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“机械互易性”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立，但细胞感知和响应其ECM机械性质的分子机制尚不清楚。已知cAMP依赖性蛋白激酶（PKA）在细胞的前沿富集和激活，这种定位对细胞迁移很重要，然而，这种激活的机制尚不清楚。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在拉伸方向。此外，PKA在迁移细胞前沿的激活被细胞外钙（Ca2+）的消耗和拉伸激活的Ca2+通道（sacc）的选择性抑制所阻断。相反，抑制PKA活性或其与a激酶锚定蛋白（AKAPs）的相互作用可显著降低sacc介导的紧张依赖性Ca2+瞬变的频率，称为“Ca2+闪烁”，发生在前缘内，对指导细胞迁移很重要。这些观察结果支持了一种假设，即PKA活性在细胞迁移过程中通过涉及sacc的机制被细胞内张力局部激活，并且这种局部PKA活性反馈控制Ca2+内流。目前提出的工作通过确定：特定目的1：局部激活PKA机械拉伸的机制来验证这一假设。具体来说，这项工作将验证机械拉伸增加细胞内张力并通过涉及肌动球蛋白收缩性、sacc、Ca2+激活的腺苷酸环化酶（ACs）和通过akap定位PKA的机制激活PKA的假设。特异性目的2：拉伸/张力在细胞迁移过程中PKA的局部激活中的作用。具体来说，这项工作将验证机械拉伸增加细胞内张力和激活PKA的能力将有助于细胞迁移过程中PKA的激活的假设。特异性目的3:PKA在细胞迁移过程中调节Ca2+和sacc的作用。具体来说，这项工作将验证PKA通过局部磷酸化和TRPM7调控细胞迁移过程中Ca2+内流的假设，TRPM7是已知产生前沿Ca2+闪烁的SACC。我们的共同努力将首次建立PKA和Ca2+之间的机械敏感“电路”，这对细胞迁移很重要。因此，所提出的工作将提供深入了解细胞用于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. PUBLIC HEALTH RELEVANCE: Cells maintain a dynamic tension with their surroundings and the balance of these forces can profoundly influence both normal and tumor cell behavior. The current application proposes work that will investigate new findings that implicate a specific molecular 'circuit' that is important for sensing this tension and translating into signals that control movement of ovarian cancer cells. A better understanding of the molecular nature of this regulation may provide clues to facilitate early detection or to prevent the spread of ovarian cancer.