PHOSPHORYLATION OF CILIARY TARGET PROTEINS

本项目的长期目标是提高我们对粘膜纤毛清除的细胞和分子调节机制的理解，粘膜纤毛清除是肺的重要宿主防御机制。纤毛活动是粘液纤毛清除的一个组成部分，纤毛搏动频率（CBF）的变化通常与粘液纤毛转运的类似变化相关。本申请的重点是两个广泛认可的第二信使细胞信号，cAMP和钙（[Ca 2 +]i），调节CBF。cAMP已显示通过cAMP依赖性激酶介导的事件增加CBF，可能是通过磷酸化命名为p26的纤毛蛋白。增加[Ca 2 +]i也刺激CBF，可能是通过睫状体Ca 2+结合蛋白。由于这两种第二信使增加CBF，问题出现了这些信号通路是否通过独立的信号转导级联调节CBF，或者这些通路是否在动力蛋白/微管相互作用之前在某种水平上会聚以影响共同的靶点。有证据表明，他们收敛，因为cAMP的升高使CBF不太敏感[Ca 2 +]1的变化。解释这种现象的最简单的假设是cAMP和Ca 2+信号通路会聚到单个纤毛蛋白（p26）或蛋白复合物（含有p26）上，其调节CBF。该靶标的磷酸化改变其Ca亲和力或解离速率。本研究旨在探索p26的身份和功能，并开始研究p26与Ca 2+通路之间的相互作用。具体目标是：1.研究p26蛋白在纤毛中的定位。2.确定外动力蛋白臂3上的钙结合蛋白。评估PKA介导的p26磷酸化与CBF增加以及p26磷酸化与Ca 2 +/CBF偶联改变之间的相关性。4.探讨p26磷酸化与脑血流量变化之间的因果关系。这些研究结果将提供新的和重要的信息，在特定的纤毛蛋白调节哺乳动物CBF的作用。此外，信号串扰的探索可能在调节某种形式的细胞运动的其他细胞类型中具有重要意义。

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.