Serine 15 Phosphorylation of p53 Directs Its Interaction with B56 and the Tumor Suppressor Activity of B56 -Specific Protein Phosphatase 2A

Article · February 2008with13 Reads
DOI: 10.1128/MCB.00983-07 · Source: PubMed
Earlier studies have demonstrated a functional link between B56γ-specific protein phosphatase 2A (B56γ-PP2A) and p53 tumor suppressor activity. Upon DNA damage, a complex including B56γ-PP2A and p53 is formed which leads to Thr55 dephosphorylation of p53, induction of the p53 transcriptional target p21, and the inhibition of cell proliferation. Although an enhanced interaction between p53 and B56γ is observed after DNA damage, the underlying mechanism and its significance in PP2A tumor-suppressive function remain unclear. In this study, we show that the increased interaction between B56γ and p53 after DNA damage requires ATM-dependent phosphorylation of p53 at Ser15. In addition, we demonstrate that the B56γ3-induced inhibition of cell proliferation, induction of cell cycle arrest in G1, and blockage of anchorage-independent growth are also dependent on Ser15 phosphorylation of p53 and p53-B56γ interaction. Taken together, our results provide a mechanistic link between Ser15 phosphorylation-mediated p53-B56γ interaction and the modulation of p53 tumor suppressor activity by PP2A. We also show an important link between ATM activity and the tumor-suppressive function of B56γ-PP2A.
    • Protein was purified from cells in culture and samples using the Thermo Scientific protein purification kit. Western Blotting was performed using antibodies against AKT (C-20, Santa Cruz Biotechnology), Phospho- AKT Ser-473 (Santa Cruz Biotechnology), Phospho-AKT Thr-308 (Santa Cruz Biotechnology), Phospho- FoxO1 (Thr24)/FoxO3A (Thr32) (Cell Signaling), PP2A A (Upstate), PP2A C (1D6, Upstate; polyclonal, Life Technologies), PP2A B55α (Upstate), PP2A B56γ [53], SET (H-120, Santa Cruz Biotechnology), and vinculin (VIN-11-5, Sigma). Immunoprecipitation was performed using protein A agarose and incubating with either AKT antibody or IgG as a negative control.
    [Show abstract] [Hide abstract] ABSTRACT: Activation of the Protein Kinase B (PKB), or AKT pathway has been shown to correlate with acute myeloid leukemia (AML) prognosis. B55α-Protein Phosphatase 2A (PP2A) has been shown to dephosphorylate AKT at Thr-308 rendering it inactive. In fact, low expression of the PP2A regulatory subunit B55α was associated with activated phospho-AKT and correlated with inferior outcomes in AML. Despite this fact, no studies have specifically demonstrated a mechanism whereby B55α expression is regulated in AML. In this study, we demonstrate novel loss of function mutations in the PPP2R2A gene identified in leukemic blasts from three AML patients. These mutations eliminate B55α protein expression thereby allowing constitutive AKT activation. In addition, leukemic blasts with PPP2R2A gene mutation were more sensitive to treatment with the AKT inhibitor MK2206, but less responsive to the PP2A activator FTY720. Using leukemia cell lines, we further demonstrate that B55α expression correlates with AKT Thr-308 phosphorylation and predicts responsiveness to AKT inhibition and PP2A activation. Together our data illustrate the importance of the B55α-PP2A-AKT pathway in leukemogenesis. Screening for disruptions in this pathway at initial AML diagnosis may predict response to targeted therapies against AKT and PP2A.
    Full-text · Article · Nov 2014
    • In regard to a specific binding domain, they all have the same amino acids from 391 to 402, which constitute a domain required for interaction with p53 [49]. Indeed, PP2A-B56γ1, γ2 nd γ3 have been reported to dephosphorylate p53 protein at Ser15 [48], [49], [74]. Similarly, all the γvariants could be plausible to bind to ERK as the present study showed that B56γ1 bound to and phosphorylated ERK.
    [Show abstract] [Hide abstract] ABSTRACT: Extracellular signal-regulated kinase (ERK) signalling plays a central role in various biological processes, including cell migration, but it remains unknown what factors directly regulate the strength and duration of ERK activation. We found that, among the B56 family of protein phosphatase 2A (PP2A) regulatory subunits, B56γ1 suppressed EGF-induced cell migration on collagen, bound to phosphorylated-ERK, and dephosphorylated ERK, whereas B56α1 and B56β1 did not. B56γ1 was immunolocalized in nuclei. The IER3 protein was immediately highly expressed in response to costimulation of cells with EGF and collagen. Knockdown of IER3 inhibited cell migration and enhanced dephosphorylation of ERK. Analysis of the time course of PP2A-B56γ1 activity following the costimulation showed an immediate loss of phosphatase activity, followed by a rapid increase in activity, and this activity then remained at a stable level that was lower than the original level. Our results indicate that the strength and duration of the nuclear ERK activation signal that is initially induced by ERK kinase (MEK) are determined at least in part by modulation of the phosphatase activity of PP2A-B56γ1 through two independent pathways.
    Full-text · Article · Dec 2013
    • The therapeutic targeting of Bcr-Abl transcripts by siRNA was demonstrated in imatinib-resistant CML cells[10,28]. PPP2R5C plays a crucial role in cell proliferation, differentiation, and transformation based on its induction of the dephosphorylation of p53 at various residues[19]and may be responsible for the tumor-suppressive function of PP2A[18]. To confirm the role of PPP2R5C down-regulation on the inhibition of CML cells, particularly TKI-resistant CML cells, we used two PPP2R5C siRNAs that target different exon sequences to analyze their effect on the inhibition of proliferation and apoptosis induction in CML cells.
    [Show abstract] [Hide abstract] ABSTRACT: Despite the success of imatinib and other tyrosine kinase inhibitors (TKIs), chronic myeloid leukemia (CML) remains largely incurable, and a number of CML patients die due to Abl mutation-related drug resistance and blast crisis. The aim of this study was to evaluate proliferation inhibition and apoptosis induction by down-regulating PPP2R5C gene expression in the imatinib-sensitive and imatinib-resistant CML cell lines K562, K562R (imatinib resistant without an Abl gene mutation), 32D-Bcr-Abl WT (imatinib-sensitive murine CML cell line with a wild type abl gene) and 32D-Bcr-Abl T315I (imatinib resistant with a T315I Abl gene mutation) and primary cells from CML patients by RNA interference. PPP2R5C siRNAs numbered 799 and 991 were obtained by chemosynthesis. Non-silencing siRNA scrambled control (SC)-treated, mock-transfected, and untreated cells were used as controls. The PPP2R5C mRNA and protein expression levels in treated CML cells were analyzed by quantitative real-time PCR and Western blotting, and in vitro cell proliferation was assayed with the cell counting kit-8 method. The morphology and percentage of apoptosis were revealed by Hoechst 33258 staining and flow cytometry (FCM). The results demonstrated that both siRNAs had the best silencing results after nucleofection in all four cell lines and primary cells. A reduction in PPP2R5C mRNA and protein levels was observed in the treated cells. The proliferation rate of the PPP2R5C-siRNA-treated CML cell lines was significantly decreased at 72 h, and apoptosis was significantly increased. Significantly higher proliferation inhibition and apoptosis induction were found in K562R cells treated with PPP2R5C-siRNA799 than K562 cells. In conclusion, the suppression of PPP2R5C by RNA interference could inhibit proliferation and effectively induce apoptosis in CML cells that were either imatinib sensitive or resistant. Down-regulating PPP2R5C gene expression might be considered as a new therapeutic target strategy for CML, particularly for imatinib-resistant CML.
    Full-text · Article · Sep 2013
    • The ranks of the relative conservation score of these 5 sites were 2, 3, 5, 6 and 19, respectively. p53 serine 15 phosphorylation could direct its interaction with B56γ and the tumor suppressor activity of B56γ-specific protein phosphatase 2A (Shouse et al., 2008). p53 serine 46 could be phosphorylated by HIPK2 upon UV irradiation, which could regulate p53 apoptotic activity and is required for acetylation by CREBBP (D'Orazi et al., 2002; Hofmann et al., 2002; Chang et al., 2005; Lee et al., 2009).
    [Show abstract] [Hide abstract] ABSTRACT: Protein phosphorylation is a ubiquitous protein post-translational modification, which plays an important role in cellular signaling systems underlying various physiological and pathological processes. Current in silico methods mainly focused on the prediction of phosphorylation sites, but rare methods considered whether a phosphorylation site is functional or not. Since functional phosphorylation sites are more valuable for further experimental research and a proportion of phosphorylation sites have no direct functional effects, the prediction of functional phosphorylation sites is quite necessary for this research area. Previous studies have shown that functional phosphorylation sites are more conserved than non-functional phosphorylation sites in evolution. Thus, in our method, we developed a web server by integrating existing phosphorylation site prediction methods, as well as both absolute and relative evolutionary conservation scores to predict the most likely functional phosphorylation sites. Using our method, we predicted the most likely functional sites of the human, rat and mouse proteomes and built a database for the predicted sites. By the analysis of overall prediction results, we demonstrated that protein phosphorylation plays an important role in all the enriched KEGG pathways. By the analysis of protein-specific prediction results, we demonstrated the usefulness of our method for individual protein studies. Our method would help to characterize the most likely functional phosphorylation sites for further studies in this research area.
    Full-text · Article · Jul 2012
    • p53, a tumor suppressor protein plays crucial role in regression of cancer progression [43]. Recent studies have revealed that phosphorylation of Ser-15 residues of p53 exhibit growth retardation in melanoma [44].
    [Show abstract] [Hide abstract] ABSTRACT: Recent understanding on cancer therapy indicated that targeting metastatic signature or angiogenic switch could be a promising and rational approach to combat cancer. Advancement in cancer research has demonstrated the potential role of various tumor suppressor proteins in inhibition of cancer progression. Current studies have shown that axonal sprouting inhibitor, semaphorin 3A (Sema 3A) acts as a potent suppressor of tumor angiogenesis in various cancer models. However, the function of Sema 3A in regulation of melanoma progression is not well studied, and yet to be the subject of intense investigation. In this study, using multiple in vitro and in vivo approaches we have demonstrated that Sema 3A acts as a potent tumor suppressor in vitro and in vivo mice (C57BL/6) models. Mouse melanoma (B16F10) cells overexpressed with Sema 3A resulted in significant inhibition of cell motility, invasiveness and proliferation as well as suppression of in vivo tumor growth, angiogenesis and metastasis in mice models. Moreover, we have observed that Sema 3A overexpressed melanoma clone showed increased sensitivity towards curcumin and Dacarbazine, anti-cancer agents. Our results demonstrate, at least in part, the functional approach underlying Sema 3A mediated inhibition of tumorigenesis and angiogenesis and a clear understanding of such a process may facilitate the development of novel therapeutic strategy for the treatment of cancer.
    Full-text · Article · Mar 2012
    • The functional PPP2R5C gene locus resides at 14q32.2, whereas a nonfunctional B56γ1 pseudogene PPP2R5C is present at 3p21.3 [4,5]. PPP2R5C plays a crucial role in cell proliferation, differentiation, and transformation, based on its induction of dephosphorylation of P53 at various residues [6]. It has been reported that the dynamic nuclear distribution of the B56γ3 regulatory subunit controls nuclear PP2A activity and may be responsible for the tumor-suppression function of PP2A [5].
    [Show abstract] [Hide abstract] ABSTRACT: Recently, we clarified at the molecular level novel chromosomal translocation t(14;14)(q11;q32) in a case of Sézary syndrome, which caused a rearrangement from TRAJ7 to the PPP2R5C gene. PPP2R5C is one of the regulatory B subunits of protein phosphatase 2A (PP2A). It plays a crucial role in cell proliferation, differentiation, and transformation. To characterize the expression and distribution of five different transcript variants of the PPP2R5C gene in leukemia, we analyzed the expression level of PPP2R5C in peripheral blood mononuclear cells from 77 patients with de novo leukemia, 26 patients with leukemia in complete remission (CR), and 20 healthy individuals by real-time PCR and identified the different variants of PPP2R5C by RT-PCR. Significantly higher expression of PPP2R5C was found in AML, CML, T-ALL, and B-CLL groups in comparison with healthy controls. High expression of PPP2R5C was detected in the B-ALL group; however, no significant difference was found compared with the healthy group. The expression level of PPP2R5C in the CML-CR group decreased significantly compared with that in the de novo CML group and was not significantly different from the level in the healthy group. By using different primer pairs that covered different exons, five transcript variants of PPP2R5C could be identified. All variants could be detected in healthy samples as well as in all the leukemia samples, and similar frequencies and distributions of PPP2R5C were indicated. Overexpression of PPP2R5C in T-cell malignancy as well as in myeloid leukemia cells might relate to its proliferation and differentiation. Investigation of the effect of target inhibition of this gene might be beneficial to further characterization of molecular mechanisms and targeted therapy in leukemia.
    Full-text · Article · May 2011
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