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

描述（由申请人提供）：所有细胞与其周围的细胞外基质（ECM）交换力，这种“机械相互作用”调节各种生理学上重要的事件，包括细胞命运、形状和运动。虽然这种调节的重要性是牢固确立的，但细胞感知和响应其ECM的机械性质的分子机制还没有很好地理解。已知cAMP依赖性蛋白激酶（PKA）在细胞的前缘富集和活化，并且这种定位对于细胞迁移是重要的：然而，这种活化的机制仍不清楚。最近的观察表明，机械拉伸对卵巢癌细胞的应用迅速和局部激活PKA的拉伸方向。此外，迁移细胞前缘内PKA的激活被细胞外钙（Ca 2+）耗竭和选择性抑制牵张激活的Ca 2+通道（SACC）阻断。相反，抑制PKA活性或其与A-激酶锚定蛋白（AKAP）的相互作用显著降低SACC介导的张力依赖性Ca 2+瞬变（称为“Ca 2+闪烁”）的频率，其发生在前缘内，对引导细胞迁移很重要。这些观察结果支持了一个假设，PKA活性是局部激活的细胞迁移过程中的细胞内张力通过一种机制，涉及SACC，这种本地化的PKA活性反馈控制Ca 2+流入。目前提出的工作与测试这一假设，通过确定：具体目标1：机械拉伸PKA的局部激活的机制。具体而言，拟议的工作将测试的假设，机械拉伸增加细胞内的张力，并通过涉及肌动球蛋白收缩，SACC，钙激活腺苷酸环化酶（AC），PKA通过AKAP的本地化的机制激活PKA。具体目标2：拉伸/张力在细胞迁移过程中PKA局部活化中的作用。具体而言，拟议的工作将测试的假设，机械拉伸，以增加细胞内的张力和激活PKA的能力将有助于激活PKA在细胞迁移过程中。具体目标3：PKA在细胞迁移过程中调节Ca 2+和SACC的作用。具体来说，拟议的工作将测试的假设，PKA调节Ca 2+内流在细胞迁移过程中通过局部磷酸化和TRPM 7，SACC已知产生前沿Ca 2+闪烁的调节。 我们的共同努力将首次建立PKA和Ca 2+之间的机械敏感性“电路”，这对细胞迁移很重要。因此，拟议的工作将提供深入了解的分子机制，细胞用于整合环境传感与控制细胞迁移的局部细胞内信号事件。

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.