| Summary: | 碩士 === 國立成功大學 === 分子醫學研究所 === 106 === 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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