RESUMEN
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.
Asunto(s)
Proteínas de Anclaje a la Quinasa A/metabolismo , Fosfatidilinositol 3-Quinasa Clase Ib/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Miocitos Cardíacos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Fosfatidilinositol 3-Quinasa Clase Ib/química , Fosfatidilinositol 3-Quinasa Clase Ib/deficiencia , Fosfatidilinositol 3-Quinasa Clase Ib/genética , Subunidad RIIalfa de la Proteína Quinasa Dependiente de AMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/metabolismo , ADN/genética , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Insuficiencia Cardíaca/tratamiento farmacológico , Insuficiencia Cardíaca/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación , Mapeo de Interacción de Proteínas , Quinoxalinas/farmacología , Receptores Adrenérgicos beta/metabolismo , Sistemas de Mensajero Secundario , Homología de Secuencia de Aminoácido , Tiazolidinedionas/farmacologíaRESUMEN
Regulatory mechanisms governing the sequence from progenitor cell proliferation to neuronal migration during corticogenesis are poorly understood. Here we report that phosphorylation of DISC1, a major susceptibility factor for several mental disorders, acts as a molecular switch from maintaining proliferation of mitotic progenitor cells to activating migration of postmitotic neurons in mice. Unphosphorylated DISC1 regulates canonical Wnt signalling via an interaction with GSK3ß, whereas specific phosphorylation at serine 710 (S710) triggers the recruitment of Bardet-Biedl syndrome (BBS) proteins to the centrosome. In support of this model, loss of BBS1 leads to defects in migration, but not proliferation, whereas DISC1 knockdown leads to deficits in both. A phospho-dead mutant can only rescue proliferation, whereas a phospho-mimic mutant rescues exclusively migration defects. These data highlight a dual role for DISC1 in corticogenesis and indicate that phosphorylation of this protein at S710 activates a key developmental switch.
Asunto(s)
Corteza Cerebral/embriología , Proteínas del Tejido Nervioso , Neuronas/citología , Neuronas/fisiología , Células Madre/citología , Animales , Células COS , Movimiento Celular/genética , Proliferación Celular , Corteza Cerebral/citología , Corteza Cerebral/fisiología , Chlorocebus aethiops , Técnicas de Silenciamiento del Gen , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Células HEK293 , Humanos , Ratones , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Células PC12 , Fosforilación , Unión Proteica , Ratas , Transducción de Señal , Proteínas Wnt/metabolismo , beta Catenina/metabolismoRESUMEN
Inhibitors of cyclic nucleotide phosphodiesterase (PDE) PDE3A have inotropic actions in human myocardium, but their long-term use increases mortality in patients with heart failure. Two isoforms in cardiac myocytes, PDE3A1 and PDE3A2, have identical amino acid sequences except for a unique N-terminal extension in PDE3A1. We expressed FLAG-tagged PDE3A1 and PDE3A2 in HEK293 cells and examined their regulation by PKA- and PKC-mediated phosphorylation. PDE3A1, which is localized to intracellular membranes, and PDE3A2, which is cytosolic, were phosphorylated at different sites within their common sequence. Exposure to isoproterenol led to phosphorylation of PDE3A1 at the 14-3-3-binding site S312, whereas exposure to PMA led to phosphorylation of PDE3A2 at an alternative 14-3-3-binding site, S428. PDE3A2 activity was stimulated by phosphorylation at S428, whereas PDE3A1 activity was not affected by phosphorylation at either site. Phosphorylation of PDE3A1 by PKA and of PDE3A2 by PKC led to shifts in elution on gel-filtration chromatography consistent with increased interactions with other proteins, and 2D electrophoresis of coimmunoprecipitated proteins revealed that the two isoforms have distinct protein interactomes. A similar pattern of differential phosphorylation of endogenous PDE3A1 and PDE3A2 at S312 and S428 is observed in human myocardium. The selective phosphorylation of PDE3A1 and PDE3A2 at alternative sites through different signaling pathways, along with the different functional consequences of phosphorylation for each isoform, suggest they are likely to have distinct roles in cyclic nucleotide-mediated signaling in human myocardium, and raise the possibility that isoform-selective inhibition may allow inotropic responses without an increase in mortality.
Asunto(s)
Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/metabolismo , Contracción Miocárdica/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Inhibidores de Fosfodiesterasa 3/farmacología , Proteínas 14-3-3/genética , Sitios de Unión/genética , Cromatografía en Gel , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Electroforesis en Gel Bidimensional , Activación Enzimática/fisiología , Células HEK293 , Humanos , Inmunoprecipitación , Isoenzimas/metabolismo , Isoproterenol/farmacología , Inhibidores de Fosfodiesterasa 3/metabolismo , Fosforilación , Proteína Quinasa C/metabolismoRESUMEN
LIS1, a WD40 repeat scaffold protein, interacts with components of the cytoplasmic dynein motor complex to regulate dynein-dependent cell motility. Here, we reveal that cAMP-specific phosphodiesterases (PDE4s) directly bind PAFAH1B1 (also known as LIS1). Dissociation of LIS1-dynein complexes is coupled with loss of dynein function, as determined in assays of both microtubule transport and directed cell migration in wounded monolayers. Such loss in dynein functioning can be achieved by upregulation of PDE4, which sequesters LIS1 away from dynein, thereby uncovering PDE4 as a regulator of dynein functioning. This process is facilitated by increased intracellular cAMP levels, which selectively augment the interaction of long PDE4 isoforms with LIS1 when they become phosphorylated within their regulatory UCR1 domain by protein kinase A (PKA). We propose that PDE4 and dynein have overlapping interaction sites for LIS1, which allows PDE4 to compete with dynein for LIS1 association in a process enhanced by the PKA phosphorylation of PDE4 long isoforms. This provides a further example to the growing notion that PDE4 itself may provide a signalling role independent of its catalytic activity, exemplified here by its modulation of dynein motor function.
Asunto(s)
1-Alquil-2-acetilglicerofosfocolina Esterasa/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/metabolismo , Dineínas/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Animales , Línea Celular , Movimiento Celular/fisiología , Chlorocebus aethiops , AMP Cíclico/biosíntesis , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/genética , Citoplasma/metabolismo , Humanos , Microtúbulos/metabolismo , Fosforilación , Pliegue de Proteína , Estructura Terciaria de Proteína , Transducción de SeñalRESUMEN
Clearance of fibrin through proteolytic degradation is a critical step of matrix remodeling that contributes to tissue repair in a variety of pathological conditions, such as stroke, atherosclerosis, and pulmonary disease. However, the molecular mechanisms that regulate fibrin deposition are not known. Here, we report that the p75 neurotrophin receptor (p75(NTR)), a TNF receptor superfamily member up-regulated after tissue injury, blocks fibrinolysis by down-regulating the serine protease, tissue plasminogen activator (tPA), and up-regulating plasminogen activator inhibitor-1 (PAI-1). We have discovered a new mechanism in which phosphodiesterase PDE4A4/5 interacts with p75(NTR) to enhance cAMP degradation. The p75(NTR)-dependent down-regulation of cAMP results in a decrease in extracellular proteolytic activity. This mechanism is supported in vivo in p75(NTR)-deficient mice, which show increased proteolysis after sciatic nerve injury and lung fibrosis. Our results reveal a novel pathogenic mechanism by which p75(NTR) regulates degradation of cAMP and perpetuates scar formation after injury.
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Fibrosis , Receptor de Factor de Crecimiento Nervioso/fisiología , Activador de Tejido Plasminógeno/antagonistas & inhibidores , Animales , Cicatriz/etiología , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4 , Fibrinólisis , Regulación de la Expresión Génica , Ratones , Ratones Noqueados , Inhibidor 1 de Activador Plasminogénico/genética , Nervio Ciático/lesiones , Heridas y LesionesRESUMEN
BACKGROUND AND AIMS: Infliximab attenuates serological responses to SARS-CoV-2 infection. Whether this is a class effect, or if anti-tumour necrosis factor [anti-TNF] level influences serological responses, remains unknown. METHODS: Seroprevalence and the magnitude of SARS-CoV-2 nucleocapsid antibody responses were measured in surplus serum from 11 422 (53.3% [6084] male; median age 36.8 years) patients with immune-mediated inflammatory diseases, stored at six therapeutic drug monitoring laboratories between January 29 and September 30, 2020. Data were linked to nationally held SARS-CoV-2 PCR results to July 11, 2021. RESULTS: Rates of PCR-confirmed SARS-CoV-2 infection were similar across treatment groups. Seroprevalence rates were lower in infliximab- and adalimumab- than vedolizumab-treated patients (infliximab: 3.0% [178/5893], adalimumab: 3.0% [152/5074], vedolizumab: 6.7% [25/375], pâ =â 0.003). The magnitude of SARS-CoV-2 reactivity was similar in infliximab- vs adalimumab-treated patients (median 4.30 cut-off index [COI] [1.94-9.96] vs 5.02 [2.18-18.70], pâ =â 0.164), but higher in vedolizumab-treated patients (median 21.60 COI [4.39-68.10, pâ <â 0.004). Compared to patients with detectable infliximab and adalimumab drug levels, patients with undetectable drug levels [<0.8 mg/L] were more likely to be seropositive for SARS-CoV-2 antibodies. One-third of patients who had PCR testing prior to antibody testing failed to seroconvert, all were treated with anti-TNF. Subsequent positive PCR-confirmed SARS-CoV-2 was seen in 7.9% [12/152] of patients after a median time of 183.5 days [129.8-235.3], without differences between drugs. CONCLUSION: Anti-TNF treatment is associated with lower SARS-CoV-2 nucleocapsid seroprevalence and antibody reactivity when compared to vedolizumab-treated patients. Higher seropositivity rates in patients with undetectable anti-TNF levels support a causal relationship, although confounding factors, such as combination therapy with a immunomodulator, may have influenced the results.
Asunto(s)
Productos Biológicos , COVID-19 , Enfermedades Inflamatorias del Intestino , Adalimumab , Adulto , Formación de Anticuerpos , Productos Biológicos/uso terapéutico , Monitoreo de Drogas , Humanos , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Infliximab , Masculino , SARS-CoV-2 , Estudios Seroepidemiológicos , Inhibidores del Factor de Necrosis Tumoral/uso terapéuticoRESUMEN
Enzymes from the PDE (phosphodiesterase) 4 cAMP-specific PDE family are crucial for the maintenance of compartmentalized cAMP responses in many cell types. Regulation of PDE activity can be achieved via post-translational modification such as phosphorylation by ERK (extracellular-signal-regulated kinase) MAPKs (mitogen-activated protein kinases) and PKA (protein kinase A). In the present paper, we report for the first time that PDE4 isoforms from the PDE4A and PDE4D subfamilies can be selectively modified by SUMO (small ubiquitin-related modifier). We have identified a single SUMO site within a consensus tetrapeptide motif, PsiKXE (where Psi represents a hydrophobic residue), which lies in the catalytic unit of these enzymes. SUMO modification of PDE4 at this site was observed upon overexpression of the SUMO E3 ligase PIASy [protein inhibitor of activated STAT (signal transducer and activator of transcription) Y] in HEK (human embryonic kidney)-293 cells and we identify PIASy as a novel binding partner for long PDE4 isoforms. Site-directed mutagenesis of the acceptor lysine residue ablated conjugation of PDE4 with SUMO, suggesting the presence of a single SUMO site in the first subdomain of the conserved PDE4 catalytic unit. This observation was supported by both cell-free in vitro SUMOylation assays and analysis of SUMOylated spot-immobilized peptide arrays. SUMO modification of long PDE4 isoforms serves to augment their activation by PKA phosphorylation and repress their inhibition by ERK phosphorylation. Following ligation of beta-adrenergic receptors, SUMOylation of PDE4 isoforms sufficiently amplified PKA-stimulated PDE4 activity to reduce markedly the PKA phosphorylation status of the beta2-adrenergic receptor. These results highlight a new means whereby cells might achieve the selective regulation of the activity of cAMP-specific PDE4 enyzmes.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Proteína SUMO-1/metabolismo , Secuencia de Aminoácidos , Animales , Células COS , Células Cultivadas , Chlorocebus aethiops , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/genética , Humanos , Datos de Secuencia Molecular , Fosforilación , Conformación Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transducción de SeñalRESUMEN
COVID-19 has dominated life in 2020 with, at the time of writing, over 4.9M global cases and >320 000 deaths. The impact has been most intensely felt in acute and critical care environments. However, with most UK elective work postponed, laboratory testing of faecal calprotectin halted due to potential risk of viral transmission and non-emergency endoscopies and surgeries cancelled, the secondary impact on chronic illnesses such as inflammatory bowel disease (IBD) is becoming apparent. Data from the Scottish Biologic Therapeutic Drug Monitoring (TDM) service shows a dramatic drop in TDM testing since the pandemic onset. April 2020 saw a 75.6% reduction in adalimumab testing and a 36.2% reduction in infliximab testing when compared with February 2020 data, a reduction coinciding with the widespread cancellation of outpatient and elective activity. It is feared that disruption to normal patterns of care and disease monitoring of biologic patients could increase the risk of disease flare and adverse clinical outcomes. Urgent changes in clinical practice have been instigated to mitigate the effects of the pandemic on routine clinical care. Further transformations are needed to maintain safe, effective, patient-centred IBD care in the future.
RESUMEN
The unique 88 amino acid N-terminal region of cAMP-specific phosphodiesterase-4D5 (PDE4D5) contains overlapping binding sites conferring interaction with the signaling scaffold proteins, betaarrestin and RACK1. A 38-mer peptide, whose sequence reflected residues 12 through 49 of PDE4D5, encompasses the entire N-terminal RACK1 Interaction Domain (RAID1) together with a portion of the beta-arrestin binding site. (1)H NMR and CD analyses indicate that this region has propensity to form a helical structure. The leucine-rich hydrophobic grouping essential for RACK1 interaction forms a discrete hydrophobic ridge located along a single face of an amphipathic alpha-helix with Arg34 and Asn36, which also play important roles in RACK1 binding. The Asn22/Pro23/Trp24/Asn26 grouping, essential for RACK1 interaction, was located at the N-terminal head of the amphipathic helix that contained the hydrophobic ridge. RAID1 is thus provided by a distinct amphipathic helical structure. We suggest that the binding of PDE4D5 to the WD-repeat protein, RACK1, may occur in a manner akin to the helix-helix interaction shown for G(gamma) binding to the WD-repeat protein, G(beta). A more extensive section of the PDE4D5 N-terminal sequence (Thr11-Ala85) is involved in beta-arrestin binding. Several residues within the RAID1 helix contribute to this interaction however. We show here that these residues form a focused band around the centre of the RAID1 helix, generating a hydrophobic patch (from Leu29, Val30 and Leu33) flanked by polar/charged residues (Asn26, Glu27, Asp28, Arg34). The interaction with beta-arrestin exploits a greater circumference on the RAID1 helix, and involves two residues (Glu27, Asp28) that do not contribute to RACK1 binding. In contrast, the interaction of RACK1 with RAID1 is extended over a greater length of the helix and includes Leu37/Leu38, which do not contribute to beta-arrestin binding. A membrane-permeable, stearoylated Val12-Ser49 38-mer peptide disrupted the interaction of both beta-arrestin and RACK1 with endogenous PDE4D5 in HEKB2 cells, whilst a cognate peptide with a Glu27Ala substitution selectively failed to disrupt PDE4D5/RACK1 interaction. The stearoylated Val12-Ser49 38-mer peptide enhanced the isoprenaline-stimulated PKA phosphorylation of the beta(2)-adrenergic receptors (beta(2)AR) and its activation of ERK, whilst the Glu27Ala peptide was ineffective in both these regards.
Asunto(s)
Arrestinas/metabolismo , Proteínas de Unión al GTP/metabolismo , Proteínas de Neoplasias/metabolismo , Resonancia Magnética Nuclear Biomolecular , Hidrolasas Diéster Fosfóricas/metabolismo , Receptores de Superficie Celular/metabolismo , Transducción de Señal , Agonistas de Receptores Adrenérgicos beta 2 , Agonistas Adrenérgicos beta/farmacología , Secuencia de Aminoácidos , Arrestinas/química , Sitios de Unión , Línea Celular , Dicroismo Circular , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3 , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4 , Células Epiteliales/efectos de los fármacos , Células Epiteliales/enzimología , Células Epiteliales/metabolismo , Proteínas de Unión al GTP/química , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Isoproterenol/farmacología , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas de Neoplasias/química , Péptidos/farmacología , Hidrolasas Diéster Fosfóricas/química , Fosforilación , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Receptores de Cinasa C Activada , Receptores Adrenérgicos beta 2/metabolismo , Receptores de Superficie Celular/química , beta-ArrestinasRESUMEN
Beta2-ARs (beta2-adrenoceptors) become desensitized rapidly upon recruitment of cytosolic beta-arrestin. PDE4D5 (family 4 cAMP-specific phosphodiesterase, subfamily D, isoform 5) can be recruited in complex with beta-arrestin, whereupon it regulates PKA (cAMP-dependent protein kinase) phosphorylation of the beta2-AR. In the present study, we have used novel technology, employing a library of overlapping peptides (25-mers) immobilized on cellulose membranes that scan the entire sequence of beta-arrestin 2, to define the interaction sites on beta-arrestin 2 for binding of PDE4D5 and the cognate long isoform, PDE4D3. We have identified a binding site in the beta-arrestin 2 N-domain for the common PDE4D catalytic unit and two regions in the beta-arrestin 2 C-domain that confer specificity for PDE4D5 binding. Alanine-scanning peptide array analysis of the N-domain binding region identified severely reduced interaction with PDE4D5 upon R26A substitution, and reduced interaction upon either K18A or T20A substitution. Similar analysis of the beta-arrestin 2 C-domain identified Arg286 and Asp291, together with the Leu215-His220 region, as being important for binding PDE4D5, but not PDE4D3. Transfection with wild-type beta-arrestin 2 profoundly decreased isoprenaline-stimulated PKA phosphorylation of the beta2-AR in MEFs (mouse embryo fibroblasts) lacking both beta-arrestin 1 and beta-arrestin 2. This effect was negated using either the R26A or the R286A mutant form of beta-arrestin 2 or a mutant with substitution of an alanine cassette for Leu215-His220, which showed little or no PDE4D5 binding, but was still recruited to the beta2-AR upon isoprenaline challenge. These data show that the interaction of PDE4D5 with both the N- and C-domains of beta-arrestin 2 are essential for beta2-AR regulation.
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , Arrestinas/química , Hidrolasas Diéster Fosfóricas/química , Análisis por Matrices de Proteínas , Arrestinas/genética , Arrestinas/metabolismo , Sitios de Unión , Línea Celular , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3 , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4 , Humanos , Riñón , Modelos Moleculares , Hidrolasas Diéster Fosfóricas/genética , Conformación Proteica , Proteínas Recombinantes/química , Transfección , beta-Arrestina 1 , Arrestina beta 2 , beta-ArrestinasRESUMEN
PDE4 family cAMP phosphodiesterases play a pivotal role in determining compartmentalised cAMP signalling through targeted cAMP breakdown. Expressing the widely found PDE4D5 isoform, as both bait and prey in a yeast 2-hybrid system, we demonstrated interaction consistent with the notion that long PDE4 isoforms form dimers. Four potential dimerization sites were uncovered using a scanning peptide array approach, where a recombinant purified PDE4D5 fusion protein was used to probe a 25-mer library of overlapping peptides covering the entire PDE4D5 sequence. Key residues involved in PDE4D5 dimerization were defined using a site-directed mutagenesis programme directed by an alanine scanning peptide array approach. Critical residues stabilising PDE4D5 dimerization were defined within the regulatory UCR1 region found in long, but not short, PDE4 isoforms, namely the Arg(173), Asn(174) and Asn(175) (DD1) cluster. Disruption of the DD1 cluster was not sufficient, in itself, to destabilise PDE4D5 homodimers. Instead, disruption of an additional interface, located on the PDE4 catalytic unit, was also required to convert PDE4D5 into a monomeric form. This second dimerization site on the conserved PDE4 catalytic unit is dependent upon a critical ion pair interaction. This involves Asp(463) and Arg(499) in PDE4D5, which interact in a trans fashion involving the two PDE4D5 molecules participating in the homodimer. PDE4 long isoforms adopt a dimeric state in living cells that is underpinned by two key contributory interactions, one involving the UCR modules and one involving an interface on the core catalytic domain. We propose that short forms do not adopt a dimeric configuration because, in the absence of the UCR1 module, residual engagement of the remaining core catalytic domain interface provides insufficient free energy to drive dimerization. The functioning of PDE4 long and short forms is thus poised to be inherently distinct due to this difference in quaternary structure.
Asunto(s)
Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/química , Multimerización de Proteína , Secuencia de Aminoácidos , Dominio Catalítico , AMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/genética , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/metabolismo , Células HEK293 , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Péptidos/química , Péptidos/metabolismo , Conformación Proteica , Estructura Terciaria de ProteínaRESUMEN
Cotinine is the primary metabolite of nicotine and the preferred biomarker for assessing cigarette smoke exposure. Several liquid chromatography-tandem mass spectrometry (LC-MS-MS) methods have been described for measuring cotinine in biological fluids. Sample preparation typically involves manual solvent evaporation and reconstitution steps. This study describes a novel LC-MS-MS method for the quantification of cotinine by using electrospray ionization with multiple reaction monitoring and cotinine-d3 as internal standard, coupled with an automated solid-phase extraction (SPE) procedure. The assay was linear over the analytical range of 0.5-1,000 ng/mL. The limits of detection and quantification were 0.13 and 0.20 ng/mL, respectively. Intra-assay and inter-assay imprecision of cotinine in all samples was <5 and <10% (coefficient of variation), respectively. The analytical recovery of cotinine spiked into plasma was >95-100%. Matrix effects in serum and plasma were <10%. A rapid, sensitive and specific LC-MS-MS method was developed and validated for the determination of cotinine in human plasma, using a straightforward automated SPE protocol. The application of this method to an epidemiological study has demonstrated its utility for batch analyses of a large sample set (>500 samples).
Asunto(s)
Cromatografía Liquida/métodos , Cotinina/sangre , Extracción en Fase Sólida/métodos , Espectrometría de Masas en Tándem/métodos , Cotinina/química , Estabilidad de Medicamentos , Humanos , Modelos Lineales , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
In Rat-1 cells, the dramatic decrease in the levels of both intracellular cyclic 3'5' adenosine monophosphate (cyclic AMP; cAMP) and in the activity of cAMP-activated protein kinase A (PKA) observed in mitosis was paralleled by a profound increase in cAMP hydrolyzing phosphodiesterase-4 (PDE4) activity. The decrease in PKA activity, which occurs during mitosis, was attributable to PDE4 activation as the PDE4 selective inhibitor, rolipram, but not the phosphodiesterase-3 (PDE3) inhibitor, cilostamide, specifically ablated this cell cycle-dependent effect. PDE4 inhibition caused Rat-1 cells to move from S phase into G2/M more rapidly, to transit through G2/M more quickly and to remain in G1 for a longer period. Inhibition of PDE3 elicited no observable effects on cell cycle dynamics. Selective immunopurification of each of the four PDE4 sub-families identified PDE4D as being selectively activated in mitosis. Subsequent analysis uncovered PDE4D9, an isoform whose expression can be regulated by Disrupted-In-Schizophrenia 1 (DISC1)/activating transcription factor 4 (ATF4) complex, as the sole PDE4 species activated during mitosis in Rat-1 cells. PDE4D9 becomes activated in mitosis through dual phosphorylation at Ser585 and Ser245, involving the combined action of ERK and an unidentified 'switch' kinase that has previously been shown to be activated by H2O2. Additionally, in mitosis, PDE4D9 also becomes phosphorylated at Ser67 and Ser81, through the action of MK2 (MAPKAPK2) and AMP kinase (AMPK), respectively. The multisite phosphorylation of PDE4D9 by all four of these protein kinases leads to decreased mobility (band-shift) of PDE4D9 on SDS-PAGE. PDE4D9 is predominantly concentrated in the perinuclear region of Rat-1 cells but with a fraction distributed asymmetrically at the cell margins. Our investigations demonstrate that the diminished levels of cAMP and PKA activity that characterise mitosis are due to enhanced cAMP degradation by PDE4D9. PDE4D9, was found to locate primarily not only in the perinuclear region of Rat-1 cells but also at the cell margins. We propose that the sequestration of PDE4D9 in a specific complex together with AMPK, ERK, MK2 and the H2O2-activatable 'switch' kinase allows for its selective multi-site phosphorylation, activation and regulation in mitosis.
Asunto(s)
Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/metabolismo , Mitosis , Proteínas del Tejido Nervioso/metabolismo , Factor de Transcripción Activador 4/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Activación Enzimática , Interfase , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Datos de Secuencia Molecular , Fosforilación , Isoformas de Proteínas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , RatasRESUMEN
The specification of biological decisions by signaling pathways is encoded by the interplay between activation dynamics and network topologies. Although we can describe complex networks, we cannot easily determine which topology the cell actually uses to transduce a specific signal. Experimental testing of all plausible topologies is infeasible because of the combinatorially large number of experiments required to explore the complete hypothesis space. Here, we demonstrate that Bayesian inference-based modeling provides an approach to explore and constrain this hypothesis space,permitting the rational ranking of pathway models. Our approach can use measurements of a limited number of biochemical species when combined with multiple perturbations. As proof of concept, we examined the activation of the extracellular signal-regulated kinase (ERK) pathway by epidermal growth factor. The predicted and experimentally validated model shows that both Raf-1 and, unexpectedly,B-Raf are needed to fully activate ERK in two different cell lines. Thus, our formal methodology rationally infers evidentially supported pathway topologies even when a limited number of biochemical and kinetic measurements are available.
Asunto(s)
Algoritmos , Biología Computacional/métodos , Modelos Biológicos , Transducción de Señal/genética , Teorema de Bayes , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Inmunoprecipitación , Proteínas Proto-Oncogénicas B-raf/metabolismo , Proteínas Proto-Oncogénicas c-raf/metabolismo , ARN Interferente Pequeño/genéticaRESUMEN
The specification of biological decisions by signaling pathways is encoded by the interplay between activation dynamics and network topologies. Although we can describe complex networks, we cannot easily determine which topology the cell actually uses to transduce a specific signal. Experimental testing of all plausible topologies is infeasible because of the combinatorially large number of experiments required to explore the complete hypothesis space. Here, we demonstrate that Bayesian inference-based modeling provides an approach to explore and constrain this hypothesis space, permitting the rational ranking of pathway models. Our approach can use measurements of a limited number of biochemical species when combined with multiple perturbations. As proof of concept, we examined the activation of the extracellular signal-regulated kinase (ERK) pathway by epidermal growth factor. The predicted and experimentally validated model shows that both Raf-1 and, unexpectedly, B-Raf are needed to fully activate ERK in two different cell lines. Thus, our formal methodology rationally infers evidentially supported pathway topologies even when a limited number of biochemical and kinetic measurements are available.
Asunto(s)
Biología Computacional/métodos , Modelos Biológicos , Transducción de Señal/fisiología , Animales , Teorema de Bayes , Factor de Crecimiento Epidérmico/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Inmunoprecipitación , Células PC12 , Fosforilación , Proteínas Proto-Oncogénicas B-raf/metabolismo , Proteínas Proto-Oncogénicas c-raf/metabolismo , ARN Interferente Pequeño/genética , RatasRESUMEN
Synaptic spines are dynamic structures that regulate neuronal responsiveness and plasticity. We examined the role of the schizophrenia risk factor DISC1 in the maintenance of spine morphology and function. We found that DISC1 anchored Kalirin-7 (Kal-7), regulating access of Kal-7 to Rac1 and controlling the duration and intensity of Rac1 activation in response to NMDA receptor activation in both cortical cultures and rat brain in vivo. These results explain why Rac1 and its activator (Kal-7) serve as important mediators of spine enlargement and why constitutive Rac1 activation decreases spine size. This mechanism likely underlies disturbances in glutamatergic neurotransmission that have been frequently reported in schizophrenia that can lead to alteration of dendritic spines with consequential major pathological changes in brain function. Furthermore, the concept of a signalosome involving disease-associated factors, such as DISC1 and glutamate, may well contribute to the multifactorial and polygenetic characteristics of schizophrenia.
Asunto(s)
Espinas Dendríticas/fisiología , Ácido Glutámico/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Sinapsis/fisiología , Proteína de Unión al GTP rac1/metabolismo , Animales , Células Cultivadas , Corteza Cerebral/citología , Corteza Cerebral/fisiología , Potenciales Postsinápticos Excitadores/fisiología , Técnicas de Silenciamiento del Gen , Proteínas del Tejido Nervioso/genética , Neuronas/citología , Neuronas/fisiología , Interferencia de ARN , Ratas , Ratas Sprague-Dawley , Receptores AMPA/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Transducción de Señal , Factores de Tiempo , Técnicas de Cultivo de TejidosRESUMEN
Beta arrestins are molecular scaffolds that can bring together three-component mitogen-activated protein kinase signalling modules to promote signal compartmentalisation. We use peptide array technology to define novel interfaces between components within the c-Jun N-terminal kinase (JNK)/beta arrestin signalling complex. We show that beta arrestin 1 and beta arrestin 2 associate with JNK3 via the kinase N-terminal domain in a region that, surprisingly, does not harbour a known 'common docking' motif. In the N-domain and C-terminus of beta arrestin 1 and beta arrestin 2 we identify two novel apoptosis signal-regulating kinase 1 binding sites and in the N-domain of the beta arrestin 1 and beta arrestin 2 we identify a novel MKK4 docking site.
Asunto(s)
Arrestinas/química , Arrestinas/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Proteína Quinasa 10 Activada por Mitógenos/química , Proteína Quinasa 10 Activada por Mitógenos/metabolismo , Péptidos/metabolismo , Análisis por Matrices de Proteínas/métodos , Secuencia de Aminoácidos , Animales , Arrestinas/genética , MAP Quinasa Quinasa 4/química , MAP Quinasa Quinasa 4/genética , MAP Quinasa Quinasa 4/metabolismo , Proteína Quinasa 10 Activada por Mitógenos/genética , Modelos Moleculares , Datos de Secuencia Molecular , Biblioteca de Péptidos , Péptidos/genética , Unión Proteica , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , beta-ArrestinasRESUMEN
The involvement of the Nuclear distribution element-like (Ndel1; Nudel) protein in the recruitment of the dynein complex is critical for neurodevelopment and potentially important for neuronal disease states. The PDE4 family of phosphodiesterases specifically degrades cAMP, an important second messenger implicated in learning and memory functions. Here we show for the first time that Ndel1 can interact directly with PDE4 family members and that the interaction of Ndel1 with the PDE4D3 isoform is uniquely disrupted by elevation of intracellular cAMP levels. While all long PDE4 isoforms are subject to stimulatory PKA phosphorylation within their conserved regulatory UCR1 domain, specificity for release of PDE4D3 is conferred due to the PKA-dependent phosphorylation of Ser13 within the isoform-specific, unique amino-terminal domain of PDE4D3. Scanning peptide array analyses identify a common region on Ndel1 for PDE4 binding and an additional region that is unique to PDE4D3. The common site lies within the stutter region that links the second coiled-coil region to the unstable third coiled-coil regions of Ndel1. The additional binding region unique to PDE4D3 penetrates into the start of the third coiled-coil region that can undergo tail-to-tail interactions between Ndel1 dimers to form a 4 helix bundle. We demonstrate Ndel1 self-interaction in living cells using a BRET approach with luciferase- and GFP-tagged forms of Ndel1. BRET assessed Ndel1-Ndel1 self-interaction is amplified through the binding of PDE4 isoforms. For PDE4D3 this effect is ablated upon elevation of intracellular cAMP due to PKA-mediated phosphorylation at Ser13, while the potentiating effects of PDE4B1 and PDE4D5 are resistant to cAMP elevation. PDE4D long isoforms and Ndel1 show a similar sub-cellular distribution in hippocampus and cortex and locate to post-synaptic densities. We show that Ndel1 sequesters EPAC, but not PKA, in order to form a cAMP signalling complex. We propose that a key function of the Ndel1 signalling scaffold is to signal through cAMP by sequestering EPAC, whose activity may thus be specifically regulated by sequestered PDE4 that also stabilizes Ndel1-Ndel1 self-interaction. In the case of PDE4D3, its association with Ndel1 is dynamically regulated by PKA input through its ability to phosphorylate Ser13 in the unique N-terminal region of this isoform, triggering the specific release of PDE4D3 from Ndel1 when cAMP levels are elevated. We propose that Ser13 may act as a redistribution trigger in PDE4D3, allowing it to dynamically re-shape cAMP gradients in distinct intracellular locales upon its phosphorylation by PKA.