Isoform-specific subcellular localization and function of protein kinase A identified by mosaic imaging of mouse brain
Isoform-specific subcellular localization and function of protein kinase A identified by mosaic imaging of mouse brain
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DOI：
10.7554/elife.17681
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发表时间：
2017-01-12
期刊：
影响因子：
7.7
通讯作者：
Taylor, Susan S.
中科院分区：
文献类型：
--
作者：
Ilouz, Ronit;Lev-Ram, Varda;Taylor, Susan S.
蛋白激酶A（PKA）在神经元功能中起关键作用，由不同的调节（R）亚基介导。RI β或RII β亚基的缺陷导致不同的神经元表型。虽然RI β有助于突触可塑性，但它是研究最少的同种型。使用同种型特异性抗体，我们生成了小鼠大脑的高分辨率大规模免疫组织化学马赛克图像，提供了几个大脑区域的全局视图，包括海马和小脑。这些异构体集中在离散的大脑区域，我们能够放大以显示亚细胞定位的不同模式。RI β富集在树突中并与MAP 2共定位，而RII β集中在轴突中。使用相关的光学和电子显微镜，我们证实了RI β在培养的神经元的线粒体和核定位。为了显示核定位的功能意义，我们证明了RI β的下调，而不是RII β，减少CREB磷酸化。我们的研究揭示了PKA亚型特异性如何通过精确定位来定义。
Protein kinase A (PKA) plays critical roles in neuronal function that are mediated by different regulatory (R) subunits. Deficiency in either the RI beta or the RII beta subunit results in distinct neuronal phenotypes. Although RI beta contributes to synaptic plasticity, it is the least studied isoform. Using isoform-specific antibodies, we generated high-resolution large-scale immunohistochemical mosaic images of mouse brain that provided global views of several brain regions, including the hippocampus and cerebellum. The isoforms concentrate in discrete brain regions, and we were able to zoom-in to show distinct patterns of subcellular localization. RI beta is enriched in dendrites and co-localizes with MAP2, whereas RII beta is concentrated in axons. Using correlated light and electron microscopy, we confirmed the mitochondrial and nuclear localization of RI beta in cultured neurons. To show the functional significance of nuclear localization, we demonstrated that downregulation of RI beta, but not of RII beta, decreased CREB phosphorylation. Our study reveals how PKA isoform specificity is defined by precise localization.