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

细胞迁移过程中 PKA、细胞张力和 Ca2 通道之间的串扰

• 批准号: 8503067
• 负责人: Alan K Howe
• 金额: $ 0.39万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8503067
• 关键词: A kinase anchoring protein; Actomyosin; Adenylate Cyclase; Back; Behavior; Calcium; Cells; Cyclic AMP-Dependent Protein Kinases; Data; Early Diagnosis; Environment; Equilibrium; Event; Extracellular Matrix; Feeds; Fluorescence Resonance Energy Transfer; Frequencies; Malignant neoplasm of ovary; Measures; Mechanics; Mediating; Microscopy; Molecular; Movement; Nature; Normal Cell; Pathway interactions; Phosphorylation; Protein Kinase; Regulation; Role; Shapes; Signal Transduction; Stretching; Testing; Time; Tissues; Translating; Work; cancer cell; cell behavior; cell motility; extracellular; genetic inhibitor; inhibitor/antagonist; insight; neoplastic cell; prevent

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.

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得到充分证实，但细胞感知和响应 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) 在细胞前缘富集和激活，这种定位对于细胞迁移很重要：然而，这种激活的机制仍不清楚。最近的观察表明，对卵巢癌细胞应用机械拉伸可以在拉伸方向上快速局部激活 PKA。此外，迁移细胞前缘内 PKA 的激活会被细胞外钙 (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 (AKAP) 的相互作用可显着降低 SACC 介导的张力依赖性 Ca2+ 瞬变（称为“Ca2+ 闪烁”）的频率，这种瞬变发生在前缘内，对于指导细胞迁​​移。这些观察结果支持这样一种假设，即细胞迁移过程中细胞内张力通过涉及 SACC 的机制局部激活 PKA 活性，并且这种局部 PKA 活性反馈以控制 Ca2+ 流入。目前提出的工作通过确定以下内容来测试这一假设： 具体目标 1：通过机械拉伸局部激活 PKA 的机制。具体来说，拟议的工作将测试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（AC）以及通过 AKAP 定位 PKA 的机制来激活 PKA 的假设。具体目标 2：拉伸/张力在细胞迁移过程中 PKA 局部激活中的作用。具体来说，拟议的工作将测试以下假设：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。具体目标 3：PKA 在细胞迁移过程中调节 Ca2+ 和 SACC 的作用。具体来说，拟议的工作将测试以下假设：PKA 通过局部磷酸化和 TRPM7（已知会产生前沿 Ca2+ 闪烁的 SACC）的调节来调节细胞迁移过程中的 Ca2+ 流入。 我们的共同努力将首次在 PKA 和 Ca2+ 之间建立对细胞迁移非常重要的机械敏感“回路”。因此，所提出的工作将深入了解细胞用于将环境传感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。