RESUMO
Although protein kinase A (PKA) activation is known to increase ciliary beat frequency in humans the molecular mechanisms involved are unknown. We demonstrate that PKA is associated with ciliary axonemes where it specifically phosphorylates a 23-kDa protein. Because PKA is often localized to subcellular compartments in proximity to its substrate(s) via interactions with A-kinase-anchoring proteins (AKAPs), we investigated whether an AKAP was also associated with ciliary axonemes. This study has identified a novel 28 kDa AKAP (AKAP28)that is highly enriched in airway axonemes. The mRNA for AKAP28 is up-regulated as primary airway cells differentiate and is specifically expressed in tissues containing cilia and/or flagella. Additionally, both Western blot and immunostaining data show that AKAP28 is enriched in airway cilia. These data demonstrate that we have identified the first human axonemal AKAP, a protein that likely plays a role in the signaling necessary for efficient modulation of ciliary beat frequency.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte , Proteínas de Transporte/química , Cílios/metabolismo , Proteínas de Membrana , Proteínas de Ancoragem à Quinase A , Adenoviridae/genética , Sequência de Aminoácidos , Axônios/metabolismo , Sítios de Ligação , Northern Blotting , Brônquios/citologia , Proteínas de Transporte/metabolismo , Células Cultivadas , Clonagem Molecular , Proteínas Quinases Dependentes de AMP Cíclico/química , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , DNA Complementar/metabolismo , Células Epiteliais/metabolismo , Biblioteca Gênica , Vetores Genéticos , Glutationa Transferase/metabolismo , Proteínas de Fluorescência Verde , Células HeLa , Humanos , Immunoblotting , Imuno-Histoquímica , Proteínas Luminescentes/metabolismo , Dados de Sequência Molecular , Testes de Precipitina , Ligação Proteica , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Fatores de Tempo , Distribuição TecidualRESUMO
Benzo[a]pyrene (BaP) is a polycyclic aromatic hydrocarbon produced during incomplete combustion of organic compounds and is one of the more carcinogenic PAHs detected in tobacco smoke. Addition of organic compounds during tobacco processing increases the likelihood of finding elevated concentrations of BaP in mainstream smoke when compared to smoke from unprocessed tobacco. To test this hypothesis, the tobacco from Marlboro "Red" (processed) cigarettes and nonprocessed Burley tobacco leaf cigarettes was combusted and the resulting combustion products were collected onto glass fiber filter pads. The quantity of BaP in the tobacco tar extracted from the pads was measured using reversed-phase liquid chromatography with fluorescence detection. The concentration of BaP in the processed tobacco smoke was significantly higher than in unprocessed tobacco smoke. These results suggest that compounds added during tobacco processing increase the concentrations of BaP and therefore the carcinogenic potential of cigarettes.