A specific PP2A regulatory subunit, B56gamma, mediates DNA damage-induced dephosphorylation of p53 at Thr55

Protein phosphatase 2A (PP2A) has been implicated to exert its tumor suppressive function via a small subset of regulatory subunits. In this study, we reported that the specific B regulatory subunits of PP2A B56gamma1 and B56gamma3 mediate dephosphorylation of p53 at Thr55. Ablation of the B56gamma protein by RNAi, which abolishes the Thr55 dephosphorylation in response to DNA damage, reduces p53 stabilization, Bax expression and cell apoptosis. To investigate the molecular mechanisms, we have shown that the endogenous B56gamma protein level and association with p53 increase after DNA damage. Finally, we demonstrate that Thr55 dephosphorylation is required for B56gamma3-mediated inhibition of cell proliferation and cell transformation. These results suggest a molecular mechanism for B56gamma-mediated tumor suppression and provide a potential route for regulation of B56gamma-specific PP2A complex function.     

The B56γ3 Regulatory Subunit of Protein Phosphatase 2A (PP2A) Regulates S Phase-specific Nuclear Accumulation of PP2A and the G1 to S Transition

Protein phosphatase 2A (PP2A) is a heterotrimeric enzyme consisting of a scaffold subunit (A), a catalytic subunit (C), and a variable regulatory subunit (B). The regulatory B subunits determine the substrate specificity and subcellular localization of the PP2A holoenzyme. Here, we demonstrate that the subcellular localization of the B56γ3 regulatory subunit is regulated in a cell cycle-specific manner. Notably, B56γ3 becomes enriched in the nucleus at the G₁/S border and in S phase. The S phase-specific nuclear enrichment of B56γ3 is accompanied by increases of nuclear A and C subunits and nuclear PP2A activity. Overexpression of B56γ3 promotes nuclear localization of the A and C subunits, whereas silencing both B56γ2 and B56γ3 blocks the S phase-specific increase in the nuclear localization and activity of PP2A. In NIH3T3 cells, B56γ3 overexpression reduces p27 phosphorylation at Thr-187, concomitantly elevates p27 protein levels, delays the G₁ to S transition, and retards cell proliferation. Consistently, knockdown of endogenous B56γ3 expression reduces p27 protein levels and increases cell proliferation in HeLa cells. These findings demonstrate that the dynamic nuclear distribution of the B56γ3 regulatory subunit controls nuclear PP2A activity, which regulates cell cycle controllers, such as p27, to restrain cell cycle progression, and may be responsible for the tumor suppressor function of PP2A.     

Genomic organization and mapping of the gene encoding the PP2A B56gamma regulatory subunit

Protein phosphatase 2A (PP2A) is a major serine/threonine phosphatase that regulates a wide variety of cellular processes. The enzymatic activity and intracellular localization of PP2A are determined by three distinct families of cellular regulatory subunits (B, B', and B'). The B' subunit, also known as B56, is the most diverse, consisting of five isoforms (alpha, beta, gamma, delta, and epsilon). The gene encoding B56gamma has been designated as PPP2R5C and encodes three differentially spliced variants: B56gamma1, -gamma2, and -gamma3. However, conflicting chromosomal loci have been reported in human genomic databases. The original cytogenetic mapping placed the gene on chromosome 3p21.3, whereas subsequent studies using radiation hybrid analysis localized PPP2R5C to chromosome 14q. In this study, by radiation hybrid mapping, FISH analysis, BAC clone sequencing, and RT-PCR analysis, we show that the functional gene PPP2R5C exists at 14q32.2 and gives rise to three splicing variants, B56gamma1, -gamma2, and -gamma3, whereas a nonfunctional B56gamma1 pseudogene, PPP2R5CP, is present at 3p21.3. We also report the genomic organization of both the functional gene and the pseudogene.     

Transgenic expression of protein phosphatase 2A regulatory subunit B56gamma disrupts distal lung differentiation

The distal epithelium of the developing lung exhibits high-level expression of protein phosphatase 2A (PP2A), a vital signaling enzyme. Here we report the discovery that in the lung, the PP2A regulatory subunit B56gamma is expressed in a discrete developmental period, with the highest protein levels at embryonic day (e) 17, but no detectable protein in the newborn or adult. By in situ hybridization, B56gamma was highly expressed in the distal epithelium of newly forming airways and in mesenchymal cells. In contrast, expression of B56gamma was quite low in the bronchial epithelium and vascular smooth muscle. Transgenic expression of B56gamma using the lung-specific promoter for surfactant protein C (SP-C) resulted in neonatal death. Examination of lungs from SP-C-B56gamma transgenic e18 fetuses revealed proximal airways and normal blood vessels, but the tissue was densely populated with epithelial-type cells and was devoid of normal peripheral lung structure. A component of the Wnt signaling pathway, beta-catenin, was developmentally regulated in the normal lung and was absent in lung tissue from B-56gamma transgenic fetuses. We propose that B56gamma is expressed at a particular stage of lung development to modulate PP2A action on the Wnt/beta-catenin signaling pathway during lung airway morphogenesis.     

含B56调节亚基的PP2A全酶在肿瘤信号通路中的调控作用

蛋白磷酸酶2A（protein phosphatase 2A,PP2A）是细胞中广泛表达的异三聚体全酶,调节许多重要的信号通路,它的表达异常所致的信号通路紊乱会引发肿瘤和促进肿瘤的发展.PP2A在特定的状态下能够发挥抑癌因子的作用,这种抑癌特性由B调节亚基与底物的相互作用来决定,因此B调节亚基在PP2A的抑癌功能中起关键作用.     

A truncated isoform of the PP2A B56 subunit promotes cell motility through paxillin phosphorylation

Both F10 and BL6 sublines of B16 mouse melanoma cells are metastatic after intravenous injection, but only BL6 cells are metastatic after subcutaneous injection. Retrotransposon insertion was found to produce an N-terminally truncated form (Deltagamma1) of the B56gamma1 regulatory subunit isoform of protein phosphatase (PP) 2A in BL6 cells, but not in F10 cells. We found an interaction of paxillin with PP2A C and B56gamma subunits by co-immunoprecipitation. B56gamma1 co-localized with paxillin at focal adhesions, suggesting a role for this isoform in targeting PP2A to paxillin. In this regard, Deltagamma1 behaved similarly to B56gamma1. However, the Deltagamma1-containing PP2A heterotrimer was insufficient for the dephosphorylation of paxillin. Transfection with Deltagamma1 enhanced paxillin phosphorylation on serine residues and recruitment into focal adhesions, and cell spreading with an actin network. In addition, Deltagamma1 rendered F10 cells as highly metastatic as BL6 cells. These results suggest that mutations in PP2A regulatory subunits may cause malignant progression.