Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Dephosphorylation of DBC1 by Protein Phosphatase 4 Is Important for p53-Mediated Cellular Functions.

ABSTRACT: Deleted in breast cancer-1 (DBC1) contributes to the regulation of cell survival and apoptosis. Recent studies demonstrated that DBC is phosphorylated at Thr454 by ATM/ATR kinases in response to DNA damage, which is a critical event for p53 activation and apoptosis. However, how DBC1 phosphorylation is regulated has not been studied. Here we show that protein phosphatase 4 (PP4) dephosphorylates DBC1, regulating its role in DNA damage response. PP4R2, a regulatory subunit of PP4, mediates the interaction between DBC1 and PP4C, a catalytic subunit. PP4C efficiently dephosphorylates pThr454 on DBC1 in vitro, and the depletion of PP4C/PP4R2 in cells alters the kinetics of DBC1 phosphorylation and p53 activation, and increases apoptosis in response to DNA damage, which are compatible with the expression of the phosphomimetic DBC-1 mutant (T454E). These suggest that the PP4-mediated dephosphorylation of DBC1 is necessary for efficient damage responses in cells.

SUBMITTER: Lee J

PROVIDER: S-EPMC4546941 | biostudies-literature | 2015 Aug

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Dephosphorylation of DBC1 by Protein Phosphatase 4 Is Important for p53-Mediated Cellular Functions.

Lee Jihye J Adelmant Guillaume G Marto Jarrod A JA Lee Dong-Hyun DH

Molecules and cells 20150721 8

Deleted in breast cancer-1 (DBC1) contributes to the regulation of cell survival and apoptosis. Recent studies demonstrated that DBC is phosphorylated at Thr454 by ATM/ATR kinases in response to DNA damage, which is a critical event for p53 activation and apoptosis. However, how DBC1 phosphorylation is regulated has not been studied. Here we show that protein phosphatase 4 (PP4) dephosphorylates DBC1, regulating its role in DNA damage response. PP4R2, a regulatory subunit of PP4, mediates the in ...[more]

PMID: 26194823

Similar Datasets

Reduction of protein phosphatase 2A (PP2A) complexity reveals cellular functions and dephosphorylation motifs of the PP2A/B'δ holoenzyme.

Project description:Protein phosphatase 2A (PP2A) is a large enzyme family responsible for most cellular Ser/Thr dephosphorylation events. PP2A substrate specificity, localization, and regulation by second messengers rely on more than a dozen regulatory subunits (including B/R2, B'/R5, and B″/R3), which form the PP2A heterotrimeric holoenzyme by associating with a dimer comprising scaffolding (A) and catalytic (C) subunits. Because of partial redundancy and high endogenous expression of PP2A holoenzymes, traditional approaches of overexpressing, knocking down, or knocking out PP2A regulatory subunits have yielded only limited insights into their biological roles and substrates. To this end, here we sought to reduce the complexity of cellular PP2A holoenzymes. We used tetracycline-inducible expression of pairs of scaffolding and regulatory subunits with complementary charge-reversal substitutions in their interaction interfaces. For each of the three regulatory subunit families, we engineered A/B charge-swap variants that could bind to one another, but not to endogenous A and B subunits. Because endogenous Aα was targeted by a co-induced shRNA, endogenous B subunits were rapidly degraded, resulting in expression of predominantly a single PP2A heterotrimer composed of the A/B charge-swap pair and the endogenous catalytic subunit. Using B'δ/PPP2R5D, we show that PP2A complexity reduction, but not PP2A overexpression, reveals a role of this holoenzyme in suppression of extracellular signal-regulated kinase signaling and protein kinase A substrate dephosphorylation. When combined with global phosphoproteomics, the PP2A/B'δ reduction approach identified consensus dephosphorylation motifs in its substrates and suggested that residues surrounding the phosphorylation site play roles in PP2A substrate specificity.

| S-EPMC7186168 | biostudies-literature

On-Slide Lambda Protein Phosphatase-Mediated Dephosphorylation of Fixed Samples.

Project description:Protein phosphorylation is a ubiquitous post-translational modification that regulates a plethora of intracellular processes, making its analysis crucial for understanding intracellular dynamics. The commonly used methods, such as radioactive labeling and gel electrophoresis, do not provide information about subcellular localization. Immunofluorescence using phospho-specific antibodies and subsequent analysis via microscopy allows researchers to assess subcellular localization, but it typically lacks validation whether the observed fluorescent signal is phosphorylation specific. In this study, an on-slide dephosphorylation assay coupled with immunofluorescence staining using phospho-specific antibodies on fixed samples is proposed as a fast and simple approach to validate phosphorylated proteins in their native subcellular context. The assay was validated using antibodies against two different phosphorylated target proteins, connexin 43 phosphorylated at serine 373, and phosphorylated substrates of protein kinase A, with a dramatic reduction in the signal upon dephosphorylation. The proposed approach provides a convenient way to validate phosphorylated proteins without the need for additional sample preparation steps, reducing the time and effort required for analysis, while minimizing the risk of protein loss or alteration.

| S-EPMC10304472 | biostudies-literature

DBC1 functions as a tumor suppressor by regulating p53 stability.

Project description:DBC1 (deleted in breast cancer 1), also known as CCAR2 or KIAA1967, is an important negative regulator of SIRT1 and cellular stress response. Although the Dbc1 gene localizes at a region that is homozygously deleted in breast cancer, its role in tumorigenesis remains unclear. It has been suggested to be either a tumor suppressor or an oncogene. Therefore, the function of DBC1 in cancer needs to be further explored. Here, we report that Dbc1 knockout mice are tumor prone, suggesting that DBC1 functions as a tumor suppressor in vivo. Our data suggest that the increased tumor incidence in Dbc1 knockout mice is independent of Sirt1. Instead, we found that DBC1 loss results in less p53 protein in vitro and in vivo. DBC1 directly binds p53 and stabilizes it through competition with MDM2. These studies reveal that DBC1 plays an important role in tumor suppression through p53 regulation.

| S-EPMC4351187 | biostudies-literature

REDD1 enhances protein phosphatase 2A-mediated dephosphorylation of Akt to repress mTORC1 signaling.

Project description:The protein kinase mTOR (mechanistic target of rapamycin) in complex 1 (mTORC1) promotes cell growth and proliferation in response to anabolic stimuli, including growth factors and nutrients. Growth factors activate mTORC1 by stimulating the kinase Akt, which phosphorylates and inhibits the tuberous sclerosis complex [TSC; which is composed of TSC1, TSC2, and TBC1D7 (Tre2-Bub2-Cdc16 domain family member 7)], thereby stimulating the mTORC1 activator Rheb (Ras homolog enriched in brain). We identified the mechanism through which REDD1 (regulated in DNA damage and development 1) represses the mTORC1 signaling pathway. We found that REDD1 promoted the protein phosphatase 2A (PP2A)-dependent dephosphorylation of Akt on Thr(308) but not on Ser(473). Consistent with previous studies showing that phosphorylation of Akt on Thr(308), but not on Ser(473), is necessary for phosphorylation of TSC2, we observed a REDD1-dependent reduction in the phosphorylation of TSC2 and subsequently in the activation state of Rheb. REDD1 and PP2A coimmunoprecipitated with Akt from wild-type but not REDD1 knockout mouse embryonic fibroblasts, suggesting that REDD1 may act as a targeting protein for the catalytic subunit of PP2A. Furthermore, binding to both Akt and PP2A was essential for REDD1 to repress signaling to mTORC1. Overall, the results demonstrate that REDD1 acts not only as a repressor of mTORC1 but also as a constant modulator of the phosphorylation of Akt in response to growth factors and nutrients.

| S-EPMC4145530 | biostudies-literature

Structure of a protein phosphatase 2A holoenzyme: insights into B55-mediated Tau dephosphorylation.

Project description:Protein phosphatase 2A (PP2A) regulates many essential aspects of cellular physiology. Members of the regulatory B/B55/PR55 family are thought to play a key role in the dephosphorylation of Tau, whose hyperphosphorylation contributes to Alzheimer's disease. The underlying mechanisms of the PP2A-Tau connection remain largely enigmatic. Here, we report the complete reconstitution of a Tau dephosphorylation assay and the crystal structure of a heterotrimeric PP2A holoenzyme involving the regulatory subunit Balpha. We show that Balpha specifically and markedly facilitates dephosphorylation of the phosphorylated Tau in our reconstituted assay. The Balpha subunit comprises a seven-bladed beta propeller, with an acidic, substrate-binding groove located in the center of the propeller. The beta propeller latches onto the ridge of the PP2A scaffold subunit with the help of a protruding beta hairpin arm. Structure-guided mutagenesis studies revealed the underpinnings of PP2A-mediated dephosphorylation of Tau.

| S-EPMC2818795 | biostudies-literature

The ATDC (TRIM29) protein binds p53 and antagonizes p53-mediated functions.

Project description:The ataxia telangiectasia group D-complementing (ATDC) gene product, also known as TRIM29, is a member of the tripartite motif (TRIM) protein family. ATDC has been proposed to form homo- or heterodimers and to bind nucleic acids. In cell cultures, ATDC expression leads to rapid growth and resistance to ionizing radiation (IR), whereas silencing of ATDC expression decreases growth rates and increases sensitivity to IR. Although ATDC is overexpressed in many human cancers, the biological significance of ATDC overexpression remains obscure. We report here that ATDC increases cell proliferation via inhibition of p53 nuclear activities. ATDC represses the expression of p53-regulated genes, including p21 and NOXA. Mechanistically, ATDC binds p53, and this interaction is potentially fine-tuned by posttranslational acetylation of lysine 116 on ATDC. The association of p53 and ATDC results in p53 sequestration outside of the nucleus. Together, these results provide novel mechanistic insights into the function of ATDC and offer an explanation for how ATDC promotes cancer cell proliferation.

| S-EPMC2876676 | biostudies-literature

Protein phosphatase 2A-mediated flotillin-1 dephosphorylation up-regulates endothelial cell migration and angiogenesis regulation.

Project description:Endothelial cells have key functions in endothelial barrier integrity and in responses to angiogenic signals that promote cell proliferation, cell migration, cytoskeletal reorganization, and formation of new blood vessels. These functions highly depend on protein-protein interactions in cell-cell junction and cell attachment complexes and on interactions with cytoskeletal proteins. Protein phosphatase 2A (PP2A) dephosphorylates several target proteins involved in cytoskeletal dynamics and cell adhesion. Our goal was to find new interacting and substrate proteins of the PP2A-B55α holoenzyme in bovine pulmonary endothelial cells. Using LC-MS/MS analysis, we identified flotillin-1 as a protein that binds recombinant GSH S-transferase-tagged PP2A-B55α. Immunoprecipitation experiments, proximity ligation assays, and immunofluorescent staining confirmed the interaction between these two endogenous proteins in endothelial cells. Originally, flotillins were described as regulatory proteins for axon regeneration, but they appear to function in many cellular processes, such as membrane receptor signaling, endocytosis, and cell adhesion. Ser315 is a known PKC-targeted site in flotillin-1. Utilizing phosphomutants of flotillin-1 and the NanoBiT luciferase assay, we show here that phosphorylation/dephosphorylation of Ser315 in flotillin-1 significantly affects its interaction with PP2A-B55α and that PP2A-B55α dephosphorylates phospho-Ser315 Spreading, attachment, migration, and in vitro tube formation rates of S315A variant-overexpressing cells were faster than those of nontransfected or S315D-transfected cells. These results indicate that the PP2A-flotillin-1 interaction identified here affects major physiological activities of pulmonary endothelial cells.

| S-EPMC6937586 | biostudies-literature

Allosteric Activators of Protein Phosphatase 2A Display Broad Antitumor Activity Mediated by Dephosphorylation of MYBL2.

Project description:Protein phosphatase 2A (PP2A) enzymes can suppress tumors, but they are often inactivated in human cancers overexpressing inhibitory proteins. Here, we identify a class of small-molecule iHAPs (improved heterocyclic activators of PP2A) that kill leukemia cells by allosterically assembling a specific heterotrimeric PP2A holoenzyme consisting of PPP2R1A (scaffold), PPP2R5E (B56ε, regulatory), and PPP2CA (catalytic) subunits. One compound, iHAP1, activates this complex but does not inhibit dopamine receptor D2, a mediator of neurologic toxicity induced by perphenazine and related neuroleptics. The PP2A complex activated by iHAP1 dephosphorylates the MYBL2 transcription factor on Ser241, causing irreversible arrest of leukemia and other cancer cells in prometaphase. In contrast, SMAPs, a separate class of compounds, activate PP2A holoenzymes containing a different regulatory subunit, do not dephosphorylate MYBL2, and arrest tumor cells in G1 phase. Our findings demonstrate that small molecules can serve as allosteric switches to activate distinct PP2A complexes with unique substrates.

| S-EPMC7397863 | biostudies-literature

Dual-specificity phosphatase 26 is a novel p53 phosphatase and inhibits p53 tumor suppressor functions in human neuroblastoma.

Project description:Chemoresistance is a major cause of treatment failure and poor outcome in neuroblastoma. In this study, we investigated the expression and function of dual-specificity phosphatase 26 (DUSP26), also known as mitogen-activated protein kinase phophatase-8, in human neuroblastoma. We found that DUSP26 was expressed in a majority of neuroblastoma cell lines and tissue specimens. Importantly, we found that DUSP26 promotes the resistance of human neuroblastoma to doxorubicin-induced apoptosis by acting as a p53 phosphatase to downregulate p53 tumor suppressor function in neuroblastoma cells. Inhibiting DUSP26 expression in the IMR-32 neuroblastoma cell line enhanced doxorubicin-induced p53 phosphorylation at Ser20 and Ser37, p21, Puma, Bax expression as well as apoptosis. In contrast, DUSP26 overexpression in the SK-N-SH cell line inhibited doxorubicin-induced p53 phosphorylation at Ser20 and Ser37, p21, Puma, Bax expression and apoptosis. Using in vitro and in vivo assays, we found that DUSP26 binds to p53 and dephosphorylates p53 at Ser20 and Ser37. In this report, we show that DUSP26 functions as a p53 phosphatase, which suppresses downstream p53 activity in response to genotoxic stress. This suggests that inhibition of this phosphatase may increase neuroblastoma chemosensitivity and DUSP26 is a novel therapeutic target for this aggressive pediatric malignancy.

| S-EPMC7580258 | biostudies-literature

Dephosphorylation of CDK9 by protein phosphatase 2A and protein phosphatase-1 in Tat-activated HIV-1 transcription.

Project description:BackgroundHIV-1 Tat protein recruits human positive transcription elongation factor P-TEFb, consisting of CDK9 and cyclin T1, to HIV-1 transactivation response (TAR) RNA. CDK9 is maintained in dephosphorylated state by TFIIH and undergo phosphorylation upon the dissociation of TFIIH. Thus, dephosphorylation of CDK9 prior to its association with HIV-1 preinitiation complex might be important for HIV-1 transcription. Others and we previously showed that protein phosphatase-2A and protein phosphatase-1 regulates HIV-1 transcription. In the present study we analyze relative contribution of PP2A and PP1 to dephosphorylation of CDK9 and to HIV-1 transcription in vitro and in vivo.ResultsIn vitro, PP2A but not PP1 dephosphorylated autophosphorylated CDK9 and reduced complex formation between P-TEFb, Tat and TAR RNA. Inhibition of PP2A by okadaic acid inhibited basal as well as Tat-induced HIV-1 transcription whereas inhibition of PP1 by recombinant nuclear inhibitor of PP1 (NIPP1) inhibited only Tat-induced transcription in vitro. In cultured cells, low concentration of okadaic acid, inhibitory for PP2A, only mildly inhibited Tat-induced HIV-1 transcription. In contrast Tat-mediated HIV-1 transcription was strongly inhibited by expression of NIPP1. Okadaic acid induced phosphorylation of endogenous as well transiently expressed CDK9, but this induction was not seen in the cells expressing NIPP1. Also the okadaic acid did not induce phosphorylation of CDK9 with mutation of Thr 186 or with mutations in Ser-329, Thr-330, Thr-333, Ser-334, Ser-347, Thr-350, Ser-353, and Thr-354 residues involved in autophosphorylation of CDK9.ConclusionOur results indicate that although PP2A dephosphorylates autophosphorylated CDK9 in vitro, in cultured cells PP1 is likely to dephosphorylate CDK9 and contribute to the regulation of activated HIV-1 transcription.

| S-EPMC1187922 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data