Protein Phosphatase 5 Expression Research Articles

MP04-09 NOVEL INHIBITOR OF PRO-SURVIVAL PROTEIN PHOSPHATASE 5 (PP5) LEADS TO APOPTOSIS IN CLEAR CELL RENAL CELL CARCINOMA

You have accessJournal of UrologyCME1 Apr 2023MP04-09 NOVEL INHIBITOR OF PRO-SURVIVAL PROTEIN PHOSPHATASE 5 (PP5) LEADS TO APOPTOSIS IN CLEAR CELL RENAL CELL CARCINOMA Elham Ahanin, Rebecca A. Sager, Sarah J. Backe, Natela Dushukyan, Michael Daneshvar, Gennady Bratslavsky, John Chisholm, Mark R. Woodford, Dimitra Bourboulia, and Mehdi Mollapour Elham AhaninElham Ahanin More articles by this author , Rebecca A. SagerRebecca A. Sager More articles by this author , Sarah J. BackeSarah J. Backe More articles by this author , Natela DushukyanNatela Dushukyan More articles by this author , Michael DaneshvarMichael Daneshvar More articles by this author , Gennady BratslavskyGennady Bratslavsky More articles by this author , John ChisholmJohn Chisholm More articles by this author , Mark R. WoodfordMark R. Woodford More articles by this author , Dimitra BourbouliaDimitra Bourboulia More articles by this author , and Mehdi MollapourMehdi Mollapour More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003215.09AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Despite good survival rates for localized renal cell carcinoma a proportion of patients present with or will progress to metastatic disease, which portends worse prognosis and poor overall survival. Survival has been improved with the use of new targeted therapies and immunotherapy regimens; many patients, however, ultimately develop resistance. There is therefore a need for identification and development of new therapeutics. Clear cell renal cell carcinoma (ccRCC), the most common histologic subtype, generally harbors loss of the tumor suppressor von Hippel Lindau (VHL). The serine/threonine protein phosphatase-5 (PP5) plays a role in the regulation of numerous signaling pathways essential for cancer growth. Previously we have shown that PP5 is targeted for ubiquitination and degradation by a VHL-containing E3 ubiquitin ligase complex in a hypoxia-independent manner. Expression and activity of PP5 is consequently increased in ccRCC, and it plays a pro-survival role. Knock-down of PP5 expression or targeting its activity through inhibition of the kinase casein kinase-1δ (CK1δ) caused apoptosis in VHL-null ccRCC. The objective of this study was to develop a novel PP5 inhibitor for ccRCC. METHODS: An in silico docking screen using the crystal structure of the PP5 active site and a large compound library was used to screen for candidate inhibitors. A series of candidates were then evaluated using both in vitro and cell-based assays for their ability to inhibit PP5 activity and cause apoptosis in ccRCC cells. In vitro PP5 activity was assessed using specific phospho-peptide substrates and in cells by immunoblotting to assess phosphorylation levels of known substrates. Two primary candidates were conjugated to biotin for PP5 pulldown experiments to assess specificity. RESULTS: Our in silico screen identified approximately 200 candidate inhibitors. Further screening of about 20 of these compounds identified two compounds, P5 and P13, which inhibit PP5 activity both in vitro and in ccRCC cells and were further refined based on structure. Pulldown using biotin-conjugated drugs demonstrated good inhibitor specificity. Furthermore, treatment with these inhibitors leads to apoptosis specifically in VHL-null ccRCC cell lines. CONCLUSIONS: PP5 promotes cell survival in ccRCC and knockdown or inhibition of PP5 leads to cellular apoptosis. We have identified a small molecule which specifically binds to and inhibits PP5. This novel PP5 inhibitor causes apoptosis in VHL-null ccRCC cells and may serve as a new therapeutic strategy for treatment of advanced ccRCC. Source of Funding: This work was supported by the National Institute of General Medical Sciences of the National Institutes of Health under award numbers R35GM139584 (M.M.) and DoD KC190038 (M.M.) © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e36 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Elham Ahanin More articles by this author Rebecca A. Sager More articles by this author Sarah J. Backe More articles by this author Natela Dushukyan More articles by this author Michael Daneshvar More articles by this author Gennady Bratslavsky More articles by this author John Chisholm More articles by this author Mark R. Woodford More articles by this author Dimitra Bourboulia More articles by this author Mehdi Mollapour More articles by this author Expand All Advertisement PDF downloadLoading ...

Increased protein phosphatase 5 expression in inflammation-induced left ventricular dysfunction in rats

Titin phosphorylation contributes to left ventricular (LV) diastolic dysfunction. The independent effects of inflammation on the molecular pathways that regulate titin phosphorylation are unclear. We investigated the effects of collagen-induced inflammation and subsequent tumor necrosis factor-α (TNF-α) inhibition on mRNA expression of genes involved in regulating titin phosphorylation in 70 Sprague-Dawley rats. LV diastolic function was assessed with echocardiography. Circulating inflammatory markers were quantified by enzyme-linked immunosorbent assay and relative LV gene expression was assessed by Taqman® polymerase chain reaction. Differences in normally distributed variables between the groups were determined by two-way analysis of variance (ANOVA), followed by Tukey post-hoc tests. For non-normally distributed variables, group differences were determined by Kruskal-Wallis tests. Collagen inoculation increased LV relative mRNA expression of vascular cell adhesion molecule 1 (VCAM1), pentraxin 3 (PTX3), and inducible nitric oxide synthase (iNOS) compared to controls, indicating local microvascular inflammation. Collagen inoculation decreased soluble guanylate cyclase alpha-2 (sGCα2) and soluble guanylate cyclase beta-2 (sGCβ2) expression, suggesting downregulation of nitric oxide-soluble guanylate cyclase-cyclic guanosine monophosphate (NO-sGC-cGMP) signaling. Inhibiting TNF-α prevented collagen-induced changes in VCAM1, iNOS, sGCα2 and sGCβ2 expression. Collagen inoculation increased protein phosphatase 5 (PP5) expression. Like LV diastolic dysfunction, increased PP5 expression was not prevented by TNF-α inhibition. Inflammation-induced LV diastolic dysfunction may be mediated by a TNF-α-independent increase in PP5 expression and dephosphorylation of the N2-Bus stretch element of titin, rather than by TNF-α-induced downregulation of NO-sGC-cGMP pathway-dependent titin phosphorylation. The steady rise in number of patients with inflammation-induced diastolic dysfunction, coupled with low success rates of current therapies warrants a better understanding of the systemic signals and molecular pathways responsible for decreased titin phosphorylation in development of LV diastolic dysfunction. The therapeutic potential of inhibiting PP5 upregulation in LV diastolic dysfunction requires investigation.

Comparison of endogenous development, invasion ability and apoptotic features between diclazuril resistant and sensitive strains of Eimeria tenella

Diclazuril (DIC) is widely used in the poultry industry to control coccidiosis. However, drug resistance makes it less effective, and the underlying mechanism remains unclear. One DIC-resistant E. tenella (RE) isolate and one sensitive E. tenella (SE) isolate were used to compare the differences in their endogenous development, pathogenicity, invasion-related gene expression and apoptotic characteristics. Chickens were allocated into four groups to receive RE or SE strain and their corresponding DIC treatment or not. Caeca tissues were sampled at 96 h, 120 h and 144 h post-infection (PI) for pathological analysis. Meanwhile, second-generation merozoites (Mz2) were separated at 120 h PI to detect alterations in mitochondrial membrane potential (MMP), apoptotic rate and caspase-3 activity and mRNA expression of protein phosphatase 5 (PP5), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), actin depolymerizing factor (ADF) and microneme proteins (MICs). Haematoxylin and eosin staining revealed that DIC treatment strictly blocked the development of the SE strain but slightly affected the RE strain. Meanwhile, the number of SE Mz2 and their MMP decreased at the same time the apoptotic rate increased after DIC treatment. Real-time quantitative PCR and caspase-3 activity studies demonstrated that Mz2 from the RE strain had higher mRNA expression of ADF and MICs along with no significant changes in GAPDH and caspase-3 activity under DIC pressure compared to its control; in contrast, the mRNA expression of ADF, MICs and PP5 was markedly suppressed in Mz2 from SE with upregulated caspase-3 activity and GAPDH transcription. In addition, the mRNA expression of GAPDH and PP5 in Mz2 from RE was remarkably higher than that of SE. Taken together, the higher mRNA expression of invasion-related genes and almost unaffected endogenous development provide a better understanding of coccidian resistance to DIC.

Abstract 2891: Protein phosphatase 5 regulation of cell motility

Abstract Metastasis has been considered as one of the cancer hallmarks. Approximately 90% of cancer-related death is due to cancer metastasis. Tumor cell migration is a prerequisite for cancer metastasis. Protein phosphatase 5 (PP5) is a serine/threonine protein phosphatase that belongs to the phosphoprotein phosphatase (PPP) family including PP1 and PP2A. It is well known that PP5 regulates apoptosis, proliferation, and stress response. However, the role of PP5 in the regulation of cell motility is only beginning to be recognized, and the mechanism of PP5 regulation of cell motility is largely unknown. In this study, our tissue microarray (TMA) studies showed the expression of PP5 increased with the stages of prostate cancer, which is consistent with the previous findings that there is a positive correlation between PP5 overexpression and breast cancer metastases. Furthermore, by the single-cell motility assay, we found that overexpression of PP5 increased the motility of various tumor cells, whereas knockdown of PP5 decreased the cell motility, suggesting that PP5 positively regulates cell motility. As focal adhesion kinase (FAK) is a key regulator of cell motility, and a report has shown that PP5 forms a complex with ASK1, FAK, CXCR4 in wounded alveolar epithelial cancer cells, we examined whether PP5 activates FAK. The Western blotting results showed that overexpression of PP5 did not obviously affect the phosphorylation of FAK. The results suggest that PP5 regulates cell motility through a new mechanism. Further research is on the way to identify how PP5 positively regulates cell motility. (Supported by the Feist-Weiller Cancer Center, LSU Health Sciences Center, Shreveport, LA, USA.) Citation Format: n Li, Shile Huang. Protein phosphatase 5 regulation of cell motility [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2891.

Protein phosphatase 5 and the tumor suppressor p53 down-regulate each other's activities in mice

Protein phosphatase 5 (PP5), a serine/threonine phosphatase, has a wide range of biological functions and exhibits elevated expression in tumor cells. We previously reported that pp5-deficient mice have altered ataxia-telangiectasia mutated (ATM)-mediated signaling and function. However, this regulation was likely indirect, as ATM is not a known PP5 substrate. In the current study, we found that pp5-deficient mice are hypersensitive to genotoxic stress. This hypersensitivity was associated with the marked up-regulation of the tumor suppressor tumor protein p53 and its downstream targets cyclin-dependent kinase inhibitor 1A (p21), MDM2 proto-oncogene (MDM2), and phosphatase and tensin homolog (PTEN) in pp5-deficient tissues and cells. These observations suggested that PP5 plays a role in regulating p53 stability and function. Experiments conducted with p53+/-pp5+/- or p53+/-pp5-/- mice revealed that complete loss of PP5 reduces tumorigenesis in the p53+/- mice. Biochemical analyses further revealed that PP5 directly interacts with and dephosphorylates p53 at multiple serine/threonine residues, resulting in inhibition of p53-mediated transcriptional activity. Interestingly, PP5 expression was significantly up-regulated in p53-deficient cells, and further analysis of pp5 promoter activity revealed that p53 strongly represses PP5 transcription. Our results suggest a reciprocal regulatory interplay between PP5 and p53, providing an important feedback mechanism for the cellular response to genotoxic stress.

Inhibition of protein phosphatase 5 suppresses non-small cell lung cancer through AMP-activated kinase activation

ObjectivesNon-small cell lung cancer (NSCLC) continues to be the top cause of cancer death. To improve the treatment of lung cancer, there is necessity to identify novel oncogenes and investigate their effects on lung carcinogenesis. Protein phosphatase 5 (PP5) has long been known to regulate stress-induced apoptosis and cell proliferation. Recently, PP5 has been found overexpressed and emerged as a viable therapeutic target in various human cancers, but its role in NSCLC remains elusive. Materials and methodsThe expression of PP5 in NSCLC cell lines (A549, H358, and H460) and human tumor samples were examined. Protein phosphatase inhibitors, cantharidin and norcantharidin, were used as proof-of-concept compounds to investigate the pathological function of PP5 in NSCLC. Apoptosis and cellular signaling were analyzed. In vivo efficacy was determined in nude mice with H460 xenograft. Results and conclusionWe found that PP5 was more highly expressed in human lung tumor samples than in adjacent normal tissues. Overexpression of PP5 promoted cell proliferation, colony formation, and sphere-forming ability of A549 cells. Inhibition of PP5 phosphatase activity by cantharidin induced significant apoptosis and upregulated AMP‐activated protein kinase (AMPK) signaling. Importantly, we found that PP5-mediated dephosphorylation of AMPK determines the in vitro anti‐NSCLC effects of cantharidin. Consistent with our in vitro data, PP5 inhibition suppressed H460 tumor growth and upregulated p‐AMPK in tumor samples. Our results demonstrate that PP5 inhibition suppresses tumor growth via activating AMPK signaling. Targeting oncogenic PP5 represents an attractive therapeutic strategy for treating lung cancer.

A heteromeric molecular complex regulates the migration of lung alveolar epithelial cells during wound healing

Alveolar type II epithelial cells (ATII) are instrumental in early wound healing in response to lung injury, restoring epithelial integrity through spreading and migration. We previously reported in separate studies that focal adhesion kinase-1 (FAK) and the chemokine receptor CXCR4 promote epithelial repair mechanisms. However, potential interactions between these two pathways were not previously considered. In the present study, we found that wounding of rat ATII cells promoted increased association between FAK and CXCR4. In addition, protein phosphatase-5 (PP5) increased its association with this heteromeric complex, while apoptosis signal regulating kinase-1 (ASK1) dissociated from the complex. Cell migration following wounding was decreased when PP5 expression was decreased using shRNA, but migration was increased in ATII cells isolated from ASK1 knockout mice. Interactions between FAK and CXCR4 were increased upon depletion of ASK1 using shRNA in MLE-12 cells, but unaffected when PP5 was depleted. Furthermore, we found that wounded rat ATII cells exhibited decreased ASK1 phosphorylation at Serine-966, decreased serine phosphorylation of FAK, and decreased association of phosphorylated ASK1 with FAK. These changes in phosphorylation were dependent upon expression of PP5. These results demonstrate a unique molecular complex comprising CXCR4, FAK, ASK1, and PP5 in ATII cells during wound healing.

Palbociclib induces activation of AMPK and inhibits hepatocellular carcinoma in a CDK4/6-independent manner.

Palbociclib, a CDK4/6 inhibitor, has recently been approved for hormone receptor‐positive breast cancer patients. The effects of palbociclib as a treatment for other malignancies, including hepatocellular carcinoma (HCC), are of great clinical interest and are under active investigation. Here, we report the effects and a novel mechanism of action of palbociclib in HCC. We found that palbociclib induced both autophagy and apoptosis in HCC cells through a mechanism involving 5′ AMP‐activated protein kinase (AMPK) activation and protein phosphatase 5 (PP5) inhibition. Blockade of AMPK signals or ectopic expression of PP5 counteracted the effect of palbociclib, confirming the involvement of the PP5/AMPK axis in palbociclib‐mediated HCC cell death. However, CDK4/6 inhibition by lentivirus‐mediated shRNA expression did not reproduce the effect of palbociclib‐treated cells, suggesting that the anti‐HCC effect of palbociclib is independent of CDK4/6. Moreover, two other CDK4/6 inhibitors (ribociclib and abemaciclib) had minimal effects on HCC cell viability and the PP5/AMPK axis. Palbociclib also demonstrated significant tumor‐suppressive activity in a HCC xenograft model, which was associated with upregulation of pAMPK and PP5 inhibition. Finally, we analyzed 153 HCC clinical samples and found that PP5 expression was highly tumor specific and was associated with poor clinical features. Taken together, we conclude that palbociclib exerted antitumor activity against HCC through the PP5/AMPK axis independent of CDK4/6. Our findings provide a novel mechanistic basis for palbociclib and reveal the therapeutic potential of targeting PP5/AMPK signaling with a PP5 inhibitor for the treatment of hepatocellular carcinoma.

Celastrol ameliorates Cd-induced neuronal apoptosis by targeting NOX2-derived ROS-dependent PP5-JNK signaling pathway.

Celastrol, a plant-derived triterpene, has neuroprotective benefit in the models of neurodegenerative disorders that are characterized by overproduction of reactive oxygen species (ROS). Recently, we have reported that cadmium (Cd) activates c-Jun N-terminal kinase (JNK) pathway leading to neuronal cell death by inducing ROS inactivation of protein phosphatase 5 (PP5), and celastrol prevents Cd-activated JNK pathway against neuronal apoptosis. Therefore, we hypothesized that celastrol could hinder Cd induction of ROS-dependent PP5-JNK signaling pathway from apoptosis in neuronal cells. Here, we show that celastrol attenuated Cd-induced expression of NADPH oxidase 2 (NOX2) and its regulatory proteins (p22phox , p40phox , p47phox , p67phox , and Rac1), as well as the generation of ROS in PC12 cells and primary neurons. Also, N-acetyl-l-cysteine, a ROS scavenger, potentiated celastrol's inhibition of the events in the cells triggered by Cd, implying neuroprotection by celastrol via blocking Cd-evoked NOX2-derived ROS. Further research revealed that celastrol was involved in the regulation of PP5 inactivation and JNK/c-Jun activation induced by Cd, as celastrol prevented Cd from reducing PP5 expression, and over-expression of wild-type PP5 or dominant negative c-Jun strengthened celastrol's inhibition of Cd-induced phosphorylation of JNK and/or c-Jun, as well as apoptosis in neuronal cells. Of importance, inhibiting NOX2 with apocynin or silencing NOX2 by RNA interference enhanced the inhibitory effects of celastrol on Cd-induced inactivation of PP5, activation of JNK/c-Jun, ROS, and apoptosis in the cells. Furthermore, we noticed that expression of wild-type PP5 or dominant negative c-Jun, or pretreatment with JNK inhibitor SP600125 reinforced celastrol's suppression of Cd-induced NOX2 and its regulatory proteins, and consequential ROS in neuronal cells. These findings indicate that celastrol ameliorates Cd-induced neuronal apoptosis via targeting NOX2-derived ROS-dependent PP5-JNK signaling pathway. Our data highlight a beneficial role of celastrol in the prevention of Cd-induced oxidative stress and neurodegenerative diseases.

Cytokine-Induced Glucocorticoid Resistance from Eosinophil Activation: Protein Phosphatase 5 Modulation of Glucocorticoid Receptor Phosphorylation and Signaling.

The mechanisms contributing to persistent eosinophil activation and poor eosinopenic response to glucocorticoids in severe asthma are poorly defined. We examined the effect of cytokines typically overexpressed in the asthmatic airways on glucocorticoid signaling in in vitro activated eosinophils. An annexin V assay used to measure eosinophil apoptosis showed that cytokine combinations of IL-2 plus IL-4 as well as TNF-α plus IFN-γ, or IL-3, GM-CSF, and IL-5 alone significantly diminished the proapoptotic response to dexamethasone. We found that IL-2 plus IL-4 resulted in impaired phosphorylation and function of the nuclear glucocorticoid receptor (GCR). Proteomic analysis of steroid sensitive and resistant eosinophils identified several differentially expressed proteins, namely protein phosphatase 5 (PP5), formyl peptide receptor 2, and annexin 1. Furthermore, increased phosphatase activity of PP5 correlated with impaired phosphorylation of the GCR. Importantly, suppression of PP5 expression with small interfering RNA restored proper phosphorylation and the proapoptotic function of the GCR. We also examined the effect of lipoxin A4 on PP5 activation by IL-2 plus IL-4. Similar to PP5 small interfering RNA inhibition, pretreatment of eosinophils with lipoxin A4 restored GCR phosphorylation and the proaptoptotic function of GCs. Taken together, our results showed 1) a critical role for PP5 in cytokine-induced resistance to GC-mediated eosinophil death, 2) supported the dependence of GCR phosphorylation on PP5 activity, and 3) revealed that PP5 is a target of the lipoxin A4-induced pathway countering cytokine-induced resistance to GCs in eosinophils.

Serine/Threonine Protein Phosphatase-5 Accelerates Cell Growth and Migration in Human Glioma.

Glioma is the most common type of primary central nervous system tumor. Ser/Thr protein phosphatase 5 (PP5) has been shown to regulate multiple signaling cascades that suppress growth and facilitate apoptosis in several human cancer cells. However, the role of PP5 in human gliomas remains unclear. Herein, the relationship between PP5 expression and glioma cell growth was investigated, and the therapeutic value of PP5 in glioma was further evaluated. We employed a short hairpin RNA targeting PPP5C gene to knock down PP5 expression in human glioma cell lines U251 and U373. Depletion of PPP5C via RNAi remarkably inhibited glioma cell proliferation and colony formation, and arrested cell cycle in the G0/G1 phase. Moreover, knockdown of PP5 markedly suppressed glioma cell migration, as determined by Transwell assay. Our findings suggest that PPP5C could be essential for glioma cell growth and serve as a promising therapeutic target in human gliomas.

Inhibition of protein phosphatase 5 (PP5) suppresses survival and growth of colorectal cancer cells.

Protein phosphatase 5 (PP5) is a unique member of the protein phosphatases family that functions in multiple signaling pathways involved in DNA damage, cell cycle control, cell growth, and apoptosis. Recent evidence indicated that PP5 may play a role in cancer progression. In this study, we aimed to examine the biological effect of PP5 on cell growth and apoptosis in human colorectal cancer (CRC). We first knocked down PP5 expression in RKO cells via a short hairpin RNA containing lentivirus system. Then, methylthiazoletetrazolium assay, colony formation assay, and flow cytometry analysis were performed. The proliferation and colony formation ability of RKO cells were remarkably suppressed in PP5-silenced groups, as compared with control groups. Moreover, downregulation of PP5 resulted in a significant G0/G1 phase cell cycle arrest and an induction of apoptosis. In all, these results demonstrated the importance of PP5 in CRC cell growth, and it might be used as a potential therapeutic target for the treatment of CRC.

Protein Phosphatase-5 is a Novel Titin Ligand Which Dephosphorylates Cardiac Titin and Reduces Passive Myocyte Stiffness

Titin's elastic I-band region is a hotspot for protein-protein interactions and important for myocyte extensibility and passive stiffness. The titin springs can be phosphorylated at the N2B-unique sequence (N2Bus) by protein kinases (PK)A or PKG and at the PEVK-domain by PKCalpha, which affects passive stiffness. We searched for protein phosphatase(s) acting on the N2Bus. A yeast-2-hybrid (Y2H) screen with the human N2Bus (bait) and a human heart cDNA library (prey) detected the catalytic domain of the serine/threonine protein phosphatase-5 (PP5) as a binding partner of the cardiac N2Bus. The interaction was confirmed in forced Y2H screens with the N2Bus and full-length PP5 or PP5 catalytic subunit (PP5c), and also in GST-pulldown assays. In cardiomyocytes, PP5 was mainly in the cytosol but also in the nucleus and at the sarcomeric I-bands. Recombinant PP5 was found by autoradiography to dephosphorylate recombinant, PKG-phosphorylated, N2Bus, and PP5 bound more strongly to (PKA-/PKG-)phosphorylated N2Bus than to non-phosphorylated N2Bus. Phosphorylation of titin could be reduced in human heart tissue treated ex vivo with recombinant PP5c, an effect detected with phospho-N2Bus (S469) specific antibodies. A transgenic mouse model with PP5 overexpression revealed reduced cardiac titin phosphorylation levels compared to wildtype mouse hearts. PP5 expression was elevated in human heart failure, while titin phosphorylation was depressed. PP5c treatment of enzymatically skinned single human cardiomyocytes significantly reduced passive stiffness, which can potentially be explained by dephosphorylation of the PEVK-domain adjacent to the N2Bus. In conclusion, PP5 is a novel binding partner of cardiac titin at the N2Bus and acts to reduce passive myocyte stiffness by dephosphorylating the titin springs. PP5 may participate in mechanical signaling pathways converging on the titin springs.

Protein phosphatase 5 protects neurons against amyloid‐β toxicity

Amyloid-beta (Abeta) is thought to promote neuronal cell loss in Alzheimer's disease, in part through the generation of reactive oxygen species (ROS) and subsequent activation of mitogen-activated protein kinase (MAPK) pathways. Protein phosphatase 5 (PP5) is a ubiquitously expressed serine/threonine phosphatase which has been implicated in several cell stress response pathways and shown to inactivate MAPK pathways through key dephosphorylation events. Therefore, we examined whether PP5 protects dissociated embryonic rat cortical neurons in vitro from cell death evoked by Abeta. As predicted, neurons in which PP5 expression was decreased by small-interfering RNA treatment were more susceptible to Abeta toxicity. In contrast, over-expression of PP5, but not the inactive mutant, PP5(H304Q), prevented MAPK phosphorylation and neurotoxicity induced by Abeta. PP5 also prevented cell death caused by direct treatment with H(2)O(2), but did not prevent Abeta-induced production of ROS. Thus, the neuroprotective effect of PP5 requires its phosphatase activity and lies downstream of Abeta-induced generation of ROS. In summary, our data indicate that PP5 plays a pivotal neuroprotective role against cell death induced by Abeta and oxidative stress. Consequently, PP5 might be an effective therapeutic target in Alzheimer's disease and other neurodegenerative disorders in which oxidative stress is implicated.

Elevated levels of Ser/Thr protein phosphatase 5 (PP5) in human breast cancer

Ser/Thr protein phosphatase 5 (PP5) regulates several signaling-cascades that suppress growth and/or facilitate apoptosis in response to genomic stress. The expression of PP5 is responsive to hypoxia inducible factor-1 (HIF-1) and estrogen, which have both been linked to the progression of human breast cancer. Still, it is not clear if PP5 plays a role in the development of human cancer. Here, immunostaining of breast cancer tissue-microarrays (TMAs) revealed a positive correlation between PP5 over-expression and ductal carcinoma in situ (DCIS; P value 0.0028), invasive ductal carcinoma (IDC; P value 0.012) and IDC with metastases at the time of diagnosis ( P value 0.0001). In a mouse xenograft model, the constitutive over-expression of PP5 was associated with an increase in the rate of tumor growth. In a MCF-7 cell culture model over-expression correlated with both an increase in the rate of proliferation and protection from cell death induced by oxidative stress, UVC-irradiation, adriamycin, and vinblastine. PP5 over-expression had no apparent effect on the sensitivity of MCF-7 cells to taxol or rapamycin. Western analysis of extracts from cells over-expressing PP5 revealed a decrease in the phosphorylation of known substrates for PP5. Together, these studies indicate that elevated levels of PP5 protein occur in human breast cancer and suggest that PP5 over-expression may aid tumor progression.

Induction of abnormal nuclear shapes in two distinct modes by overexpression of serine/threonine protein phosphatase 5 in Hela cells

Okadaic acid-sensitve serine/threonine protein phosphatase 5 (PP5) is expressed ubiquitously in various tissues and is considered to participate in many cellular processes. PP5 has a catalytic domain in the C-terminal region and three tetratricopeptide repeat (TPR) motifs in the N-terminal region, which are suspected to function as a protein-protein interaction domain. Physiological roles of PP5 are still largely unknown, although several PP5-binding proteins were reported and a few in vivo functions of PP5 were suggested. In the present study, the effects of expression of the full-length wild-type PP5 fused with EGFP (EGFP-PP5(WT)) and its phosphatase-dead mutant EGFP-PP5(H304A) were investigated. Transient expression of either EGFP-PP5(WT) or EGFP-PP5(H304A) in HeLa cells induced deformed nuclei with a 10-fold frequency compared to that of EGFP. Abnormal-shaped nuclei were also substantially increased by induced moderate expression of PP5 in tet-on HeLa cells. Many HeLa cells expressing EGFP-PP5(WT) possessed multi-nuclei separated from each other by nuclear membrane, while expression of EGFP-PP5(H304A) induced deformed nuclei which were multiple-like in shape, but not separated completely and were surrounded by one nuclear membrane. These results suggest that PP5 plays important roles at the M-phase of the cell cycle, especially in separation of chromosomes and formation of nuclear membrane.

Basal adrenocorticotropin (ACTH) secretion is negatively modulated by protein phosphatase 5 in mouse pituitary corticotropin AtT20 cells

siRNA oligonucleotides for protein phosphatase 5 (PP5) were designed and transfected into mouse corticotroph AtT20 cells to induce lower PP5 expression levels. PP5-siRNA transfections (at 3 days) produced a ∼50% down-regulation in targeted protein levels. PP5-underexpressing cells released significantly more ir-ACTH (10–12-fold) relative to baseline levels and promoted POMC release into the media. Neither CRF-mediated ACTH release nor dexamethasone-induced ACTH repression were affected in PP5-siRNA transfected cells. In summary, our observations suggest that endogenous PP5 can exert a negative modulatory effect on basal ACTH release in neurosecretion-competent AtT20 cells through a mechanism as yet unknown but which does not directly involve regulated CRF or glucocorticoid receptor-dependent pathways. However, PP5 may cause mis-sorting of POMC and POMC-derived peptides at the constitutive-like secretory pathway level in an unregulated manner. Such a missorting could lead to impaired processing of POMC.

Constitutive over expression of serine/threonine protein phosphatase 5 (PP5) augments estrogen-dependent tumor growth in mice

Serine/threonine protein phosphatase 5 (PP5) appears to play an underappreciated role in the regulation of cellular proliferation. In estrogen-responsive cells, PP5 expression is stimulated by 17 β-estradiol, and in a variety of p53 wild-type tumor cells the suppression of PP5 expression with ISIS 15534 inhibits growth. To further explore the relationship between PP5 and the development of human cancer, here we tested the effect of elevated PP5 expression on tumor growth using a mouse xenograph model and a stable MCF-7 cell line in which the expression of wild-type PP5 was placed under the control of tetracycline-off regulated transactivator and operator plasmids. In the xenograph model a modest two fold increase in PP5 protein levels significantly enhanced the growth rate of estrogen-dependent tumors, suggesting PP5 plays a positive role in tumor development.

Dephosphorylation of microtubule-associated protein tau by protein phosphatase 5.

Protein phosphatase 5 (PP5) is a 58-kDa novel phosphoseryl/phosphothreonyl protein phosphatase. It is ubiquitously expressed in all mammalian tissues examined, with a high level in the brain, but little is known about its physiological substrates. We found that this phosphatase dephosphorylated recombinant tau phosphorylated with cAMP-dependent protein kinase and glycogen synthase kinase-3beta, as well as abnormally hyperphosphorylated tau isolated from brains of patients with Alzheimer's disease. The specific activity of PP5 toward tau was comparable to those reported with other protein substrates examined to date. The PP5 activity toward tau was stimulated by arachidonic acid by 30- to 45-fold. Immunostaining demonstrated that PP5 was primarily cytoplasmic in PC12 cells and in neurons of postmortem human brain tissue. A small pool of PP5 associated with microtubules. Expression of active PP5 in PC12 cells resulted in reduced phosphorylation of tau, suggesting that PP5 can also dephosphorylate tau in cells. These results suggest that PP5 plays a role in the dephosphorylation of tau and might be involved in the molecular pathogenesis of Alzheimer's disease.

Serine / threonine protein phosphatase 5 (PP5) participates in the regulation of glucocorticoid receptor nucleocytoplasmic shuttling

BackgroundIn most cells glucocorticoid receptors (GR) reside predominately in the cytoplasm. Upon hormone binding, the GR translocates into the nucleus, where the hormone-activated GR-complex regulates the transcription of GR-responsive genes. Serine/threonine protein phosphatase type 5 (PP5) associates with the GR-heat-shock protein-90 complex, and the suppression of PP5 expression with ISIS 15534 stimulates the activity of GR-responsive reporter plasmids, without affecting the binding of hormone to the GR.ResultsTo further characterize the mechanism by which PP5 affects GR-induced gene expression, we employed immunofluorescence microscopy to track the movement of a GR-green fluorescent fusion protein (GR-GFP) that retained hormone binding, nuclear translocation activity and specific DNA binding activity, but is incapable of transactivation. In the absence of glucocorticoids, GR-GFP localized mainly in the cytoplasm. Treatment with dexamethasone results in the efficient translocation of GR-GFPs into the nucleus. The nuclear accumulation of GR-GFP, without the addition of glucocorticoids, was also observed when the expression of PP5 was suppressed by treatment with ISIS 15534. In contrast, ISIS 15534 treatment had no apparent effect on calcium induced nuclear translocation of NFAT-GFP.ConclusionThese studies suggest that PP5 participates in the regulation of glucocorticoid receptor nucleocytoplasmic shuttling, and that the GR-induced transcriptional activity observed when the expression of PP5 is suppressed by treatment with ISIS 15534 results from the nuclear accumulation of GR in a form that is capable of binding DNA yet still requires agonist to elicit maximal transcriptional activation.