RESUMO
We show that the antibody, clone mAb(D38C6), of the α isoform of the catalytic subunit of PKA (PKAcα) inhibits the kinase-catalyzed phosphorylation with low-nanomolar inhibitory potency (Ki = 2.4 nM). This property of the antibody was established by its capacity to displace a synthetic small-molecule active site-binding (orthosteric) photoluminescent ARC-Lum(Fluo) probe from the complex with PKAcα. Likely, the competitiveness of association of the two binders with the protein is coming from two excluding conformations of PKAcα to which the binders bind. mAb(D38C6) possesses a linear peptide epitope and it binds to the disordered C-tail of unliganded inactive conformer of PKAcα. ARC-Lum(Fluo) probes bind to the ordered and active conformation of PKAcα with Phe327 residue from the C-tail taking part in the formation of the active core. Consecutive application of these competitive PKAcα binders was used to develop an immunoassay allowing the determination of PKAcα concentration in complex biological solutions. At first, PKAcα was captured from the solution by the isoform-specific antibody and thereafter a high-affinity ARC-Lum(Fluo) probe was used to displace PKAcα from the binary complex. The developed immunoassay could be used for quantification of small amounts (starting from 93 pg, 2.3 fmol) of PKAcα in cell lysates.
Assuntos
Anticorpos Monoclonais/química , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/análise , Imunoensaio , Sondas Moleculares/química , Peptídeos/química , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Especificidade de Anticorpos , Sítios de Ligação , Ligação Competitiva , Linhagem Celular Tumoral , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/química , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Células HeLa , Humanos , Cinética , Medições Luminescentes , Modelos Moleculares , Peptídeos/metabolismo , Fosforilação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Estrutura Terciária de ProteínaRESUMO
Somatic mutations in protein kinase A catalytic α subunit (PRKACA) were found to be causative for 30-40% of cortisol-producing adenomas (CPA) of the adrenal gland, rendering PKA signalling constitutively active. In its resting state, PKA is a stable and inactive heterotetramer, consisting of two catalytic and two regulatory subunits with the latter inhibiting PKA activity. The human genome encodes three different PKA catalytic subunits and four different regulatory subunits that are preferentially expressed in different organs. In normal adrenal glands all regulatory subunits are expressed, while CPA exhibit reduced protein levels of the regulatory subunit IIß. In this study, we linked for the first time the loss of RIIß protein levels to the PRKACA mutation status and found the down-regulation of RIIß to arise post-transcriptionally. We further found the PKA subunit expression pattern of different tumours is also present in the zones of the normal adrenal cortex and demonstrate that the different PKA subunits have a differential expression pattern in each zone of the normal adrenal gland, indicating potential specific roles of these subunits in the regulation of different hormones secretion.
Assuntos
Glândulas Suprarrenais/fisiologia , Adenoma Adrenocortical/patologia , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/análise , Subunidade RIIbeta da Proteína Quinase Dependente de AMP Cíclico/análise , Perfilação da Expressão Gênica , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/genética , HumanosRESUMO
The cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMP-dependent protein kinase (PKA)-regulated Cl(-) channel, crucial for epithelial cell regulation of salt and water transport. Previous studies showed that ezrin, an actin binding and A-kinase anchoring protein (AKAP), facilitates association of PKA with CFTR. We used immunohistochemistry and immunogold transmission electron microscopy to localize CFTR, ezrin, and PKA type II regulatory (RII) and catalytic (C) subunits in striated duct cells of human parotid and submandibular glands. Immunohistochemistry localized the four proteins mainly to the apical membrane and the apical cytoplasm of striated duct cells. In acinar cells, ezrin localized to the luminal membrane, and PKA RII subunits were present in secretory granules, as previously described. Immunogold labeling showed that CFTR and PKA RII and C subunits were localized to the luminal membrane and associated with apical granules and vesicles of striated duct cells. Ezrin was present along the luminal membrane, on microvilli and along the junctional complexes between cells. Double labeling showed specific protein associations with apical granules and vesicles and along the luminal membrane. Ezrin, CFTR, and PKA RII and C subunits are co-localized in striated duct cells, suggesting the presence of signaling complexes that serve to regulate CFTR activity.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/análise , Regulador de Condutância Transmembrana em Fibrose Cística/análise , Proteínas do Citoesqueleto/análise , Glândula Parótida/química , Ductos Salivares/química , Glândula Submandibular/química , Proteínas de Ancoragem à Quinase A/análise , Membrana Celular/química , Membrana Celular/ultraestrutura , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/análise , Proteína Quinase Tipo II Dependente de AMP Cíclico/análise , Citoplasma/química , Citoplasma/ultraestrutura , Humanos , Imuno-Histoquímica , Junções Intercelulares/química , Junções Intercelulares/ultraestrutura , Microscopia Eletrônica de Transmissão , Microvilosidades/química , Microvilosidades/ultraestrutura , Glândula Parótida/citologia , Ductos Salivares/citologia , Vesículas Secretórias/química , Vesículas Secretórias/ultraestrutura , Glândula Submandibular/citologia , Vacúolos/química , Vacúolos/ultraestruturaRESUMO
The protein composition of oral fluid is modulated by environmental factors and physiological states, i.e. chemical, mechanical and pharmacologic stimuli, pathologic conditions, and psychological stress. Secretory protein concentrations in samples of whole saliva (WS) from children were measured and the results were subjected to statistical analysis. Protein expression was determined using electrophoresis and Western blotting. Protein profiles of children were significantly different from those of adults (n = 50, P < 0.05). All samples of saliva from children contained a group of high-molecular-weight (>90 kDa) proteins, whereas fewer than 5% of samples from adults had comparable bands. The ratio of the regulatory subunits (RII) of type II protein kinase A (an enzyme that regulates secretion) to total protein was stable in children's saliva, but variable in saliva from adults. Alpha amylase (alpha-amylase), an enzyme that digests carbohydrates, was less degraded in WS of children than in that of adults. Gingival crevicular fluid of both children and adults did not contain alpha-amylase or RII. No significant gender-based differences were found, but Caucasian children had higher salivary protein levels than children with an African background. Saliva collection is rapid, painless, non-invasive, economical, and yields findings that are reproducible. Objective, biochemical monitoring of secretory proteins in oral fluid of children may reveal responses to stressful stimuli.
Assuntos
Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/biossíntese , Saliva/enzimologia , Proteínas e Peptídeos Salivares/biossíntese , alfa-Amilases/biossíntese , Adolescente , Adulto , Western Blotting , Criança , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/análise , Eletroforese em Gel de Poliacrilamida , Feminino , Líquido do Sulco Gengival/enzimologia , Humanos , Masculino , Proteínas e Peptídeos Salivares/análise , Estresse Fisiológico/fisiologia , alfa-Amilases/análiseRESUMO
Major depression (MDD) is a common and potentially life-threatening condition. Widespread neurobiological abnormalities suggest abnormalities in fundamental cellular mechanisms as possible physiological mediators. Cyclic AMP-dependent protein kinase [also known as protein kinase A (PKA)] and protein kinase C (PKC) are important components of intracellular signal transduction cascades that are linked to G-coupled receptors. Previous research using both human peripheral and post-mortem brain tissue specimens suggests that a subset of depressed patients exhibit reduced PKA and PKC activity, which has been associated with reduced levels of specific protein isoforms. Prior research also suggests that specific clinical phenotypes, particularly melancholia and suicide, may be particularly associated with low activity. This study examined PKA and PKC protein levels in human post-mortem brain tissue samples from persons with MDD (n=20) and age- and sex-matched controls (n=20). Specific PKA subunits and PKC isoforms were assessed using Western blot analysis in post-mortem samples from Brodmann area 10, which has been implicated in reinforcement and reward mechanisms. The MDD sample exhibited significantly lower protein expression of PKA regulatory Ialpha (RIalpha), PKA catalytic alpha (Calpha) and Cbeta, PKCbeta1, and PKCepsilon relative to controls. The melancholic subgroup showed low PKA RIalpha and PKA Cbeta, while the portion of the MDD sample who died by suicide had low PKA RIalpha and PKA Calpha. These data continue to support the significance of abnormalities of these two key kinases, and suggest linkages between molecular endophenotypes and specific clinical phenotypes.