The type 2C phosphatase Wip1: an oncogenic regulator of tumor suppressor and DNA damage response pathways.
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ABSTRACT:
SUBMITTER: Lu X
PROVIDER: S-EPMC2362138 | biostudies-other | 2008 Jun
REPOSITORIES: biostudies-other
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Project description:Wild-type (wt) p53-induced phosphatase 1 (Wip1), encoded by the protein phosphatase, Mg2+/Mn2+ dependent 1D (PPM1D) gene, is a serine/threonine phosphatase induced upon genotoxic stress in a p53-dependent manner. Wip1/PPM1D is frequently overexpressed, amplified and mutated in human solid tumors harboring wt p53 and is thus currently recognized as an oncogene. Oncogenic Wip1 dampens cellular stress responses, such as cell cycle checkpoints, apoptosis and senescence, and consequently increases resistance to anticancer therapeutics. Targeting Wip1 has emerged as a therapeutic strategy for tumors harboring wt p53. However, little is known about the efficacy of Wip1-targeted therapies in tumors lacking p53. The present study aimed to investigate the potential role of oncogenic Wip1 in p53 mutant (mt) Jurkat cells. In the present study, it was demonstrated that p53 mt Jurkat cells exhibited PPM1D/Wip1 gene amplification and expressed relatively high levels of Wip1, as confirmed by gene copy number and RNA expression analysis. In addition, Jurkat cells underwent G2 cell cycle arrest, apoptotic cell death and senescence in response to etoposide and doxorubicin, although the phosphorylation levels of DNA damage response (DDR) elements, including ataxia-telangiectasia mutated, ataxia-telangiestasia and Rad3-related, checkpoint kinase (Chk)1 and Chk2 were significantly low. Accordingly, the targeting of Wip1 phosphatase by RNA interference increased the phosphorylation of DDR elements, but decreased the rate of apoptosis in response to etoposide or doxorubicin in Jurkat cells. The induction of senescence or cell cycle arrest was not affected by the knockdown of Wip1. The results suggest that increased Wip1 expression enhances the apoptotic sensitivity of Jurkat cells in response to chemotherapeutic agents by attenuating DDR signaling. The present study highlights the possible pro-apoptotic role of Wip1 in a p53 mt T-cell acute lymphoblastic leukemia cell line. The data suggest the careful consideration of future treatment strategies aiming to manipulate or target Wip1 in human cancers lacking p53.
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Project description:Wild-type p53-induced phosphatase 1 (Wip1) was identified as an oncogene amplified and overexpressed in several human cancers. Recent evidence suggested that Wip1 is a critical inhibitor in the ATM/ATR-p53 DNA damage signaling pathway. Wip1 dephosphorylates several key DNA damage-responsive proteins and reverses DNA damage-induced cell cycle checkpoints. Previous reports showed that Wip1 was transcriptionally induced by p53 at the early stage of the DNA damage response. To investigate the temporal and functional regulation of Wip1, we identified a microRNA, miR-16, that specifically targets the mRNA of Wip1 and thus negatively regulates the expression level of Wip1. miR-16 itself is induced immediately after DNA damage. Therefore, the increase in Wip1 protein level is significantly postponed compared with that of its mRNA level, preventing a premature inactivation of ATM/ATR signaling and allowing a functional completion of the early DNA damage response. To better understand miR-16 biological functions in the context of cancer cells, we examined its expression in mammary tumor stem cells and found it to be markedly downregulated in mammary tumor stem cells. Overexpression of miR-16 or inhibition of Wip1 suppresses the self-renewal and growth of mouse mammary tumor stem cells and sensitizes MCF-7 human breast cancer cells to the chemotherapeutic drug doxorubicin. Together, our results suggest an important role of miR-16 in the regulation of Wip1 phosphatase in the DNA damage response and mammary tumorigenesis.
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Project description:MdmX and Mdm2 regulate p53 tumor suppressor functions by controlling p53 transcriptional activity and/or stability in cells exposed to DNA damage. Accumulating evidence indicates that ATM-mediated phosphorylation and degradation of Mdm2 and MdmX may be the initial driving force that induces p53 activity during the early phase of the DNA damage response. We have recently determined that a novel protein phosphatase, Wip1 (or PPM1D), contributes to p53 regulation by dephosphorylating Mdm2 to close the p53 activation loop initiated by the ATM/ATR kinases. In the present study, we determine that Wip1 directly dephosphorylates MdmX at the ATM-targeted Ser403 and indirectly suppresses phosphorylation of MdmX at Ser342 and Ser367. Wip1 inhibits the DNA damage-induced ubiquitination and degradation of MdmX, leading to the stabilization of MdmX and reduction of p53 activities. Our data suggest that Wip1 is an important component in the ATM-p53-MdmX regulatory loop.
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Project description:Deregulation of SHP2 is associated with malignant diseases as well as developmental disorders. Although SHP2 is required for full activation of RAS signaling, other potential roles in cell physiology have not been elucidated. Here we show that SHP2 dephosphorylates parafibromin/Cdc73, a core component of the RNA polymerase II-associated factor (PAF) complex. Parafibromin is known to act as a tumor suppressor that inhibits cyclin D1 and c-myc by recruiting SUV39H1 histone methyltransferase. However, parafibromin can also act in the opposing direction by binding ?-catenin, thereby activating promitogenic/oncogenic Wnt signaling. We found that, on tyrosine dephosphorylation by SHP2, parafibromin acquires the ability to stably bind ?-catenin. The parafibromin/?-catenin interaction overrides parafibromin/SUV39H1-mediated transrepression and induces expression of Wnt target genes, including cyclin D1 and c-myc. Hence, SHP2 governs the opposing functions of parafibromin, deregulation of which may cause the development of tumors or developmental malformations.
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Project description:cAMP-dependent phosphorylation activates the cystic fibrosis transmembrane conductance regulator (CFTR) in epithelia. However, the protein phosphatase (PP) that dephosphorylates and inactivates CFTR in airway and intestinal epithelia, two major sites of disease, is not certain. We found that in airway and colonic epithelia, neither okadaic acid nor FK506 prevented inactivation of CFTR when cAMP was removed. These results suggested that a phosphatase distinct from PP1, PP2A, and PP2B was responsible. Because PP2C is insensitive to these inhibitors, we tested the hypothesis that it regulates CFTR. We found that PP2Calpha is expressed in airway and T84 intestinal epithelia. To test its activity on CFTR, we generated recombinant human PP2Calpha and found that it dephosphorylated CFTR and an R domain peptide in vitro. Moreover, in cell-free patches of membrane, addition of PP2Calpha inactivated CFTR Cl- channels; reactivation required readdition of kinase. Finally, coexpression of PP2Calpha with CFTR in epithelia reduced the Cl- current and increased the rate of channel inactivation. These results suggest that PP2C may be the okadaic acid-insensitive phosphatase that regulates CFTR in human airway and T84 colonic epithelia. It has been suggested that phosphatase inhibitors could be of therapeutic value in cystic fibrosis; our data suggest that PP2C may be an important phosphatase to target.
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Project description:Death domain-associated protein 6 (DAXX) is a histone chaperone, putative regulator of apoptosis and transcription, and candidate modulator of p53-mediated gene expression following DNA damage. DAXX becomes phosphorylated upon DNA damage, however regulation of this modification, and its relationship to p53 remain unclear. Here we show that in human cells exposed to ionizing radiation or genotoxic drugs etoposide and neocarzinostatin, DAXX became rapidly phosphorylated in an ATM kinase-dependent manner. Our deletion and site-directed mutagenesis experiments identified Serine 564 (S564) as the dominant ATM-targeted site of DAXX, and immunofluorescence experiments revealed localization of S564-phosphorylated DAXX to PML nuclear bodies. Furthermore, using a panel of human cell types, we identified the p53-regulated Wip1 protein phosphatase as a key negative regulator of DAXX phosphorylation at S564, both in vitro and in cells. Consistent with the emerging oncogenic role of Wip1, its DAXX-dephosphorylating impact was most apparent in cancer cell lines harboring gain-of-function mutant and/or overexpressed Wip1. Unexpectedly, while Wip1 depletion increased DAXX phosphorylation both before and after DNA damage and increased p53 stability and transcriptional activity, knock-down of DAXX impacted neither p53 stabilization nor p53-mediated expression of Gadd45a, Noxa, Mdm2, p21, Puma, Sesn2, Tigar or Wip1. Consistently, analyses of cells with genetic, TALEN-mediated DAXX deletion corroborated the notion that neither phosphorylated nor non-phosphorylated DAXX is required for p53-mediated gene expression upon DNA damage. Overall, we identify ATM kinase and Wip1 phosphatase as opposing regulators of DAXX-S564 phosphorylation, and propose that the role of DAXX phosphorylation and DAXX itself are independent of p53-mediated gene expression.
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Project description:Cells are constantly challenged by DNA damage and protect their genome integrity by activation of an evolutionary conserved DNA damage response pathway (DDR). A central core of DDR is composed of a spatiotemporally ordered net of post-translational modifications, among which protein phosphorylation plays a major role. Activation of checkpoint kinases ATM/ATR and Chk1/2 leads to a temporal arrest in cell cycle progression (checkpoint) and allows time for DNA repair. Following DNA repair, cells re-enter the cell cycle by checkpoint recovery. Wip1 phosphatase (also called PPM1D) dephosphorylates multiple proteins involved in DDR and is essential for timely termination of the DDR. Here we have investigated how Wip1 is regulated in the context of the cell cycle. We found that Wip1 activity is downregulated by several mechanisms during mitosis. Wip1 protein abundance increases from G(1) phase to G(2) and declines in mitosis. Decreased abundance of Wip1 during mitosis is caused by proteasomal degradation. In addition, Wip1 is phosphorylated at multiple residues during mitosis, and this leads to inhibition of its enzymatic activity. Importantly, ectopic expression of Wip1 reduced ?H2AX staining in mitotic cells and decreased the number of 53BP1 nuclear bodies in G(1) cells. We propose that the combined decrease and inhibition of Wip1 in mitosis decreases the threshold necessary for DDR activation and enables cells to react adequately even to modest levels of DNA damage encountered during unperturbed mitotic progression.
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Project description:By interference of the yeast pheromone mitogen-activated protein kinase (MAPK) pathway with an alfalfa cDNA expression library, we have isolated the MP2C gene encoding a functional protein phosphatase type 2C. Epistasis analysis in yeast indicated that the molecular target of the MP2C phosphatase is Ste11, a MAPK kinase kinase that is a central regulator of the pheromone and osmosensing pathways. In plants, MP2C functions as a negative regulator of the stress-activated MAPK (SAMK) pathway that is activated by cold, drought, touch, and wounding. Although activation of the SAMK pathway occurs by a posttranslational mechanism, de novo transcription and translation of protein factor(s) are necessary for its inactivation. MP2C is likely to be this or one of these factors, because wound-induced activation of SAMK is followed by MP2C gene expression and recombinant glutathione S-transferase-MP2C is able to inactivate extracts containing wound-induced SAMK. Wound-induced MP2C expression is a transient event and correlates with the refractory period, i.e., the time when restimulation of the SAMK pathway is not possible by a second stimulation. These data suggest that MP2C is part of a negative feedback mechanism that is responsible for resetting the SAMK cascade in plants.
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Project description:Although a great deal of progress has been made toward understanding the role of abscisic acid (ABA) in fruit ripening, many components in the ABA signalling pathway remain to be elucidated. Here, a strawberry gene homologous to the Arabidopsis gene ABI1, named FaABI1, was isolated and characterized. The 1641bp cDNA includes an intact open reading frame that encodes a deduced protein of 546 amino acids, in which putative conserved domains were determined by homology analysis. Transcriptional analysis showed that the levels of FaABI1 mRNA expression declined rapidly during strawberry fruit development as evidenced by real-time PCR, semi-quantitative reverse transcription-PCR, and northern blotting analyses, suggesting that the Ser/Thr protein phosphatase PP2C1 encoded by FaABI1 may be involved in fruit ripening as a negative regulator. The results of Tobacco rattle virus-induced gene silencing and PBI121 vector-mediated overexpression suggested that the down- and up-regulation of FaABI1 mRNA expression levels in degreening strawberry fruit could promote and inhibit ripening, respectively. Furthermore, alteration of FaABI1 expression could differentially regulate the transcripts of a set of both ABA-responsive and ripening-related genes, including ABI3, ABI4, ABI5, SnRK2, ABRE1, CHS, PG1, PL, CHI, F3H, DFR, ANS, and UFGT. Taken together, the data provide new evidence for an important role for ABA in regulating strawberry fruit ripening in the processes of which the type 2C protein phosphatase ABI1 serves as a negative regulator. Finally, a possible core mechanism underlying ABA perception and signalling transduction in strawberry fruit ripening is discussed.
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