishes that the Ca(2+)-sensing C2 domain interacts intramolecularly with the kinase domain and the carboxyl-terminal tail, locking PKC in an inactive conformation. Ca(2+)-dependent bridging of the C2 domain to membranes provides the first step in activating PKC via conformational selection. Although the placement of the C1 domains remains to be determined, elucidation of the structural basis for autoinhibition of PKCßII unveils a unique direction for therapeutically targeting PKC.


Assuntos
Proteína Quinase C beta/química , Sequência de Aminoácidos , Animais , Células COS , Cálcio/metabolismo , Chlorocebus aethiops , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Proteína Quinase C beta/metabolismo , Estrutura Terciária de Proteína
12.
Cell ; 160(3): 489-502, 2015 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-25619690

RESUMO

Protein kinase C (PKC) isozymes have remained elusive cancer targets despite the unambiguous tumor promoting function of their potent ligands, phorbol esters, and the prevalence of their mutations. We analyzed 8% of PKC mutations identified in human cancers and found that, surprisingly, most were loss of function and none were activating. Loss-of-function mutations occurred in all PKC subgroups and impeded second-messenger binding, phosphorylation, or catalysis. Correction of a loss-of-function PKCß mutation by CRISPR-mediated genome editing in a patient-derived colon cancer cell line suppressed anchorage-independent growth and reduced tumor growth in a xenograft model. Hemizygous deletion promoted anchorage-independent growth, revealing that PKCß is haploinsufficient for tumor suppression. Several mutations were dominant negative, suppressing global PKC signaling output, and bioinformatic analysis suggested that PKC mutations cooperate with co-occurring mutations in cancer drivers. These data establish that PKC isozymes generally function as tumor suppressors, indicating that therapies should focus on restoring, not inhibiting, PKC activity.


Assuntos
Proteína Quinase C/química , Proteína Quinase C/genética , Animais , Linhagem Celular Tumoral , Transferência Ressonante de Energia de Fluorescência , Genes Supressores de Tumor , Xenoenxertos , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Camundongos Nus , Modelos Moleculares , Mutação , Transplante de Neoplasias , Neoplasias/tratamento farmacológico , Neoplasias/genética , Proteína Quinase C/metabolismo , Estrutura Terciária de Proteína
13.
Biochem Soc Trans ; 42(6): 1477-83, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25399557

RESUMO

Precise control of the amplitude of protein kinase C (PKC) signalling is essential for cellular homoeostasis, and disruption of this control leads to pathophysiological states such as cancer, neurodegeneration and diabetes. For conventional and novel PKC, this amplitude is meticulously tuned by multiple inputs that regulate the amount of enzyme in the cell, its ability to sense its allosteric activator diacylglycerol, and protein scaffolds that co-ordinate access to substrates. Key to regulation of the signalling output of most PKC isoenzymes is the ability of cytosolic enzyme to respond to the membrane-embedded lipid second messenger, diacylglycerol, in a dynamic range that prevents signalling in the absence of agonists but allows efficient activation in response to small changes in diacylglycerol levels. The present review discusses the regulatory inputs that control the spatiotemporal dynamics of PKC signalling, with a focus on conventional and novel PKC isoenzymes.


Assuntos
Proteína Quinase C/metabolismo , Transdução de Sinais , Regulação Alostérica , Humanos , Conformação Proteica , Proteína Quinase C/química , Sistemas do Segundo Mensageiro
14.
Chem Biol ; 21(4): 459-469, 2014 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-24631122

RESUMO

Optimal tuning of enzyme signaling is critical for cellular homeostasis. We use fluorescence resonance energy transfer reporters in live cells to follow conformational transitions that tune the affinity of a multidomain signal transducer, protein kinase C (PKC), for optimal response to second messengers. This enzyme comprises two diacylglycerol sensors, the C1A and C1B domains, that have a sufficiently high intrinsic affinity for ligand so that the enzyme would be in a ligand-engaged, active state if not for mechanisms that mask its domains. We show that both diacylglycerol sensors are exposed in newly synthesized PKC and that conformational transitions following priming phosphorylations mask the domains so that the lower affinity sensor, the C1B domain, is the primary diacylglycerol binder. The conformational rearrangements of PKC serve as a paradigm for how multimodule transducers optimize their dynamic range of signaling.


Assuntos
Proteína Quinase C/química , Proteína Quinase C/metabolismo , Transdução de Sinais , Animais , Células COS , Bovinos , Células Cultivadas , Chlorocebus aethiops , Diglicerídeos/química , Diglicerídeos/metabolismo , Transferência Ressonante de Energia de Fluorescência , Camundongos , Modelos Moleculares , Fosforilação , Conformação Proteica , Ratos
15.
Mol Cell Proteomics ; 12(12): 3498-508, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23788531

RESUMO

The plasma membrane serves as a dynamic interface that relays information received at the cell surface into the cell. Lipid second messengers coordinate signaling on this platform by recruiting and activating kinases and phosphatases. Specifically, diacylglycerol and phosphatidylinositol 3,4,5-trisphosphate activate protein kinase C and Akt, respectively, which then phosphorylate target proteins to transduce downstream signaling. This review addresses how the spatiotemporal dynamics of protein kinase C and Akt signaling can be monitored using genetically encoded reporters and provides information on how the coordination of signaling at protein scaffolds or membrane microdomains affords fidelity and specificity in phosphorylation events.


Assuntos
Diglicerídeos/metabolismo , Microdomínios da Membrana/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Proteína Quinase C/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-akt/metabolismo , Sistemas do Segundo Mensageiro , Animais , Células Eucarióticas/citologia , Células Eucarióticas/metabolismo , Regulação da Expressão Gênica , Humanos , Microdomínios da Membrana/química , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação , Proteína Quinase C/genética , Proteínas Proto-Oncogênicas c-akt/genética
16.
J Biol Chem ; 288(23): 16905-16915, 2013 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-23589289

RESUMO

The cellular activation of conventional protein kinase C (PKC) isozymes is initiated by the binding of their C2 domains to membranes in response to elevations in intracellular Ca(2+). Following this C2 domain-mediated membrane recruitment, the C1 domain binds its membrane-embedded ligand diacylglycerol, resulting in activation of PKC. Here we explore the molecular mechanisms by which the C2 domain controls the initial step in the activation of PKC. Using stopped-flow fluorescence spectroscopy to measure association and dissociation rate constants, we show that hydrophobic interactions are the major driving force in the binding of the C2 domain to anionic membranes, whereas electrostatic interactions dominate in membrane retention. Specifically, mutation of select hydrophobic or select basic residues in the Ca(2+)-binding loops reduces membrane affinity by distinct mechanisms; mutation of hydrophobic residues primarily alters association rate constants, whereas mutation of charged residues affects dissociation rate constants. Live cell imaging reveals that introduction of these mutations into full-length PKCα not only reduces the Ca(2+)-dependent translocation to plasma membrane but, by impairing the plasma membrane-sensing role of the C2 domain, causes phorbol ester-triggered redistribution of PKCα to other membranes, such as the Golgi. These data underscore the key role of the C2 domain in driving conventional PKC isozymes to the plasma membrane and reveal that not only the amplitude but also the subcellular location of conventional PKC signaling can be tuned by altering the affinity of this module for membranes.


Assuntos
Cálcio/metabolismo , Membrana Celular/enzimologia , Proteína Quinase C-alfa/metabolismo , Animais , Células COS , Membrana Celular/genética , Chlorocebus aethiops , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Interações Hidrofóbicas e Hidrofílicas , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Mutação , Proteína Quinase C-alfa/genética , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Ratos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Eletricidade Estática
17.
Chem Biol ; 19(8): 994-1000, 2012 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-22921066

RESUMO

To identify small molecules that selectively control hematopoietic stem cell differentiation, we performed an unbiased screen using primary human CD34(+) cells. We identified a plant-derived natural product, euphohelioscopin A, capable of selectively differentiating CD34(+) cells down the granulocyte/monocytic lineage. Euphohelioscopin A also inhibits proliferation and induces differentiation of the myeloid leukemia cell lines THP-1 and HL-60. Mechanistic studies revealed that euphohelioscopin A is an activator of protein kinase C (PKC), and that the promonocytic effects of this natural product are mediated by PKC activation. In addition to shedding insights into normal hematopoiesis, this work may ultimately facilitate the application of stem cell therapies to a host of myeloid dysfunctions.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Diterpenos/farmacologia , Proteína Quinase C/metabolismo , Antígenos CD34/metabolismo , Linhagem da Célula , Células Cultivadas , Diterpenos/química , Granulócitos/citologia , Células HEK293 , Células HL-60 , Células HeLa , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Indóis/farmacologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Maleimidas/farmacologia , Proteína Quinase C/química , Acetato de Tetradecanoilforbol/análogos & derivados , Acetato de Tetradecanoilforbol/farmacologia
18.
J Exp Zool A Ecol Genet Physiol ; 317(1): 9-23, 2012 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-22021243

RESUMO

The long-term effect of hypoxia is to decrease both the production and use of ATP and thus decrease the reliance on mitochondrial oxidative energy production. Yet, recent studies include more immediate affects of hypoxia on gene expression and these data suggest the maintenance of mitochondrial function. To better understand the short-term physiological response to hypoxia, we quantified metabolic mRNA expression in the heart ventricles and livers of the teleost fish Fundulus grandis exposed to partial oxygen pressure of 2.8 kPa (-13.5% air saturation).Twenty-eight individuals from a single population were exposed to hypoxia for 0, 4, 8, 12, 24, 48, and 96 hr. Liver and cardiac tissues were sampled from the same individuals at 0-48 hr. At 96 hr, only cardiac tissue was assayed. Gene expression was significantly different (ANOVA, P < 0.05) for 17 of 226 metabolic genes (7.5%) in cardiac tissue and for 20 of 256 (7.8%) metabolic genes in hepatic tissue. For the two tissues examined in this study, the maximum response occurred at different times. For cardiac tissue, using Dunnett's post hoc test, most of these significant differences occurred at 96 hr of exposure. For liver, all but one significant difference occurred at 4 hr. Surprisingly, too many (relative to random expectations) of the genes with significant increase in mRNA are involved in the oxidative phosphorylation pathway: 44% of the significant genes at 96 hr in the heart and 33% of the significant genes at 4 hr in the liver are involved in the oxidative phosphorylation pathway. These data indicate that there are tissue-specific differences in the timing of the response to hypoxia, yet both cardiac and hepatic tissues have increases in mRNA that code for enzyme in the oxidative phosphorylation pathway. If these changes in mRNA produce a similar change in protein, then these data suggest that the initial response to hypoxia involves an increase in the oxidative pathway potentially as a mechanism to maintain ATP production.


Assuntos
Regulação da Expressão Gênica , Hipóxia/metabolismo , Animais , Fundulidae/metabolismo , Fundulidae/fisiologia , Ventrículos do Coração/metabolismo , Fígado/metabolismo , Hibridização de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos , Oxirredução , RNA Mensageiro/metabolismo , Fatores de Tempo
19.
J Biol Chem ; 286(33): 28922-28930, 2011 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-21715334

RESUMO

Conformational changes acutely control protein kinase C (PKC). We have previously shown that the autoinhibitory pseudosubstrate must be removed from the active site in order for 1) PKC to be phosphorylated by its upstream kinase phosphoinositide-dependent kinase 1 (PDK-1), 2) the mature enzyme to bind and phosphorylate substrates, and 3) the mature enzyme to be dephosphorylated by phosphatases. Here we show an additional level of conformational control; binding of active site inhibitors locks PKC in a conformation in which the priming phosphorylation sites are resistant to dephosphorylation. Using homogeneously pure PKC, we show that the active site inhibitor Gö 6983 prevents the dephosphorylation by pure protein phosphatase 1 (PP1) or the hydrophobic motif phosphatase, pleckstrin homology domain leucine-rich repeat protein phosphatase (PHLPP). Consistent with results using pure proteins, treatment of cells with the competitive inhibitors Gö 6983 or bisindolylmaleimide I, but not the uncompetitive inhibitor bisindolylmaleimide IV, prevents the dephosphorylation and down-regulation of PKC induced by phorbol esters. Pulse-chase analyses reveal that active site inhibitors do not affect the net rate of priming phosphorylations of PKC; rather, they inhibit the dephosphorylation triggered by phorbol esters. These data provide a molecular explanation for the recent studies showing that active site inhibitors stabilize the phosphorylation state of protein kinases B/Akt and C.


Assuntos
Domínio Catalítico/fisiologia , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Animais , Células COS , Chlorocebus aethiops , Estabilidade Enzimática/efeitos dos fármacos , Estabilidade Enzimática/genética , Humanos , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Proteína Quinase C/química , Proteína Quinase C/genética , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Piruvato Desidrogenase Quinase de Transferência de Acetil
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