PHOSPHORYLATION OF CILIARY TARGET PROTEINS

本项目的长期目标是提高我们对黏毛清除的细胞和分子调控机制的认识，这是肺部重要的宿主防御机制。纤毛活动是纤毛黏液清除不可分割的一部分，纤毛搏动频率（CBF）的变化通常与纤毛黏液运输的类似变化相关。本应用侧重于细胞信号传导中两个广泛认可的第二信使，cAMP和钙（[Ca2+]i）如何调节CBF。cAMP已被证明通过cAMP依赖性激酶介导的事件增加CBF，可能是通过磷酸化指定为p26的纤毛蛋白。增加[Ca2+]i也刺激CBF，可能是通过纤毛Ca2+结合蛋白。由于这两种第二信使都增加了脑血流，因此问题就出现了，这些信号通路是通过独立的信号转导级联来调节脑血流，还是在动力蛋白/微管相互作用之前，这些信号通路在某种程度上汇聚以影响一个共同的目标。有证据表明，它们会聚在一起，因为cAMP的升高使CBF对[Ca2+]1的变化不那么敏感。解释这一现象的最简单的假设是cAMP和Ca2+信号通路汇聚到一个单一的纤毛蛋白（p26）或蛋白复合物（含有p26）上，从而调节CBF。这个靶标的磷酸化改变了它的钙亲和率或脱配率。本研究旨在探索p26的身份和功能，并开始研究p26与Ca2+通路之间的相互作用。具体目标是：1。鉴定p26的特征并将其定位于纤毛内。2. 鉴定动力蛋白外臂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Ca2+/CBF偶联改变之间的相关性。4. 评估p26磷酸化与脑血流变化之间的因果关系。这些研究结果将为研究特定纤毛蛋白在哺乳动物脑血流调节中的作用提供新的重要信息。此外，信号串扰的探索可能对调节某种形式的细胞运动的其他细胞类型具有重要意义。

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.