A B56 regulatory subunit of protein phosphatase 2A localizes to nuclear speckles in cardiomyocytes
A B56 regulatory subunit of protein phosphatase 2A localizes to nuclear speckles in cardiomyocytes
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DOI：
10.1152/ajpheart.01291.2004
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发表时间：
2005-07-01
影响因子：
4.8
通讯作者：
Rogers, TB
中科院分区：
文献类型：
--
作者：
Gigena, MS;Ito, A;Rogers, TB
蛋白磷酸酶2A（PP 2A）广泛分布于心脏组织中，但其确切的细胞功能知之甚少。这项研究是基于这样的概念，即PP 2A的行动是由核心酶与B靶向/调节亚基的相互作用。两个B亚基，B56 α和B56 γ 1的亚细胞定位进行了评估，使用腺病毒驱动的表达表位标记（血凝素，HA）在培养的新生和成年大鼠心室肌细胞。共聚焦成像显示，HA-B56 α被排除在细胞核和装饰的条纹状结构，而HA-B56 γ 1主要被发现在细胞核中。精确的免疫标记研究表明，B56 γ 1集中在称为核斑点的核内结构中，这是一种积累转录和剪接因子的大分子结构。Western印迹分析显示，任何B亚基的过度表达对培养的新生心肌细胞中其他PP 2A亚基的水平没有影响。然而，当在细胞提取物中测量时，仅B56 γ 1的过表达使全细胞PP 2A活性增加40%。最后，B56 γ 1没有改变整体基因表达或肥大基因标记物如α-骨骼肌动蛋白的表达。然而，共聚焦图像的形态学分析显示，B56 γ 1改变了心脏细胞核斑点的动态组装/拆卸过程。这些研究为PP 2A靶向心肌细胞亚核结构的机制以及这种磷酸酶在核信号传导中的作用提供了新的见解。
Protein phosphatase 2A (PP2A) is widely distributed in heart tissues, yet its precise cellular functions are poorly understood. This study is based on the notion that PP2A action is governed by interactions of the core enzyme with B targeting/ regulatory subunits. The subcellular localizations of two B subunits, B56 alpha and B56 gamma 1, were assessed using adenovirus-driven expression of epitope-tagged ( hemagglutinin, HA) in cultured neonatal and adult rat ventricular myocytes. Confocal imaging revealed that HA-B56 alpha was excluded from the nucleus and decorated striated structures, whereas HA-B56 gamma 1 was principally found in the nucleus. Precise immunolabeling studies showed that B56 gamma 1 was concentrated in intranuclear structures known as nuclear speckles, macromolecular structures that accumulate transcription and splicing factors. Western blot analyses revealed that overexpression of either B subunit had no effect on the levels of other PP2A subunits in cultured neonatal cardiac cells. However, overexpression of only B56 gamma 1 increased whole cell PP2A activity by 40% when measured in cell extracts. Finally, B56 gamma 1 did not alter global gene expression or expression of hypertrophic gene markers such as alpha-skeletal actin. However, morphometric analyses of confocal images revealed that B56 gamma 1 alters the dynamic assembly/disassembly process of nuclear speckles in heart cells. These studies provide new insight into mechanisms of PP2A targeting in the subnuclear architecture in cardiomyocytes and into the role of this phosphatase in nuclear signaling.