Jorge Piedrahita (College of Veterinary Medicine, North Carolina State University) for offering generous access to fluorescence microscopes

Jorge Piedrahita (College of Veterinary Medicine, North Carolina State University) for offering generous access to fluorescence microscopes. wild-type construct increased PKC and enhanced mucin secretion and MARCKS phosphorylation. Similar transfections of a dominant-negative or wild-type PKC construct did not affect either mucin secretion or MARCKS phosphorylation. The results suggest that PKC plays an important role in mucin secretion by airway epithelium via regulation of MARCKS phosphorylation. Mucus produced by epithelium of respiratory, gastrointestinal, and reproductive tracts provides a barrier between the external environment and cellular components of the epithelial layer. Mucins, the glycoprotein component of mucus, constitute a family of large, highly glycosylated macromolecules that impart physical (aggregation, viscosity, viscoelasticity, and lubrication) and biological (protection) properties to mucus (reviewed in Ref. 1). Airway mucus is an integral component of the mucociliary clearance system in the trachea and bronchi ABT-492 (Delafloxacin) and thus serves to protect the lower airways and alveoli from impingement of particulate matter and pathogens. However, mucin secretion is abnormally augmented in disease states, such as chronic bronchitis, asthma, and cystic fibrosis, increasing morbidity and mortality in these patients (reviewed in Refs. 1 and 2). Mucin hypersecretion is potentiated by many pathophysiological mediators, such as bacterial proteinases and endotoxin, adenine and guanine nucleotides, cytokines, inflammatory mediators, and eicosanoids (reviewed in Ref. 3). Intracellular mechanisms Rabbit polyclonal to CCNA2 and signaling molecules involved in the secretory process have not been fully elucidated. Protein kinase C (PKC) is a serine/threonine kinase involved in various exocytotic events in different cell types, including secretion of mucin,4,5 insulin,6 neurotransmitters,7 and platelet dense granules.8 Previously, we demonstrated that mucin secretion in airway epithelial cells is regulated by PKC via phosphorylation of the myristoylated alanine-rich C kinase substrate (MARCKS).9,10 In addition, we demonstrated that mucin hypersecretion in human airway epithelial cells in response to human neutrophil elastase (HNE) appears to be mediated by the -isoform of PKC (PKC).11 Not surprisingly, PKC, a novel PKC isoform, has a strong affinity for MARCKS and can phosphorylate MARCKS both and (Eppendorf 5417 centrifuge) for 40 minutes. The supernatant was collected and kept as the cytosolic fraction at ?80C until used. The remaining pellet was resuspended in lysis buffer containing 1% Triton X-100, sonicated, and centrifuged at 20,000 for 40 minutes. The supernatant membrane fraction was stored at ?80C until analyzed by Western blot. Western Blot Analysis Total MARCKS, phosphorylated MARCKS, PKC, and PKC protein levels were measured via Western blot. The protein concentrations of cell lysates were quantified by a Bradford assay (Bio-Rad Laboratories, Hercules, CA). Sample lysates were prepared by boiling in 2 SDS sample buffer [125 mmol/L Tris-Cl (pH 6.8), 25% glycerol, 4% SDS, 10% -mercaptoethanol, and 0.04% bromphenol blue] for 10 minutes. Sample lysates (30 to 60 g) were loaded on 10 or 12% SDS-polyacrylamide gels and then transferred to a polyvinylidene difluoride membrane (Schleicher & Schuell BioScience, Inc., Keene, NH) following electrophoresis. Polyvinylidene difluoride membranes were blocked with 5% nonfat milk and then probed with an appropriate dilution of primary antibody followed by horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibodies. Chemiluminescent detection was performed using ECL detection ABT-492 (Delafloxacin) reagents (GE Health care Life Sciences, Piscataway, NJ) following the manufacturers protocol. Amounts of specific proteins in bands were quantified using Labworks image acquisition and analysis software 4.0. (Ultra Violet Products, Ltd., Upland, CA). Antibodies against -tubulin (Santa Cruz Biotechnology, Inc., Santa Cruz, ABT-492 (Delafloxacin) CA) and E-cadherin (BD Biosciences, San Jose, CA) were used as loading controls for cytosolic and membrane fractions, respectively. Phosphorylated MARCKS (at serine 152/156) was detected with a specific antibody (Cell Signaling Technology, Inc.). After detection, the membrane was stripped in 62.5 mmol/L Tris-Cl (pH 6.5), 10% SDS, and 100 mmol/L -mercaptoethanol for 10 minutes at room temperature and reprobed with a monoclonal antibody against total MARCKS protein (clone no. 2F12; Upstate, Charlottesville, VA) to verify equal loading. Transient Transfection of.