We have made the novel discovery that MARCKS protein (Myristoylated Alanine-Rich C Kinase Substrate) is a key molecule regulating mucin secretion from human bronchial epithelial ceils in vitro, and in mice in vivo. In this renewal, we propose to elucidate the precise molecular mechanisms whereby MARCKS controls the secretory process in airway epithelium, utilizing both in vitro and in vivo models. The hypothetical mechanism is a new paradigm for exocytotic release of preformed, membrane-bound secretory granules from airway epithelium. In this mechanism, membrane-bound secretory granules containing pre-formed mucin exist in the cell, associated with a number of specific fusion and docking proteins, as """"""""secretory modules."""""""" Each secretory module is fully equipped for fusion with the plasma membrane and resultant exocytotic release of the granule contents into the airway lumen. However, the module lacks the necessary apparatus to translocate to the plasmalemma. This is where MARCKS is critical: MARCKS, phosphorylated by PKC, moves to the cytoplasm, where it is brought to the secretory module by binding to the chaperone, heat shock protein 70 (Hsp 70). The Hsp 70/MARCKS complex binds to the secretory module via the N-terminus of MARCKS by interaction with a specific granule membrane protein, cysteine string protein (CSP). After its PKG-mediated dephosphorylation by protein phosphatase 2A (PP2A), MARCKS binds to actin/myosin while still attached to the secretory module. Thus, MARCKS links mucin granules to the contractile cytoskeleton. Upon translocation to the plasma membrane, fusion and docking proteins associated with the secretory module (Rab 3, VAMP 2 and VAMP 8) in conjunction with target proteins in the plasmlemma, position the granules for exocytosis. Critical aspects of this mechanism will be addressed utilizing 3 model systems: 1. primary human airway epithelial cells; 2. a human airway epithelial cell line (HBE1); and 3. an in vivo murine model of mucin hypersecretion.
The aims will define the mechanisms whereby MARCKS binds to Hsp70, how the MARCKS/Hsp70 complex binds to CSP on granule membranes, and specific fusion and docking proteins of the secretory module involved in exocytotic release of mucin both in vitro and in vivo.
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