The long-term goal of this project is to improve our understanding of the cellular and molecular regulation mechanisms of mucociliary clearance, an important host defense mechanism of the lung. Ciliary activity is an integral part of mucociliary clearance and changes in ciliary beat frequency (CBF) are often associated with similar changes in mucociliary transport. This application focuses on how two widely recognized second messengers in cell signaling, cAMP and calcium ([Ca2+]i), regulate CBF. cAMP has been shown to increase CBF through a cAMP-dependent kinase-mediated event, possibly by phosphorylating a ciliary protein designated p26. Increasing [Ca2+]i also stimulates CBF, likely through a ciliary Ca2+-binding protein. Because both second messengers increase CBF, the question arises whether these signaling pathways regulate CBF through independent signal transduction cascades or whether the pathways converge at some level to affect a common target prior to dynein/microtubule interaction. Evidence suggests that they converge because elevations in cAMP render CBF less sensitive to [Ca2+]1 changes. The simplest hypothesis to explain this phenomenon is that the cAMP and Ca2+ signaling pathways converge onto a single ciliary protein (p26) or protein complex (containing p26), which regulates CBF. The phosphorylation of this target changes its Ca - affinity or off-rate. This proposal seeks to explore the identity and function of p26 and begin to examine interactions between p26 and the Ca2+ pathway.
The specific aims are: 1. To characterize p26 and localize it within the cilium. 2. To identify a calcium-binding protein on outer dynein arms 3. To evaluate the correlation between PKA-mediated phosphorylation of p26 and increases in CBF as well as p26 phosphorylation and altered Ca2+/CBF coupling. 4. To evaluate a cause-effect relationship between p26 phosphorylation and CBF changes. The results of these studies will provide new and important information on the role of specific ciliary proteins in regulating mammalian CBF. In addition, the exploration of signaling crosstalk may have important implications in other cell types that regulate some form of cell motility.
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