Visualization of PKA activity in plasma membrane microdomains
Visualization of PKA activity in plasma membrane microdomains
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DOI：
10.1039/c0mb00079e
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发表时间：
2011-01-01
影响因子：
--
通讯作者：
Zhang, Jin
中科院分区：
文献类型：
--
作者：
Depry, Charlene;Allen, Michael D.;Zhang, Jin
膜筏是富含鞘脂和胆固醇的微区，含有信号蛋白的动态排列。值得注意的是，普遍存在的cAMP/蛋白激酶A（PKA）途径的各种组分，包括β-肾上腺素能受体（β-AR）、G蛋白和腺苷酸环化酶（AC），已显示在质膜的膜筏和非筏区域之间差异定位。由于PKA参与调节多种基本细胞功能，其中一些需要膜筏，重要的是要了解PKA活性是如何在这些膜微区具体调节。为此，我们开发了一种改进的基于FRET的PKA活性生物传感器，并将其定位于质膜的膜筏和非筏区域，以检测不同质膜微区中PKA活性的动态变化。通过胆固醇耗竭破坏膜筏显示增强β-AR刺激的质膜PKA活性，表明膜筏在β-AR刺激的PKA活化中起负面作用。此外，我们发现，膜筏具有更高的基础PKA活性在静息状态下相比，非筏区域，这取决于膜筏的完整性和PKA的适当定位。本研究表明膜筏在调节质膜PKA活性中起着重要作用，并证明了基于活细胞FRET的测定能够揭示质膜微区之间的动态差异，为进一步解剖膜调节信号转导奠定了基础。
Membrane rafts are sphingolipid-and cholesterol-rich microdomains that contain dynamic arrangements of signaling proteins. Notably, various components of the ubiquitous cAMP/Protein Kinase A (PKA) pathway, including beta-adrenergic receptors (beta-ARs), G proteins, and adenylyl cyclases (ACs), have been shown to localize differentially between membrane rafts and non-raft regions of the plasma membrane. As PKA participates in regulating diverse fundamental cellular functions, a number of which require membrane rafts, it is important to understand how PKA activity is specifically regulated in these membrane microdomains. To this end, we developed an improved FRET-based PKA activity biosensor, and targeted it to both membrane raft and non-raft regions of the plasma membrane to examine PKA activity dynamics in different plasma membrane microdomains. Disruption of membrane rafts via cholesterol depletion was shown to enhance beta-AR stimulated PKA activity at the plasma membrane, suggesting that membrane rafts play a negative role in beta-AR stimulated PKA activation. Furthermore, we found that membrane rafts possess higher basal PKA activity in the resting state compared to non-raft regions, which depends on the integrity of membrane rafts and proper localization of PKA. This study shows that membrane rafts play an important role in regulating the activity of PKA at the plasma membrane, and demonstrates the ability of live-cell FRET-based assays to reveal dynamic differences amongst plasma membrane microdomains, laying a foundation for further dissection of membrane regulated signal transduction.