Cross-talk between PKA, cellular tension, and Ca2+ channels during cell migration

描述(申请人提供)：所有细胞与其周围的细胞外基质(ECM)交换力，这种‘机械力相互作用’调节各种重要的生理事件，包括细胞命运、形状和运动。虽然这种调节的重要性已经确定，但细胞感知和响应其ECM的机械性质的分子机制还没有被很好地理解。CAMP依赖的蛋白激酶(PKA)在细胞的前沿被丰富和激活，这种定位对细胞迁移很重要：然而，这种激活的机制还不清楚。最近的观察表明，对卵巢癌细胞施加机械拉伸可以迅速并局部地激活沿拉伸方向的PKA。此外，细胞外钙(Ca~(2+))的耗竭和拉伸激活的Ca~(2+)通道(SACCS)的选择性抑制也阻碍了迁移细胞前沿内PKA的激活。相反，抑制PKA活性或其与A-激酶锚定蛋白(AKAP)的相互作用显著减少SACC介导的、张力依赖的钙瞬变的频率，这种瞬间发生在前沿，对引导细胞迁移非常重要。这些观察结果支持一种假说，即在细胞迁移过程中，通过涉及SACCS的机制，PKA活性被细胞内张力局部激活，并且这种局部的PKA活性反馈控制钙内流。目前提出的工作通过确定：特定目标1：机械拉伸局部激活PKA的机制来验证这一假说。具体地说，这项拟议的工作将检验机械拉伸增加细胞内张力和激活PKA的假说，该假说涉及肌动蛋白收缩、SACCS、钙激活的腺苷环酶(ACS)以及通过AKAPs定位PKA的机制。特定目标2：拉伸/张力在细胞迁移过程中PKA局部激活中的作用。具体地说，这项拟议的工作将检验这样一种假设，即机械拉伸增加细胞内张力并激活PKA的能力将有助于在细胞迁移过程中激活PKA。具体目标3：PKA在细胞迁移过程中调节钙离子和SACCS的作用。具体地说，拟议的工作将检验这一假设，即PKA通过局部磷酸化和调节TRPM7来调节细胞迁移过程中的钙内流。TRPM7是已知产生前沿钙闪烁的SACC。我们的共同努力将首次在PKA和Ca~(2+)之间建立一个机械敏感的‘回路’，这对细胞迁移是重要的。因此，这项拟议的工作将深入了解细胞用来将环境感知与控制细胞迁移的局部细胞内信号事件相结合的分子机制。

DESCRIPTION (provided by applicant): All cells exchange forces with their surrounding extracellular matrix (ECM) and this 'mechanoreciprocity' regulates a variety of physiologically important events, including cell fate, shape, and movement. While the importance of this regulation is firmly established, the molecular mechanisms through which cells sense and respond to the mechanical nature of their ECM are not well understood. The cAMP- dependent protein kinase (PKA) is known to be enriched and activated in the leading edge of cells and this localization is important for cell migration: however, the mechanism for this activation remains unclear. Recent observations demonstrate that application of mechanical stretch to ovarian cancer cells rapidly and locally activates PKA in the direction of the stretch. In addition, activation of PKA within the leading edge of migrating cells is blocked by depletion of extracellular calcium (Ca2+) and by selective inhibition of stretch-activated Ca2+ channels (SACCs). Conversely, inhibition of PKA activity or its interaction with A-kinase anchoring proteins (AKAPs) significantly reduces the frequency of SACC-mediated, tension-dependent Ca2+ transients, known as 'Ca2+ flickers', that occur within the leading edge and are important for steering cell migration. These observations support a hypothesis in which PKA activity is locally activated by intracellular tension during cell migration through a mechanism that involves SACCs, and that this localized PKA activity feeds back to control Ca2+ influx. The currently proposed work with test this hypothesis by determining: Specific Aim 1: The mechanism of localized activation of PKA by mechanical stretch. Specifically, the proposed work will test the hypothesis that Mechanical stretch increases intracellular tension and activates PKA through a mechanism involving actomyosin contractility, SACCs, Ca2+-activated adenylyl cyclases (ACs), and localization of PKA through AKAPs. Specific Aim 2: The role of stretch/tension in localized activation of PKA during cell migration. Specifically, the proposed work will test the hypothesis that the ability of mechanical stretch to increase intracellular tension and activate PKA will contribute to the activation of PKA during cell migration. Specific Aim 3: The role of PKA in regulating Ca2+ and SACCs during cell migration. Specifically, the proposed work will test the hypothesis that PKA regulates Ca2+ influx during cell migration through localized phosphorylation and regulation of TRPM7, the SACC known to generate leading edge Ca2+ flickers. Our combined efforts will establish, for the first time, a mechanosensitive 'circuit' between PKA and Ca2+ that is important for cell migration. Thus, the proposed work will provide insight into the molecular mechanisms that cells use to integrate environmental sensing with localized intracellular signaling events that control cell migration.