RESUMO
Filamin A is an essential protein in the cell cytoskeleton because of its actin binding properties and unique homodimer rod-shaped structure, which organises actin into three-dimensional orthogonal networks imperative to cell motility, spreading and adhesion. Filamin A is subject to extensive posttranslational modification (PTM) which serves to co-ordinate cellular architecture and to modulate its large protein-protein interaction network which is key to the protein's role as a cellular signalling hub. Characterised PTMs include phosphorylation, irreversible cleavage, ubiquitin mediated degradation, hydroxylation and O-GlcNAcylation, with preliminary evidence of tyrosylation, carbonylation and acetylation. Each modification and its relation to filamin A function will be described here. These modifications are often aberrantly applied in a range of diseases including, but not limited to, cancer, cardiovascular disease and neurological disease and we discuss the concept of target specific PTMs with novel therapeutic modalities. In summary, our review represents a topical 'one-stop-shop' that enables understanding of filamin A function in cell homeostasis and provides insight into how a variety of modifications add an extra level of Filamin A control.
Assuntos
Filaminas , Processamento de Proteína Pós-Traducional , Filaminas/metabolismo , Humanos , Animais , Fosforilação , Neoplasias/metabolismoRESUMO
Inhibition of phosphodiesterase 4D (PDE4D) enzymes has been investigated as therapeutic strategy to treat memory problems in Alzheimer's disease (AD). Although PDE4D inhibitors are effective in enhancing memory processes in rodents and humans, severe side effects may hamper their clinical use. PDE4D enzymes comprise different isoforms, which, when targeted specifically, can increase treatment efficacy and safety. The function of PDE4D isoforms in AD and in molecular memory processes per se has remained unresolved. Here, we report the upregulation of specific PDE4D isoforms in transgenic AD mice and hippocampal neurons exposed to amyloid-ß. Furthermore, by means of pharmacological inhibition and CRISPR-Cas9 knockdown, we show that the long-form PDE4D3, -D5, -D7, and -D9 isoforms regulate neuronal plasticity and convey resilience against amyloid-ß in vitro. These results indicate that isoform-specific, next to non-selective, PDE4D inhibition is efficient in promoting neuroplasticity in an AD context. Therapeutic effects of non-selective PDE4D inhibitors are likely achieved through actions on long isoforms. Future research should identify which long PDE4D isoforms should be specifically targeted in vivo to both improve treatment efficacy and reduce side effects.
Assuntos
Doença de Alzheimer , Diester Fosfórico Hidrolases , Humanos , Animais , Camundongos , Neuritos , Peptídeos beta-Amiloides , Neurônios , Camundongos Transgênicos , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4RESUMO
Prostate cancer is one of the most common cancers in men and one of the top causes of death in men worldwide. Development and function of both normal prostate cells and early-stage prostate cancer cells are dependent on the cross-talk between androgen signalling systems and a variety of other transduction pathways which drive differentiation of these cells towards castration-resistance. One such signalling pathway is the ubiquitous cAMP signalling axis which functions to activate spatially restricted pools of cAMP effectors such as protein kinase A (PKA). The importance of both PKA and cAMP in the development of prostate cancer, and their interactions with the androgen receptor, were the focus of a review by Merkle and Hoffmann in 2010. In this updated review, we revisit this topic with analysis of current PKA-related prostate cancer literature and introduce novel information on the relevance of another cAMP effector, the exchange protein directly activated by cAMP (EPAC).
Assuntos
Fatores de Troca do Nucleotídeo Guanina , Neoplasias da Próstata , Masculino , Humanos , Fatores de Troca do Nucleotídeo Guanina/metabolismo , AMP Cíclico/metabolismo , Transdução de Sinais , Neoplasias da Próstata/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismoRESUMO
BACKGROUND: Androgen signalling remains the seminal therapeutic approach for the management of advanced prostate cancer. However, most tumours eventually shift towards an aggressive phenotype, characterised by androgen independence and treatment resistance. The cyclic adenosine monophosphate (cAMP) pathway plays a crucial role in regulating various cellular processes, with the phosphodiesterase PDE4D7 being a vital modulator of cAMP signalling in prostate cancer cells. METHODS: Using shRNA-mediated PDE4D7 knockdown in LNCaP cells and downstream analysis via RNA sequencing and phenotypic assays, we replicate clinical observations that diminished PDE4D7 expression promotes an aggressive prostate cancer phenotype. RESULTS: Our study provides evidence that loss of PDE4D7 expression represents a pivotal switch driving the transition from an androgen-sensitive state to hormone unresponsiveness and neuroendocrine differentiation. In addition, we demonstrate that PDE4D7 loss affects DNA repair pathways, conferring resistance to poly ADP ribose polymerase (PARP) inhibitors. CONCLUSION: Reinstating PDE4D7 expression sensitises prostate cancer cells to anti-androgens, DNA damage response inhibitors, and cytotoxic therapies. These findings provide significant insight into the regulatory role of PDE4D7 in the development of lethal prostate cancer and the potential of its modulation as a novel therapeutic strategy.
Assuntos
Androgênios , Neoplasias da Próstata , Masculino , Humanos , Neoplasias da Próstata/patologia , Próstata/metabolismo , Reparo do DNA , Diferenciação Celular , Receptores Androgênicos/genética , Linhagem Celular TumoralRESUMO
A robust body of evidence supports the concept that phosphodiesterase 10A (PDE10A) activity in the basal ganglia orchestrates the control of coordinated movement in human subjects. Although human mutations in the PDE10A gene manifest in hyperkinetic movement disorders that phenocopy many features of early Huntington's disease, characterization of the maladapted molecular mechanisms and aberrant signaling processes that underpin these conditions remains scarce. Recessive mutations in the GAF-A domain have been shown to impair PDE10A function due to the loss of striatal PDE10A protein levels, but here we show that this paucity is caused by irregular intracellular trafficking and increased PDE10A degradation in the cytosolic compartment. In contrast to GAF-A mutants, dominant mutations in the GAF-B domain of PDE10A induce PDE10A misfolding, a common pathological phenotype in many neurodegenerative diseases. These data demonstrate that the function of striatal PDE10A is compromised in disorders where disease-associated mutations trigger a reduction in the fidelity of PDE compartmentalization.
Assuntos
Membrana Celular/metabolismo , Doença de Huntington/genética , Neurônios/enzimologia , Diester Fosfórico Hidrolases/genética , Domínios Proteicos/genética , Animais , Autofagia/genética , Corpo Estriado/citologia , Corpo Estriado/patologia , AMP Cíclico/metabolismo , Embrião de Mamíferos , Células HEK293 , Humanos , Doença de Huntington/patologia , Hidrólise , Isoenzimas/genética , Isoenzimas/metabolismo , Mutação , Neurônios/citologia , Técnicas de Patch-Clamp , Diester Fosfórico Hidrolases/metabolismo , Cultura Primária de Células , Proteólise , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Cyclic AMP is a ubiquitous second messenger used to transduce intracellular signals from a variety of Gs-coupled receptors. Compartmentalisation of protein intermediates within the cAMP signaling pathway underpins receptor-specific responses. The cAMP effector proteins protein-kinase A and EPAC are found in complexes that also contain phosphodiesterases whose presence ensures a coordinated cellular response to receptor activation events. Popeye domain containing (POPDC) proteins are the most recent class of cAMP effectors to be identified and have crucial roles in cardiac pacemaking and conduction. We report the first observation that POPDC proteins exist in complexes with members of the PDE4 family in cardiac myocytes. We show that POPDC1 preferentially binds the PDE4A sub-family via a specificity motif in the PDE4 UCR1 region and that PDE4s bind to the Popeye domain of POPDC1 in a region known to be susceptible to a mutation that causes human disease. Using a cell-permeable disruptor peptide that displaces the POPDC1-PDE4 complex we show that PDE4 activity localized to POPDC1 modulates cycle length of spontaneous Ca2+ transients firing in intact mouse sinoatrial nodes.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico , AMP Cíclico , Animais , Proteínas de Transporte/metabolismo , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Camundongos , Diester Fosfórico Hidrolases/metabolismo , Sistemas do Segundo Mensageiro , Transdução de SinaisRESUMO
Cyclic AMP (cAMP) phosphodiesterase-4 (PDE4) enzymes degrade cAMP and underpin the compartmentalization of cAMP signaling through their targeting to particular protein complexes and intracellular locales. We describe the discovery and characterization of a small-molecule compound that allosterically activates PDE4 long isoforms. This PDE4-specific activator displays reversible, noncompetitive kinetics of activation (increased Vmax with unchanged Km), phenocopies the ability of protein kinase A (PKA) to activate PDE4 long isoforms endogenously, and requires a dimeric enzyme assembly, as adopted by long, but not by short (monomeric), PDE4 isoforms. Abnormally elevated levels of cAMP provide a critical driver of the underpinning molecular pathology of autosomal dominant polycystic kidney disease (ADPKD) by promoting cyst formation that, ultimately, culminates in renal failure. Using both animal and human cell models of ADPKD, including ADPKD patient-derived primary cell cultures, we demonstrate that treatment with the prototypical PDE4 activator compound lowers intracellular cAMP levels, restrains cAMP-mediated signaling events, and profoundly inhibits cyst formation. PDE4 activator compounds thus have potential as therapeutics for treating disease driven by elevated cAMP signaling as well as providing a tool for evaluating the action of long PDE4 isoforms in regulating cAMP-mediated cellular processes.
Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Animais , Linhagem Celular , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/efeitos dos fármacos , Cães , Ativação Enzimática/efeitos dos fármacos , Humanos , Células Madin Darby de Rim Canino , Fosforilação , Doenças Renais Policísticas/metabolismo , Isoformas de ProteínasRESUMO
The E-protein transcription factors guide immune cell differentiation, with E12 and E47 (hereafter called E2A) being essential for B-cell specification and maturation. E2A and the oncogenic chimera E2A-PBX1 contain three transactivation domains (ADs), with AD1 and AD2 having redundant, independent, and cooperative functions in a cell-dependent manner. AD1 and AD2 both mediate their functions by binding to the KIX domain of the histone acetyltransferase paralogues CREB-binding protein (CBP) and E1A-binding protein P300 (p300). This interaction is necessary for B-cell maturation and oncogenesis by E2A-PBX1 and occurs through conserved ΦXXΦΦ motifs (with Φ denoting a hydrophobic amino acid) in AD1 and AD2. However, disruption of this interaction via mutation of the KIX domain in CBP/p300 does not completely abrogate binding of E2A and E2A-PBX1. Here, we determined that E2A-AD1 and E2A-AD2 also interact with the TAZ2 domain of CBP/p300. Characterization of the TAZ2:E2A-AD1(1-37) complex indicated that E2A-AD1 adopts an α-helical structure and uses its ΦXXΦΦ motif to bind TAZ2. Whereas this region overlapped with the KIX recognition region, key KIX-interacting E2A-AD1 residues were exposed, suggesting that E2A-AD1 could simultaneously bind both the KIX and TAZ2 domains. However, we did not detect a ternary complex involving E2A-AD1, KIX, and TAZ2 and found that E2A containing both intact AD1 and AD2 is required to bind to CBP/p300. Our findings highlight the structural plasticity and promiscuity of E2A-AD1 and suggest that E2A binds both the TAZ2 and KIX domains of CBP/p300 through AD1 and AD2.
Assuntos
Proteína de Ligação a CREB/química , Proteína p300 Associada a E1A/genética , Domínios Proteicos/genética , Fator 3 de Transcrição/química , Linfócitos B/química , Linfócitos B/metabolismo , Proteína de Ligação a CREB/genética , Proteína de Ligação a CREB/ultraestrutura , Proteína p300 Associada a E1A/química , Proteína p300 Associada a E1A/ultraestrutura , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/ultraestrutura , Humanos , Mutação/genética , Proteínas de Fusão Oncogênica/química , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/ultraestrutura , Ligação Proteica/genética , Conformação Proteica , Fator 3 de Transcrição/genética , Fator 3 de Transcrição/ultraestruturaRESUMO
OBJECTIVE: Transient receptor potential (TRP) melastatin 7 (TRPM7) cation channel, a dual-function ion channel/protein kinase, regulates vascular smooth muscle cell (VSMC) Mg2+ homeostasis and mitogenic signaling. Mechanisms regulating vascular growth effects of TRPM7 are unclear, but epidermal growth factor (EGF) may be important because it is a magnesiotropic hormone involved in cellular Mg2+ regulation and VSMC proliferation. Here we sought to determine whether TRPM7 is a downstream target of EGF in VSMCs and if EGF receptor (EGFR) through TRPM7 influences VSMC function. Approach and results: Studies were performed in primary culture VSMCs from rats and humans and vascular tissue from mice deficient in TRPM7 (TRPM7+/Δkinase and TRPM7R/R). EGF increased expression and phosphorylation of TRPM7 and stimulated Mg2+ influx in VSMCs, responses that were attenuated by gefitinib (EGFR inhibitor) and NS8593 (TRPM7 inhibitor). Co-immunoprecipitation (IP) studies, proximity ligation assay (PLA) and live-cell imaging demonstrated interaction of EGFR and TRPM7, which was enhanced by EGF. PP2 (c-Src inhibitor) decreased EGF-induced TRPM7 activation and prevented EGFR-TRPM7 association. EGF-stimulated migration and proliferation of VSMCs were inhibited by gefitinib, PP2, NS8593 and PD98059 (ERK1/2 inhibitor). Phosphorylation of EGFR and ERK1/2 was reduced in VSMCs from TRPM7+/Δkinase mice, which exhibited reduced aortic wall thickness and decreased expression of PCNA and Notch 3, findings recapitulated in TRPM7R/R mice. CONCLUSIONS: We show that EGFR directly interacts with TRPM7 through c-Src-dependent processes. Functionally these phenomena regulate [Mg2+]i homeostasis, ERK1/2 signaling and VSMC function. Our findings define a novel signaling cascade linking EGF/EGFR and TRPM7, important in vascular homeostasis.
Assuntos
Fator de Crescimento Epidérmico/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Canais de Cátion TRPM/metabolismo , Animais , Proteína Tirosina Quinase CSK/metabolismo , Cálcio/metabolismo , Proteínas de Transporte de Cátions/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células HEK293 , Humanos , Magnésio/metabolismo , Camundongos Endogâmicos C57BL , Morfogênese , Músculo Liso Vascular/crescimento & desenvolvimento , Fosforilação , Cultura Primária de Células , Ratos Endogâmicos WKYRESUMO
Recent evidence has revealed that heterotrimeric G-proteins can be activated by cytoplasmic proteins that share an evolutionarily conserved sequence called the Gα-binding-and-activating (GBA) motif. This mechanism provides an alternative to canonical activation by G-protein-coupled receptors (GPCRs) and plays important roles in cell function, and its dysregulation is linked to diseases such as cancer. Here, we describe a discovery pipeline that uses biochemical and genetic approaches to validate GBA candidates identified by sequence similarity. First, putative GBA motifs discovered in bioinformatics searches were synthesized on peptide arrays and probed in batch for Gαi3 binding. Then, cDNAs encoding proteins with Gαi3-binding sequences were expressed in a genetically-modified yeast strain that reports mammalian G-protein activity in the absence of GPCRs. The resulting GBA motif candidates were characterized by comparison of their biochemical, structural, and signaling properties with those of all previously described GBA motifs in mammals (GIV/Girdin, DAPLE, Calnuc, and NUCB2). We found that the phospholipase Cδ4 (PLCδ4) GBA motif binds G-proteins with high affinity, has guanine nucleotide exchange factor activity in vitro, and activates G-protein signaling in cells, as indicated by bioluminescence resonance energy transfer (BRET)-based biosensors of G-protein activity. Interestingly, the PLCδ4 isoform b (PLCδ4b), which lacks the domains required for PLC activity, bound and activated G-proteins more efficiently than the full-length isoform a, suggesting that PLCδ4b functions as a G-protein regulator rather than as a PLC. In summary, we have identified PLCδ4 as a nonreceptor activator of G-proteins and established an experimental pipeline to discover and characterize GBA motif-containing proteins.
Assuntos
Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Fosfolipase C delta/química , Fosfolipase C delta/genética , Motivos de Aminoácidos , Cristalografia por Raios X , Subunidades alfa de Proteínas de Ligação ao GTP/química , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/química , Proteínas Heterotriméricas de Ligação ao GTP/genética , Humanos , Fosfolipase C delta/metabolismo , Ligação Proteica , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de SinaisRESUMO
Spatio-temporal regulation of localised cAMP nanodomains is highly dependent upon the compartmentalised activity of phosphodiesterase (PDE) cyclic nucleotide degrading enzymes. Strategically positioned PDE-protein complexes are pivotal to the homeostatic control of cAMP-effector protein activity that in turn orchestrate a wide range of cellular signalling cascades in a variety of cells and tissue types. Unsurprisingly, dysregulated PDE activity is central to the pathophysiology of many diseases warranting the need for effective therapies that target PDEs selectively. This short review focuses on the importance of activating compartmentalised cAMP signalling by displacing the PDE component of signalling complexes using cell-permeable peptide disrupters.
Assuntos
AMP Cíclico/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Transdução de Sinais , Animais , Sítios de Ligação , Diester Fosfórico Hidrolases/química , Domínios Proteicos , Relação Estrutura-AtividadeRESUMO
Phosphodiesterases (PDEs) have long been considered as targets for the treatment of Alzheimer's disease (AD) and a substantial body of evidence suggests that one sub-family from the super-family of PDEs, namely PDE4D, has particular significance in this context. This review discusses the role of PDE4 in the orchestration of cAMP response element binding signaling in AD and outlines the benefits of targeting PDE4D specifically. We examine the limited available literature that suggests PDE4 expression does not change in AD brains together with reports that show PDE4 inhibition as an effective treatment in this age-related neurodegenerative disease. Actually, aging induces changes in PDE4 expression/activity in an isoform and brain-region specific manner that proposes a similar complexity in AD brains. Therefore, a more detailed account of AD-related alterations in cellular/tissue location and the activation status of PDE4 is required before novel therapies can be developed to target cAMP signaling in this disease.
Assuntos
Doença de Alzheimer/enzimologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Doença de Alzheimer/tratamento farmacológico , Animais , Proteína de Ligação a CREB/metabolismo , AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Humanos , Transdução de SinaisRESUMO
BACKGROUND: Recent advances in the treatment of melanoma that involve immunotherapy and B-Raf inhibition have revolutionised cancer care for this disease. However, an un-met clinical need remains in B-Raf inhibitor resistant patients where first-generation B-Raf inhibitors provide only short-term disease control. In these cases, B-Raf inhibition leads to paradoxical activation of the C-Raf - MEK - ERK signalling pathway, followed by metastasis. PDE8A has been shown to directly interact with and modulate the cAMP microdomain in the vicinity of C-Raf. This interaction promotes C-Raf activation by attenuating the PKA-mediated inhibitory phosphorylation of the kinase. METHODS: We have used a novel cell-penetrating peptide agent (PPL-008) that inhibits the PDE8A - C-Raf complex in a human malignant MM415 melanoma cell line and MM415 melanoma xenograft mouse model to investigate ERK MAP kinase signalling. RESULTS: We have demonstrated that the PDE8A - C-Raf complex disruptor PPL-008 increased inhibitory C-Raf-S259 phosphorylation and significantly reduced phospho-ERK signalling. We have also discovered that the ability of PPL-008 to dampen ERK signalling can be used to counter B-Raf inhibitor-driven paradoxical activation of phospho-ERK in MM415 cells treated with PLX4032 (Vemurafenib). PPL-008 treatment also significantly retarded the growth of these cells. When applied to a MM415 melanoma xenograft mouse model, PPL-008C penetrated tumour tissue and significantly reduced phospho-ERK signalling in that domain. CONCLUSION: Our data suggests that the PDE8A-C-Raf complex is a promising therapeutic treatment for B-Raf inhibitor resistant melanoma.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Peptídeos Penetradores de Células/administração & dosagem , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Melanoma/tratamento farmacológico , Proteínas Proto-Oncogênicas c-raf/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Peptídeos Penetradores de Células/farmacologia , Feminino , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Melanoma/metabolismo , Camundongos , Ligação Proteica/efeitos dos fármacos , Vemurafenib/administração & dosagem , Vemurafenib/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Adrenergic stimulation of the heart engages cAMP and phosphoinositide second messenger signaling cascades. Cardiac phosphoinositide 3-kinase p110γ participates in these processes by sustaining ß-adrenergic receptor internalization through its catalytic function and by controlling phosphodiesterase 3B (PDE3B) activity via an unknown kinase-independent mechanism. We have discovered that p110γ anchors protein kinase A (PKA) through a site in its N-terminal region. Anchored PKA activates PDE3B to enhance cAMP degradation and phosphorylates p110γ to inhibit PIP(3) production. This provides local feedback control of PIP(3) and cAMP signaling events. In congestive heart failure, p110γ is upregulated and escapes PKA-mediated inhibition, contributing to a reduction in ß-adrenergic receptor density. Pharmacological inhibition of p110γ normalizes ß-adrenergic receptor density and improves contractility in failing hearts.
Assuntos
Proteínas de Ancoragem à Quinase A/metabolismo , Classe Ib de Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Miócitos Cardíacos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Classe Ib de Fosfatidilinositol 3-Quinase/química , Classe Ib de Fosfatidilinositol 3-Quinase/deficiência , Classe Ib de Fosfatidilinositol 3-Quinase/genética , Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/metabolismo , DNA/genética , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Cardíaca/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação , Mapeamento de Interação de Proteínas , Quinoxalinas/farmacologia , Receptores Adrenérgicos beta/metabolismo , Sistemas do Segundo Mensageiro , Homologia de Sequência de Aminoácidos , Tiazolidinedionas/farmacologiaRESUMO
Scaffolding proteins organize the information flow from activated G protein-coupled receptors (GPCRs) to intracellular effector cascades both spatially and temporally. By this means, signaling scaffolds, such as A-kinase anchoring proteins (AKAPs), compartmentalize kinase activity and ensure substrate selectivity. Using a phosphoproteomics approach we identified a physical and functional connection between protein kinase A (PKA) and Gpr161 (an orphan GPCR) signaling. We show that Gpr161 functions as a selective high-affinity AKAP for type I PKA regulatory subunits (RI). Using cell-based reporters to map protein-protein interactions, we discovered that RI binds directly and selectively to a hydrophobic protein-protein interaction interface in the cytoplasmic carboxyl-terminal tail of Gpr161. Furthermore, our data demonstrate that a binary complex between Gpr161 and RI promotes the compartmentalization of Gpr161 to the plasma membrane. Moreover, we show that Gpr161, functioning as an AKAP, recruits PKA RI to primary cilia in zebrafish embryos. We also show that Gpr161 is a target of PKA phosphorylation, and that mutation of the PKA phosphorylation site affects ciliary receptor localization. Thus, we propose that Gpr161 is itself an AKAP and that the cAMP-sensing Gpr161:PKA complex acts as cilium-compartmentalized signalosome, a concept that now needs to be considered in the analyzing, interpreting, and pharmaceutical targeting of PKA-associated functions.
Assuntos
Proteínas de Ancoragem à Quinase A/metabolismo , Proteína Quinase Tipo I Dependente de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Células HEK293 , Humanos , Luciferases de Renilla , Camundongos , Fosforilação , Peixe-ZebraRESUMO
zDHHC S-acyltransferases are enzymes catalyzing protein S-acylation, a common post-translational modification on proteins frequently affecting their membrane targeting and trafficking. The ankyrin repeat (AR) domain of zDHHC17 (HIP14) and zDHHC13 (HIP14L) S-acyltransferases, which is involved in both substrate recruitment and S-acylation-independent functions, was recently shown to bind at least six proteins, by specific recognition of a consensus sequence in them. To further refine the rules governing binding to the AR of zDHHC17, we employed peptide arrays based on zDHHC AR-binding motif (zDABM) sequences of synaptosomal-associated protein 25 (SNAP25) and cysteine string protein α (CSPα). Quantitative comparisons of the binding preferences of 400 peptides allowed us to construct a position-specific scoring matrix (PSSM) for zDHHC17 AR binding, with which we predicted and subsequently validated many putative zDHHC17 interactors. We identified 95 human zDABM sequences with unexpected versatility in amino acid usage; these sequences were distributed among 90 proteins, of which 62 have not been previously implicated in zDHHC17/13 binding. These zDABM-containing proteins included all family members of the SNAP25, sprouty, cornifelin, ankyrin, and SLAIN-motif containing families; seven endogenous Gag polyproteins sharing the same binding sequence; and several proteins involved in cytoskeletal organization, cell communication, and regulation of signaling. A dozen of the zDABM-containing proteins had more than one zDABM sequence, whereas isoform-specific binding to the AR of zDHHC17 was identified for the Ena/VASP-like protein. The large number of zDABM sequences within the human proteome suggests that zDHHC17 may be an interaction hub regulating many cellular processes.
Assuntos
Aciltransferases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteoma/metabolismo , Aciltransferases/química , Proteínas Adaptadoras de Transdução de Sinal/química , Repetição de Anquirina , Linhagem Celular , Proteínas de Choque Térmico HSP40/química , Proteínas de Choque Térmico HSP40/metabolismo , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/química , Peptídeos/química , Peptídeos/metabolismo , Análise Serial de Proteínas/métodos , Ligação Proteica , Proteoma/química , Proteína 25 Associada a Sinaptossoma/química , Proteína 25 Associada a Sinaptossoma/metabolismoRESUMO
Cyclic AMP (cAMP)-specific phosphodiesterase-4 (PDE4) enzymes underpin compartmentalised cAMP signalling by localising to distinct signalling complexes. PDE4 long isoforms can be phosphorylated by mitogen-activated protein kinase-activated protein kinase 2 (MK2), which attenuates activation of such enzymes through their phosphorylation by protein kinase A. Here we show that MK2 interacts directly with PDE4 long isoforms and define the sites of interaction. One is a unique site that locates within the regulatory upstream conserved region 1 (UCR1) domain and contains a core Phe141, Leu142 and Tyr143 (FLY) cluster (PDE4A5 numbering). Located with the second site is a critical core Phe693, Glu694, Phe695 (FQF) motif that is also employed in the sequestering of PDE4 long forms by an array of other signalling proteins, including the signalling scaffold ß-arrestin, the tyrosyl kinase Lyn, the SUMOylation E2 ligase UBC9, the dynein regulator Lis1 (PAFAH1B1) and the protein kinase Erk. We propose that the FQF motif lies at the heart of a multifunctional docking (MFD) site located within the PDE4 catalytic unit. It is clear from our data that, as well as aiding fidelity of interaction, the MFD site confers exclusivity of binding between PDE4 and a single specific partner protein from the cohort of signalling proteins whose interaction with PDE4 involves the FQF motif.
Assuntos
Domínio Catalítico , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/química , Peptídeos e Proteínas de Sinalização Intracelular/química , Simulação de Acoplamento Molecular , Proteínas Serina-Treonina Quinases/química , 1-Alquil-2-acetilglicerofosfocolina Esterase/química , 1-Alquil-2-acetilglicerofosfocolina Esterase/genética , 1-Alquil-2-acetilglicerofosfocolina Esterase/metabolismo , Motivos de Aminoácidos , Animais , Células COS , Chlorocebus aethiops , Proteínas Quinases Dependentes de AMP Cíclico/química , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Expressão Gênica , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/química , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/química , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Enzimas de Conjugação de Ubiquitina/química , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , beta-Arrestinas/química , beta-Arrestinas/genética , beta-Arrestinas/metabolismo , Quinases da Família src/química , Quinases da Família src/genética , Quinases da Família src/metabolismoRESUMO
Protein kinase B (Akt) is a key enzyme in the insulin signalling cascade, required for insulin-stimulated NO production in endothelial cells (ECs). Previous studies have suggested that AMP-activated protein kinase (AMPK) activation stimulates NO synthesis and enhances insulin-stimulated Akt activation, yet these studies have largely used indirect activators of AMPK. The effects of the allosteric AMPK activator A769662 on insulin signalling and endothelial function was therefore examined in cultured human macrovascular ECs. Surprisingly, A769662 inhibited insulin-stimulated NO synthesis and Akt phosphorylation in human ECs from umbilical veins (HUVECs) and aorta (HAECs). In contrast, the AMPK activators compound 991 and AICAR had no substantial inhibitory effect on insulin-stimulated Akt phosphorylation in ECs. Inhibition of AMPK with SBI-0206965 had no effect on the inhibition of insulin-stimulated Akt phosphorylation by A769662, suggesting the inhibitory action of A769662 is AMPK-independent. A769662 decreased IGF1-stimulated Akt phosphorylation yet had no effect on VEGF-stimulated Akt signalling in HUVECs, suggesting that A769662 attenuates early insulin/IGF1 signalling. The effects of A769662 on insulin-stimulated Akt phosphorylation were specific to human ECs, as no effect was observed in the human cancer cell lines HepG2 or HeLa, as well as in mouse embryonic fibroblasts (MEFs). A769662 inhibited insulin-stimulated Erk1/2 phosphorylation in HAECs and MEFs, an effect that was independent of AMPK in MEFs. Therefore, despite being a potent AMPK activator, A769662 has effects unlikely to be mediated by AMPK in human macrovascular ECs that reduce insulin sensitivity and eNOS activation.
Assuntos
Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Aorta/enzimologia , Células Endoteliais da Veia Umbilical Humana/enzimologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Pironas/farmacologia , Tiofenos/farmacologia , Proteínas Quinases Ativadas por AMP/metabolismo , Aorta/citologia , Compostos de Bifenilo , Ativação Enzimática/efeitos dos fármacos , Células HeLa , Células Hep G2 , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
UNLABELLED: Alterations in cAMP signaling are thought to contribute to neurocognitive and neuropsychiatric disorders. Members of the cAMP-specific phosphodiesterase 4 (PDE4) family, which contains >25 different isoforms, play a key role in determining spatial cAMP degradation so as to orchestrate compartmentalized cAMP signaling in cells. Each isoform binds to a different set of protein complexes through its unique N-terminal domain, thereby leading to targeted degradation of cAMP in specific intracellular compartments. However, the functional role of specific compartmentalized PDE4 isoforms has not been examined in vivo Here, we show that increasing protein levels of the PDE4A5 isoform in mouse hippocampal excitatory neurons impairs a long-lasting form of hippocampal synaptic plasticity and attenuates hippocampus-dependent long-term memories without affecting anxiety. In contrast, viral expression of a truncated version of PDE4A5, which lacks the unique N-terminal targeting domain, does not affect long-term memory. Further, overexpression of the PDE4A1 isoform, which targets a different subset of signalosomes, leaves memory undisturbed. Fluorescence resonance energy transfer sensor-based cAMP measurements reveal that the full-length PDE4A5, in contrast to the truncated form, hampers forskolin-mediated increases in neuronal cAMP levels. Our study indicates that the unique N-terminal localization domain of PDE4A5 is essential for the targeting of specific cAMP-dependent signaling underlying synaptic plasticity and memory. The development of compounds to disrupt the compartmentalization of individual PDE4 isoforms by targeting their unique N-terminal domains may provide a fruitful approach to prevent cognitive deficits in neuropsychiatric and neurocognitive disorders that are associated with alterations in cAMP signaling. SIGNIFICANCE STATEMENT: Neurons exhibit localized signaling processes that enable biochemical cascades to be activated selectively in specific subcellular compartments. The phosphodiesterase 4 (PDE4) family coordinates the degradation of cAMP, leading to the local attenuation of cAMP-dependent signaling pathways. Sleep deprivation leads to increased hippocampal expression of the PDE4A5 isoform. Here, we explored whether PDE4A5 overexpression mimics behavioral and synaptic plasticity phenotypes associated with sleep deprivation. Viral expression of PDE4A5 in hippocampal neurons impairs long-term potentiation and attenuates the formation of hippocampus-dependent long-term memories. Our findings suggest that PDE4A5 is a molecular constraint on cognitive processes and may contribute to the development of novel therapeutic approaches to prevent cognitive deficits in neuropsychiatric and neurocognitive disorders that are associated with alterations in cAMP signaling.
Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Hipocampo/citologia , Hipocampo/fisiologia , Memória de Longo Prazo/fisiologia , Plasticidade Neuronal/fisiologia , Neurônios/fisiologia , Análise de Variância , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Células Cultivadas , Colforsina/farmacologia , Condicionamento Clássico/fisiologia , AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Estimulação Elétrica , Ensaio de Imunoadsorção Enzimática , Medo , Transferência Ressonante de Energia de Fluorescência , Proteína Glial Fibrilar Ácida/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Plasticidade Neuronal/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ratos , Reconhecimento Psicológico/fisiologia , Transdução de Sinais/genética , Transdução Genética , Vasodilatadores/farmacologiaRESUMO
The Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is a sequence-specific DNA-binding protein that plays an essential role in viral episome replication and segregation, by recruiting the cellular complex of DNA replication onto the origin (oriP) and by tethering the viral DNA onto the mitotic chromosomes. Whereas the mechanisms of viral DNA replication are well documented, those involved in tethering EBNA1 to the cellular chromatin are far from being understood. Here, we have identified regulator of chromosome condensation 1 (RCC1) as a novel cellular partner for EBNA1. RCC1 is the major nuclear guanine nucleotide exchange factor for the small GTPase Ran enzyme. RCC1, associated with chromatin, is involved in the formation of RanGTP gradients critical for nucleo-cytoplasmic transport, mitotic spindle formation and nuclear envelope reassembly following mitosis. Using several approaches, we have demonstrated a direct interaction between these two proteins and found that the EBNA1 domains responsible for EBNA1 tethering to the mitotic chromosomes are also involved in the interaction with RCC1. The use of an EBNA1 peptide array confirmed the interaction of RCC1 with these regions and also the importance of the N-terminal region of RCC1 in this interaction. Finally, using confocal microscopy and Förster resonance energy transfer analysis to follow the dynamics of interaction between the two proteins throughout the cell cycle, we have demonstrated that EBNA1 and RCC1 closely associate on the chromosomes during metaphase, suggesting an essential role for the interaction during this phase, perhaps in tethering EBNA1 to mitotic chromosomes.