蛋白磷酸酶2A型（以下見稱PP2A）是一種絲氨酸/蘇氨酸磷酸酶，在哺乳動物細胞中參與了許多細胞功能的調控。PP2A主要由三個次單元所組成，包含結構性次單元 A，催化性次單元 C，以及多樣的調節性次單元 B。其中B 次單元可以分為四個家族包括 B、B'、B'、以及 B'。目前的研究認為多樣的B 次單元可以影響PP2A 磷酸酶對受質的專一性以及細胞中的分布表現。儘管已知PP2A三聚體完全酶是PP2A的具有功能的型式，但是以探討PP2A完全酶在細胞中的座落位置及其組成的調節機制的研究仍很少。
在此，我們研究了PP2A-B55β2完全酶座落於線粒體與其組裝的調節。B55β2次單元是由PPP2R2B基因所轉錄的，並且在很多的組織中皆有表現，其中在大腦和小腦表現最多。
目前研究已知，PPP2R2B的缺是造成常染色體顯性脊髓小腦性共濟失調12型（簡稱SCA12）的主要原因。SCA12是一種小腦退化的神經退化性疾病。
此外，已知B55β2是B55β的同源異構體，在給予細胞壓力刺激下，B55β2會轉移至粒線體，並且參與調節神經元細胞死亡的過程。
我們透過螢光顯微鏡以雙分子螢光互補作用 (BiFC, Bimolecular fluorescence complementation) 結合螢光共振能量轉移 （FRET, fluorescence resonance energy transfer）的分析方法來探討B55β2次單元及B55β2完全酶在細胞中的分布型態。
我們發現，利用轉染使細胞中表現B55β2-CFP時，結果顯示在NIH3T3中B55β2主要呈現類似粒線體的點狀分布 (punctate distribution)，或均勻分布在細胞中普遍存在，而在HEK293T和SK-N-SH細胞中顯示彌散分佈(diffuse distribution)在細胞質。BiFC-FRET分析的結果顯示，PP2A-B55β2完全酶在HEK293T細胞中似乎主要是以細胞質的聚集體型式表現(cytoplasmic aggregates)，在SK-N-SH細胞中是點狀或彌散的細胞質分佈，並且在NIHT3細胞中是點狀或細胞質聚集體。為了探討完全酶型式是否是B55β2坐落於線粒體的先決條件，我們觀察一個具有與A次單元的結合缺陷的B55β2mut的表現位置。因為B55β2mut具有與A次單元的結合缺陷，因而無法整合到完全酶中，所以如預期的，B55β2mut沒有顯示出完全酶組裝的FRET信號，但仍然呈現可以座落線粒體的分佈。
已知Okadaic acid抑制C次單元上的羧基甲基化，而羧基甲基化對於形成包含B55調節次單元家族的完全酶是必需的，而對於其他B調節次單元則否。利用BiFC-FRET分析測定PP2A-B55β2完全酶組成的現象，結果顯示在細胞再添加Okadaic acid的環境下沒有FRET信號產生。然而，測定PP2A-B56γ3完全酶組裝的分析，結果顯示仍有部分FRET仍然存在於有Okadaic acid處理的環境。
為進一步研究PP2A-B55β2完全酶在細胞壓力刺激下的分佈，我們用細胞凋亡誘導劑放線菌素D(actinomycin D)處理細胞，並分析其對PP2A-B55β2完全酶的分佈和組成的影響。放線菌素D處理導致沒有FRET信號產生，並且增加了具有B55β2-CFP細胞質聚集體和類似細胞凋亡的皺縮的細胞形態的細胞數量。
綜上所述，我們的結果顯示PP2A-B55β2完全酶根據不同的細胞類型，會呈現不完全相同的細胞內分佈模式，並且形成三聚體完全酶不是B55β2座落於線粒體的先決條件。Okadaic acid完全抑制PP2A-B55β2完全酶的組成，但只降低部分PP2A-B56γ3完全酶的組成，這與C次單元上的羧基甲基化對不同B次單元家族組裝成完全酶的影響的程度差異的結果一致。另外，細胞壓力刺激下，PP2A-B55β2完全酶的形成會受抑制，並使B55β2形成大的細胞質聚集體及促使細胞凋亡發生。

Protein phosphatase 2A (PP2A) is an abundant serine/threonine phosphatase that regulates many cellular processes in mammalian cells. The heterotrimeric PP2A holoenzyme mainly comprises a structural A subunit, a variable regulatory B subunit, and a catalytic C subunit. The B subunits determine the substrate specificity, subcellular localization, and functions of PP2A. Although the trimeric PP2A holoenzyme is the functional form of PP2A, however, no study was done for investigating the regulation of subcellular localization and assembly of the holoenzyme as a whole. Herein, we report on the regulation of mitochondrial localization and assembly of the PP2A holoenzyme containing the B55β2 regulatory subunit. The B55β regulatory subunit is encoded by PPP2R2B (Bβ) and mainly expressed in neurons of cerebellum in brain. Defects of PPP2R2B have been implicated as the cause in autosomal dominant spinocerebellar ataxia 12 (SCA12), B55β2, a splice isoform of B55β, was reported to be translocated to mitochondria upon neuronal stress and plays a critical role in regulating neuronal cell death under cellular stresses. We investigated the subcellular distribution of B55β2 by fluorescence microscopy and the PP2A holoenzyme containing B55β2 (PP2A-B55β2) by bimolecular fluorescence complementation (BiFC) in conjunction with fluorescence resonance energy transfer (FRET) analysis. Transiently transfected B55β2-CFP displayed either a mainly punctate, mitochondria-like or ubiquitous distribution in NIH3T3 cells, but displayed a ubiquitous distribution in HEK293T and SK-N-SH cells. Results of BiFC-FRET analysis showed that the trimeric complexes consisting of YN-Aα, Cα-YC, and B55β2-CFP (PP2A-B55β2 holoenzymes) appear to be predominantly cytoplasmic aggregates in HEK293T cells. One the other hand, the PP2A-B55β2 holoenzymes displayed punctate or ubiquitous distribution in SK-N-SH cells and displayed either punctate or cytoplasmic aggregates in NIHT3 cells. To investigate whether assembly into a holoenzyme is a prerequisite for the mitochondrial localization of B55β2, we found that in contrast to wild-type B55β2, co-expression of YN-Aα, Cα-YC, and B55β2mut-CFP, which is defective in binding to the A subunit and cannot integrate into the holoenzyme, showed no FRET signals indicative of the holoenzyme assembly. However, B55β2mut-CFP still displayed a punctate pattern, and was co-localized with mitochondria. Okadaic acid (OA), a PP2A-selective inhibitor, which inhibits carboxyl-methylation of the C subunit almost completely abolished the assembly of the PP2A-B55β2 holoenzyme, but only partially inhibited assembly of the PP2A-B56γ3 holoenzyme using BiFC-FRET analysis. Furthermore, we investigated the distribution of PP2A-B55β2 holoenzyme under cellular stresses by treating cells with actinomycin D, a mitochondrial stress inducer, and found that cells gradually lost FRET of the PP2A-B55β2 holoenzyme and that numbers of cells with large B55β2-CFP aggregates and shrunken cell morphology were significantly increased through the time course of treatment of actinomycin D.
In summary, our results demonstrate that the PP2A-B55β2 holoenzyme displays differential subcellular distribution patterns, including both mitochondrial localization, diffuse cytosolic distribution, and cytosolic aggregates, in a cell type-dependent manner and forming a trimeric holoenzyme is not a prerequisite for the mitochondrial localization of the B55β2. OA completely inhibits the assembly of the PP2A-B55β2 holoenzyme, but only partially affects the PP2A-B56γ3 holoenzyme, consistent with differential effects of carboxyl-methylation of C subunits on the integration of different B subunits into the holoenzyme. Additionally, induction of cellular stress by actinomycin D treatment abolishes the formation of the PP2A-B55β2 holoenzyme and renders B55β2 to form large cytoplasmic aggregates.

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