RESUMEN
Nonhydrolysable stable analogues of τ-phosphohistidine (τ-pHis) and π-pHis have been designed, aided by electrostatic surface potential calculations, and subsequently synthesized. The τ-pHis and π-pHis analogues (phosphopyrazole 8 and pyridyl amino amide 13, respectively) were used as haptens to generate pHis polyclonal antibodies. Both τ-pHis and π-pHis conjugates in the form of BSA-glutaraldehyde-τ-pHis and BSA-glutaraldehyde-π-pHis were synthesized and characterized by 31 P NMR spectroscopy. Commercially available τ-pHis (SC56-2) and π-pHis (SC1-1; SC50-3) monoclonal antibodies were used to show that the BSA-G-τ-pHis and BSA-G-π-pHis conjugates could be used to assess the selectivity of pHis antibodies in a competitive ELISA. Subsequently, the selectivity of the pHis antibodies generated by using phosphopyrazole 8 and pyridyl amino amide 13 as haptens was assessed by competitive ELISA against His, pSer, pThr, pTyr, τ-pHis and π-pHis. Antibodies generated by using phosphopyrazole 8 as a hapten were found to be selective for τ-pHis, and antibodies generated by using pyridyl amino amide 13 were found to be selective for π-pHis. Both τ- and π-pHis antibodies were shown to be effective in immunological experiments, including ELISA, western blot, and immunofluorescence. The τ-pHis antibody was also shown to be useful in the immunoprecipitation of proteins containing pHis.
Asunto(s)
Anticuerpos Monoclonales , Haptenos , Glutaral , FosforilaciónRESUMEN
Since the publication of the paper the authors have noted some errors in the text (please read the full correction for more information). These errors have now been corrected in both the HTML and PDF versions of the paper.
RESUMEN
Nucleoside diphosphate kinases (NDPKs) are multifunctional proteins encoded by the nme (non-metastatic cells) genes, also called NM23. NDPKs catalyze the transfer of γ-phosphate from nucleoside triphosphates to nucleoside diphosphates by a ping-pong mechanism involving the formation of a high-energy phosphohistidine intermediate. Growing evidence shows that NDPKs, particularly NDPK-B, can additionally act as a protein histidine kinase. Protein kinases and phosphatases that regulate reversible O-phosphorylation of serine, threonine, and tyrosine residues have been studied extensively in many organisms. Interestingly, other phosphoamino acids histidine, lysine, arginine, aspartate, glutamate, and cysteine exist in abundance but remain understudied due to the paucity of suitable methods and antibodies. The N-phosphorylation of histidine by histidine kinases via the two- or multi-component signaling systems is an important mediator in cellular responses in prokaryotes and lower eukaryotes, like yeast, fungi, and plants. However, in vertebrates knowledge of phosphohistidine signaling has lagged far behind and the identity of the protein kinases and protein phosphatases involved is not well established. This article will therefore provide an overview of our current knowledge on protein histidine phosphorylation particularly the role of nm 23 gene products as protein histidine kinases.
Asunto(s)
Nucleósido Difosfato Quinasas NM23/metabolismo , Animales , Canales de Calcio/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Proteínas de Unión al GTP/metabolismo , Histidina Quinasa/metabolismo , Humanos , Metástasis de la Neoplasia , Fosforilación , Canales de Potasio Calcio-Activados/metabolismoRESUMEN
NM23 proteins NDPK-A and -B bind to the cystic fibrosis (CF) protein CFTR in different ways from kinases such as PKA, CK2 and AMPK or linkers to cell calcium such as calmodulin and annexins. NDPK-A (not -B) interacts with CFTR through reciprocal AMPK binding/control, whereas NDPK-B (not -A) binds directly to CFTR. NDPK-B can activate G proteins without ligand-receptor coupling, so perhaps NDPK-B's binding influences energy supply local to a nucleotide-binding site (NBD1) needed for CFTR to function. Curiously, CFTR (ABC-C7) is a member of the ATP-binding cassette (ABC) protein family that does not obey 'clan rules'; CFTR channels anions and is not a pump, regulates disparate processes, is itself regulated by multiple means and is so pleiotropic that it acts as a hub that orchestrates calcium signaling through its consorts such as calmodulin/annexins. Furthermore, its multiple partners make CFTR dance to different tunes in different cellular and subcellular locations as it recycles from the plasma membrane to endosomes. CFTR function in airway apical membranes is inhibited by smoking which has been dubbed 'acquired CF'. CFTR alone among family members possesses a trap for other proteins that it unfurls as a 'fish-net' and which bears consensus phosphorylation sites for many protein kinases, with PKA being the most canonical. Recently, the site of CFTR's commonest mutation has been proposed as a knock-in mutant that alters allosteric control of kinase CK2 by log orders of activity towards calmodulin and other substrates after CFTR fragmentation. This link from CK2 to calmodulin that binds the R region invokes molecular paths that control lumen formation, which is incomplete in the tracheas of some CF-affected babies. Thus, we are poised to understand the many roles of NDPK-A and -B in CFTR function and, especially lumen formation, which is defective in the gut and lungs of many CF babies.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Nucleósido Difosfato Quinasas NM23/metabolismo , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Calmodulina/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Humanos , Dominios Proteicos , Isoformas de ProteínasRESUMEN
Protein phosphorylation is an important post-translational modification that is an integral part of cellular function. The O-phosphorylated amino-acid residues, such as phosphoserine (pSer), phosphothreonine (pThr) and phosphotyrosine (pTyr), have dominated the literature while the acid labile N-linked phosphorylated amino acids, such as phosphohistidine (pHis), have largely been historically overlooked because of the acidic conditions routinely used in amino-acid detection and analysis. This review highlights some misinterpretations that have arisen in the existing literature, pinpoints outstanding questions and potential future directions to clarify the role of pHis in mammalian signalling systems. Particular emphasis is placed on pHis isomerization and the hybrid functionality for both pHis and pTyr of the proposed τ-pHis analogue bearing the triazole residue.
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Histidina/análogos & derivados , Animales , Anticuerpos , Histidina/análisis , Histidina/química , Histidina/metabolismo , Humanos , IsomerismoRESUMEN
Inhibitors of phosphodiesterase 5 (PDE5) - sildenafil citrate (Viagra; Pfizer) and vardenafil hydrochloride (Levitra; Bayer/GlaxoSmithKline) - approved for the treatment of erectile dysfunction and pulmonary arterial hypertension also rescue the loss of cystic fibrosis (CF) chloride channel function and the mislocalization of F508del-CFTR in affected tissues in CF. Can PDE5 inhibitors provide a therapeutic strategy which combines ability to correct the basic ion transport defect and to control de-regulated lung inflammation in CF?
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística , Diclorhidrato de Vardenafil , Humanos , Imidazoles , Macrófagos/efectos de los fármacos , Piperazinas , SulfonasRESUMEN
Cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel mutations cause cystic fibrosis lung disease. A better understanding of CFTR regulatory mechanisms could suggest new therapeutic strategies. AMP-activated protein kinase (AMPK) binds to and phosphorylates CFTR, attenuating PKA-activated CFTR gating. However, the requirement for AMPK binding to CFTR and the potential role of other proteins in this regulation are unclear. We report that nucleoside diphosphate kinase A (NDPK-A) interacts with both AMPK and CFTR in overlay blots of airway epithelial cell lysates. Binding studies in Xenopus oocytes and transfected HEK-293 cells revealed that a CFTR peptide fragment that binds AMPK (CFTR-1420-57) disrupted the AMPK-CFTR interaction. Introduction of CFTR-1420-57 into human bronchial Calu-3 cells enhanced forskolin-stimulated whole cell conductance in patch clamp measurements. Similarly, injection of CFTR-1420-57 into Xenopus oocytes blocked the inhibition of cAMP-stimulated CFTR conductance by AMPK in two-electrode voltage clamp studies. AMPK also inhibited CFTR conductance with co-expression of WT NDPK-A in two-electrode voltage clamp studies, but co-expression of a catalytically inactive H118F mutant or various Ser-120 NDPK-A mutants prevented this inhibition. In vitro phosphorylation of WT NDPK-A was enhanced by purified active AMPK, but phosphorylation was prevented in H118F and phosphomimic Ser-120 NDPK-A mutants. AMPK does not appear to phosphorylate NDPK-A directly but rather promotes an NDPK-A autophosphorylation event that involves His-118 and Ser-120. Taken together, these results suggest that NDPK-A exists in a functional cellular complex with AMPK and CFTR in airway epithelia, and NDPK-A catalytic function is required for the AMPK-dependent regulation of CFTR.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Regulación de la Expresión Génica , Nucleósido Difosfato Quinasas NM23/fisiología , Animales , Bronquios/citología , Catálisis , Línea Celular , Células Epiteliales/citología , Células HEK293 , Humanos , Iones/química , Modelos Biológicos , Mutación , Oocitos/citología , Técnicas de Placa-Clamp , Fosforilación , Unión Proteica , Xenopus laevisRESUMEN
There is growing evidence to suggest that phosphohistidines are present at significant levels in mammalian cells and play a part in regulating cellular activity, in particular signaling pathways related to cancer. Because of the chemical instability of phosphohistidine at neutral or acid pH, it remains unclear how much phosphohistidine is present in cells. Here we describe a protocol for extracting proteins from mammalian cells in a way that avoids loss of covalent phosphates from proteins, and use it to measure phosphohistidine concentrations in human bronchial epithelial cell (16HBE14o-) lysate using 31P NMR spectroscopic analysis. Phosphohistidine is determined on average to be approximately one third as abundant as phosphoserine and phosphothreonine combined (and thus roughly 15 times more abundant than phosphotyrosine). The amount of phosphohistidine, and phosphoserine/phosphothreonine per gram of protein from a cell lysate was determined to be 23 µmol/g and 68 µmol/g respectively. The amount of phosphohistidine, and phosphoserine/phosphothreonine per cell was determined to be 1.8 fmol/cell, and 5.8 fmol/cell respectively. Phosphorylation is largely at the N3 (tele) position. Typical tryptic digest conditions result in loss of most of the phosphohistidine present, which may explain why the amounts reported here are greater than is generally seen using mass spectroscopy assays. The results further strengthen the case for a functional role of phosphohistidine in eukaryotic cells.
Asunto(s)
Histidina , Proteínas , Animales , Línea Celular , Histidina/análogos & derivados , Histidina/metabolismo , Humanos , Mamíferos/metabolismo , Fosforilación , Fosfoserina/metabolismo , Fosfotreonina/metabolismo , Proteínas/metabolismoRESUMEN
Cystic fibrosis results from mutations in the cystic fibrosis conductance regulator protein (CFTR), a cAMP/protein kinase A (PKA) and ATP-regulated Cl(-) channel. CFTR is increasingly recognized as a component of multiprotein complexes and although several inhibitory proteins to CFTR have been identified, protein complexes that stimulate CFTR function remain less well characterized. We report that annexin 2 (anx 2)-S100A10 forms a functional cAMP/PKA/calcineurin (CaN)-dependent complex with CFTR. Cell stimulation with forskolin/3-isobutyl-1-methylxanthine significantly increases the amount of anx 2-S100A10 that reciprocally coimmunoprecipitates with cell surface CFTR and calyculin A. Preinhibition with PKA or CaN inhibitors attenuates the interaction. Furthermore, we find that the acetylated peptide (STVHEILCKLSLEG, Ac1-14), but not the nonacetylated equivalent N1-14, corresponding to the S100A10 binding site on anx 2, disrupts the anx 2-S100A10/CFTR complex. Analysis of 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and CFTR(inh172)-sensitive currents, taken as indication of the outwardly rectifying Cl(-) channels (ORCC) and CFTR-mediated currents, respectively, showed that Ac1-14, but not N1-14, inhibits both the cAMP/PKA-dependent ORCC and CFTR activities. CaN inhibitors (cypermethrin, cyclosporin A) discriminated between ORCC/CFTR by inhibiting the CFTR(inh172)-, but not the DIDS-sensitive currents, by >70%. Furthermore, peptide Ac1-14 inhibited acetylcholine-induced short-circuit current measured across a sheet of intact intestinal biopsy. Our data suggests that the anx 2-S100A10/CFTR complex is important for CFTR function across epithelia.
Asunto(s)
Anexina A2/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Proteínas S100/metabolismo , Calcineurina/metabolismo , Línea Celular , Colforsina/farmacología , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/enzimología , Humanos , Técnicas In Vitro , Activación del Canal Iónico/efectos de los fármacos , Unión Proteica/efectos de los fármacosRESUMEN
Cystic fibrosis (CF) is characterised by impaired epithelial ion transport and is caused by mutations in the cystic fibrosis conductance regulator protein (CFTR), a cAMP/PKA and ATP-regulated chloride channel. We recently demonstrated a cAMP/PKA/calcineurin (CnA)-driven association between annexin 2 (anx 2), its cognate partner -S100A10 and cell surface CFTR. The complex is required for CFTR and outwardly rectifying chloride channel function in epithelia. Since the cAMP/PKA-induced Cl(-) current is absent in CF epithelia, we hypothesized that the anx 2-S100A10/CFTR complex may be defective in CFBE41o cells expressing the commonest F508del-CFTR (DeltaF-CFTR) mutation. Here, we demonstrate that, despite the presence of cell surface DeltaF-CFTR, cAMP/PKA fails to induce anx 2-S100A10/CFTR complex formation in CFBE41o- cells homozygous for F508del-CFTR. Mechanistically, PKA-dependent serine phosphorylation of CnA, CnA-anx 2 complex formation and CnA-dependent dephosphorylation of anx 2 are all defective in CFBE41o- cells. Immunohistochemical analysis confirms an abnormal cellular distribution of anx 2 in human and CF mouse epithelia. Thus, we demonstrate that cAMP/PKA/CnA signaling pathway is defective in CF cells and suggest that loss of anx 2-S100A10/CFTR complex formation may contribute to defective cAMP/PKA-dependent CFTR channel function.
Asunto(s)
Anexina A2/metabolismo , Calcineurina/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Fibrosis Quística/metabolismo , Proteínas S100/metabolismo , Adulto , Animales , Anexina A2/análisis , Calcineurina/análisis , Línea Celular , Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Técnica del Anticuerpo Fluorescente , Humanos , Inmunohistoquímica , Ratones , Mutación , Mucosa Respiratoria/química , Mucosa Respiratoria/citología , Mucosa Respiratoria/metabolismo , Proteínas S100/análisisRESUMEN
Annexin A2 and S100A10 proteins form a heterotetrameric complex and belong to different families of Ca(2+)-binding proteins. Annexins are non-EF-hand-type Ca(2+)-binding proteins that exhibit Ca(2+)-dependent binding to phospholipids and membranes in various tissues. They have been implicated in many Ca(2+)-regulated processes, including regulation of membrane organization, trafficking and interact with many targets such as ion channels. S100 proteins comprise a family of small proteins characterised by the presence of two consecutive EF-hand type Ca(2+)-binding motifs, interact with ion channels and regulate diverse processes and play a role as Ca(2+) sensors. Several annexin-S100 complexes have been characterized and require calcium. In this regard, S100A10 binding to annexin A2 is an exception in that it is regulated by a post-translational modification of annexin A2 and occurs independently of calcium concentration. This review focuses on the regulatory mechanism behind annexin A2-S100A10 complex formation, its role in regulating chloride transport in health and cystic fibrosis and the potential of this mechanism to integrate calcium and cAMP signalling in airway epithelia. We propose that cAMP/PKA-dependent activation of chloride flux (through CFTR and ORCC) requires the mobilisation of a multi-protein complex involving calcium binding proteins from three different families (annexin 2, S100A10 and Calcineurin A).
Asunto(s)
Anexina A2/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Regulación de la Expresión Génica , Proteínas S100/metabolismo , Animales , Transporte Biológico , Calcio/metabolismo , AMP Cíclico/metabolismo , Fibrosis Quística/metabolismo , Epitelio/metabolismo , Humanos , Modelos Biológicos , Fosforilación , Transporte de ProteínasRESUMEN
The inflammasome is an intracellular multi-protein complex that orchestrates the release of the pro-inflammatory cytokines IL-1ß and IL-18, and a form of cell death known as pyroptosis. Tyrosine phosphorylation of the inflammasome sensors NLRP3, AIM2, NLRC4, and the adaptor protein, apoptosis-associated speck-like protein (ASC) has previously been demonstrated to be essential in the regulation of the inflammasome. By using the pharmacological protein tyrosine phosphatase (PTPase) inhibitor, phenylarsine oxide (PAO), we have demonstrated that tyrosine dephosphorylation is an essential step for the activation of the NLRP3 and AIM2 inflammasomes in human and murine macrophages. We have also shown that PTPase activity is required for ASC nucleation leading to caspase-1 activation, IL-1ß, and IL-18 processing and release, and cell death. Furthermore, by site-directed mutagenesis of ASC tyrosine residues, we have identified the phosphorylation of tyrosine Y60 and Y137 of ASC as critical for inflammasome assembly and function. Therefore, we report that ASC tyrosine dephosphorylation and phosphorylation are crucial events for inflammasome activation.
Asunto(s)
Proteínas Adaptadoras de Señalización CARD/metabolismo , Proteínas de Unión al ADN/metabolismo , Inflamasomas/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Fosforilación/fisiología , Tirosina/metabolismo , Animales , Caspasa 1/metabolismo , Línea Celular , Citocinas/metabolismo , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Proteínas Tirosina Fosfatasas/metabolismo , Células TH1RESUMEN
The formation of a heterotetrameric complex between annexin 2 (anx 2) and S100A10 plays an important role in regulating the cellular distribution and biochemical properties of anx 2. A major distinction between the anx 2-S100A10 complex and other annexin-S100 complexes is that S100A10 binding to anx 2 occurs independently of calcium. Here we describe a cyclic 3',5'-adenosine monophosphate (cAMP) and cAMP-dependent protein kinase (PKA, EC 2.7.1.37)-dependent mechanism regulating anx 2-S100A10 complex formation and its interaction with the transient receptor potential vanilloid type 6 channel (TRPV6) in airway and gut epithelia. In both 16HBE14o- and Caco-2 cells, forskolin (FSK) stimulated increased anx 2-S100A10 complex formation, which was attenuated by either PKA inhibitors or calcineurin A (CnA) inhibitors. The anx 2-S100A10 complex association with TRPV6 was dependent on FSK-induced CnA-dependent dephosphorylation of anx 2. Analysis of the significance of the cAMP/PKA/CnA pathway on calcium influx showed that both PKA and CnA inhibitors attenuated Ca(45) uptake in Caco-2, but not 16HBE14o-, cells. Thus, the cAMP/PKA/CnA-induced anx 2-S100A10/TRPV6 complex may require additional factors for calcium influx or play a role independent of calcium influx in airway epithelia. In conclusion, our data demonstrates that cAMP/PKA/CnA signalling is important for anx 2-S100A10 complex formation and interaction with target molecules in both absorptive and secretory epithelia.
Asunto(s)
Anexina A2/metabolismo , Calcineurina/fisiología , Canales de Calcio/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , AMP Cíclico/fisiología , Sistema Respiratorio/metabolismo , Proteínas S100/metabolismo , Canales Catiónicos TRPV/metabolismo , Western Blotting , Células CACO-2 , Calcio/metabolismo , Membrana Celular/metabolismo , Citosol/metabolismo , Epitelio/metabolismo , Humanos , Inmunoprecipitación , Mucosa Intestinal/citología , Mucosa Intestinal/metabolismo , Mucosa Respiratoria , Sistema Respiratorio/citología , Transducción de Señal/fisiologíaRESUMEN
Cystic fibrosis (CF) results from mutations within the cystic fibrosis transmembrane-conductance regulator (CFTR) protein. The AMP-activated protein kinase (AMPK) is a heterotrimer composed of different isoforms of the alphabetagamma subunits, where the alpha1 catalytic subunit binds CFTR. Nucleoside diphosphate kinase (NDPK, NM23/awd) converts nucleoside diphosphates to nucleoside triphosphates but also acts as a protein kinase. We recently showed that AMPK alpha1 binds NDPK-A in lung epithelial cytosol. Here we report that in the plasma membrane of human airway epithelial cells, NDPK-A and AMPK alpha1 associate with the plasma membrane via CFTR. We show that the regulatory domain of CFTR binds NDPK-A whereas AMPK gamma1 or gamma2 bind the first nucleotide binding domain (NBD1) and AMPK alpha1 binds the second (NBD2) of CFTR. We also show that NDPK-A specifically binds AMPK alpha1 and AMPK gamma2 subunits, thereby specifying the isozyme of AMPK heterotrimer that associates with CFTR at the membrane. Thus, the combined data provide novel insight into the subunit composition of the epithelial CFTR/AMPK/NDPK complex, such that: CFTR interacts specifically with AMPK alpha1, gamma2 and NDPK-A and not NDPK-B or AMPK gamma1.
Asunto(s)
Membrana Celular/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Células Epiteliales/metabolismo , Complejos Multienzimáticos/metabolismo , Nucleósido-Difosfato Quinasa/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Quinasas Activadas por AMP , Animales , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Humanos , Pulmón/citología , Ratones , Modelos Biológicos , Nucleósido-Difosfato Quinasa/deficiencia , Nucleótidos/metabolismo , Unión ProteicaRESUMEN
Nucleoside diphosphate kinase (NDPK, NM23/awd) belongs to a multifunctional family of highly conserved proteins (approximately 16-20 kDa) containing two well-characterized isoforms (NM23-H1 and -H2; also known as NDPK A and B). NDPK catalyses the conversion of nucleoside diphosphates into nucleoside triphosphates, regulates a diverse array of cellular events and can act as a protein histidine kinase. AMPK (AMP-activated protein kinase) is a heterotrimeric protein complex that responds to cellular energy status by switching off ATP-consuming pathways and switching on ATP-generating pathways when ATP is limiting. AMPK was first discovered as an activity that inhibited preparations of ACC1 (acetyl-CoA carboxylase), a regulator of cellular fatty acid synthesis. We report that NM23-H1/NDPK A and AMPK alpha1 are associated in cytosol from two different tissue sources: rat liver and a human lung cell line (Calu-3). Co-immunoprecipitation and binding assay data from both cell types show that the H1/A (but not H2/B) isoform of NDPK is associated with AMPK complexes containing the alpha1 (but not alpha2) catalytic subunit. Manipulation of NM23-H1/NDPK A nucleotide transphosphorylation activity to generate ATP (but not GTP) enhances the activity of AMPK towards its specific peptide substrate in vitro and also regulates the phosphorylation of ACC1, an in vivo target for AMPK. Thus novel NM23-H1/NDPK A-dependent regulation of AMPK alpha1-mediated phosphorylation is present in mammalian cells.
Asunto(s)
Hígado/enzimología , Pulmón/enzimología , Complejos Multienzimáticos/metabolismo , Nucleósido-Difosfato Quinasa/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Quinasas Activadas por AMP , Acetiltransferasas/metabolismo , Animales , Línea Celular Tumoral , Humanos , Hígado/citología , Hígado/metabolismo , Pulmón/metabolismo , Modelos Biológicos , Nucleósido Difosfato Quinasas NM23 , Fosforilación , Unión Proteica , RatasRESUMEN
Cystic fibrosis results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-dependent protein kinase A (PKA) and ATP-regulated chloride channel. Here, we demonstrate that nucleoside diphosphate kinase B (NDPK-B, NM23-H2) forms a functional complex with CFTR. In airway epithelia forskolin/IBMX significantly increases NDPK-B co-localisation with CFTR whereas PKA inhibitors attenuate complex formation. Furthermore, an NDPK-B derived peptide (but not its NDPK-A equivalent) disrupts the NDPK-B/CFTR complex in vitro (19-mers comprising amino acids 36-54 from NDPK-B or NDPK-A). Overlay (Far-Western) and Surface Plasmon Resonance (SPR) analysis both demonstrate that NDPK-B binds CFTR within its first nucleotide binding domain (NBD1, CFTR amino acids 351-727). Analysis of chloride currents reflective of CFTR or outwardly rectifying chloride channels (ORCC, DIDS-sensitive) showed that the 19-mer NDPK-B peptide (but not its NDPK-A equivalent) reduced both chloride conductances. Additionally, the NDPK-B (but not NDPK-A) peptide also attenuated acetylcholine-induced intestinal short circuit currents. In silico analysis of the NBD1/NDPK-B complex reveals an extended interaction surface between the two proteins. This binding zone is also target of the 19-mer NDPK-B peptide, thus confirming its capability to disrupt NDPK-B/CFTR complex. We propose that NDPK-B forms part of the complex that controls chloride currents in epithelia.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Nucleósido Difosfato Quinasas NM23/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Línea Celular , Membrana Celular/metabolismo , Polaridad Celular , AMP Cíclico/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Citosol/metabolismo , Células Epiteliales/citología , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Nucleósido Difosfato Quinasas NM23/química , Unión Proteica , Estructura Terciaria de Proteína , Transporte de Proteínas , Sistema Respiratorio/citología , Adulto JovenRESUMEN
Human nasal epithelium must adapt to cold climates, and yet, in vitro, human ciliary beat frequency (CBF) is zero at 4 degrees C. Similarly, hibernating mammals do not die of pneumonia despite a core body temperature as low as 6 degrees C, implying that cilia continue to beat. Here, we show that protein kinase C (PKC) and Ca(2+)/calmodulin-dependent kinase II (CaMKII) regulate the profile of human nasal CBF in response to rising temperature from 4 degrees C. Onset of ciliary beat was at 10 degrees C in Medium 199, 7 degrees C in the presence of the PKC activator phorbol 12-myristate 13-acetate (PMA), the calcium ionophore ionomycin, or the CAMKII blocker myristoylated autocamtide-2 related inhibitory peptide (MACI), and 6 degrees C for the myristoylated peptide PKC inhibitor EGF-R Fragment 651-658 (MyrPKCI). During cell warming to 32 degrees C, the thermal profile was sigmoid in all solutions except those containing MACI+PMA. Surprisingly, cilia continued to beat despite 4 degrees C and were significantly more responsive to rising temperature with either MACI+PMA, or MACI+MyrPKCI. Our data suggest that CaMKII and PKC regulate the thermal slope and profile of CBF in vitro, and that when these protein kinases are manipulated, cilia can continue to beat despite hypothermia. These findings may relate to adaptive responses to cold climates.
Asunto(s)
Depuración Mucociliar/fisiología , Mucosa Nasal/fisiología , Proteína Quinasa C/fisiología , Transducción de Señal/fisiología , Adaptación Fisiológica , Proteínas Quinasas Dependientes de Calcio-Calmodulina/fisiología , Cilios/fisiología , Frío , Humanos , Isoenzimas/fisiología , Mucosa Nasal/enzimologíaRESUMEN
We report the synthesis of a stable analogue of τ-phosphohistidine: 4-phosphopyrazol-2-yl alanine (pPza). Polyclonal antibodies generated against the mimic show high reactivity and selectivity for τ-phosphohistidine, with minor or no cross-reactivity towards non-phosphorylated histidine or O-phosphoamino acids, including phosphotyrosine.
Asunto(s)
Aminoácidos Cíclicos/química , Histidina/análogos & derivados , Organofosfonatos/química , Pirazoles/química , Aminoácidos Cíclicos/inmunología , Animales , Reacciones Cruzadas , Histidina/química , Histidina/inmunología , Hidrólisis , Sueros Inmunes/inmunología , Isomerismo , Organofosfonatos/inmunología , Pirazoles/inmunología , RatasRESUMEN
In medium 199, ciliary beat frequency (CBF) in human nasal epithelium declines to 60% of baseline by 2 h and 1 nM phorbol-12-myristate-13-acetate (PMA) doubles the rate of decline by activating protein kinase C (PKC). We find that a reported negative control for PMA, 4alpha-phorbol (1 pM-1 nM)+/-1 nM PMA, not only maintains CBF at baseline, but arrests a pre-existing PMA-induced decline in CBF and alters the profile of multiple phosphorylated PKC species. Thus, 4alpha-phorbol not only potently prevents PMA from inhibiting CBF but also has potent effects on the phosphorylation of PKC.
Asunto(s)
Cilios/efectos de los fármacos , Forboles/farmacología , Proteína Quinasa C/metabolismo , Acetato de Tetradecanoilforbol/antagonistas & inhibidores , Western Blotting , Células Cultivadas , Cilios/enzimología , Relación Dosis-Respuesta a Droga , Humanos , Indicadores y Reactivos , Mucosa Nasal/efectos de los fármacos , Mucosa Nasal/enzimología , Fosforilación , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Polyclonal antibodies raised against 4-phosphothiophen-2-yl alanine 2a, a novel five-membered ring analogue of phosphotyrosine, showed high selectivity for phosphotyrosine and no cross-reactivity with other phosphorylated amino acids. Western blots showed that the polyclonal was similarly effective, but different in selectivity, to a commercially available monoclonal antibody.