Leucine-rich Repeat Protein Phosphatase Research Articles

PH domain leucine-rich repeat protein phosphatase 2 (PHLPP2) regulates G-protein–coupled receptor kinase 5 (GRK5)-induced cardiac hypertrophy in vitro

PH domain leucine-rich repeat protein phosphatase (PHLPP) is a serine/threonine phosphatase that has been shown to regulate cell growth and survival through dephosphorylation of several members of the AGC family of kinases. G-protein-coupled receptor kinase 5 (GRK5) is an AGC kinase that regulates phenylephrine (PE)-induced cardiac hypertrophy through its noncanonical function of directly targeting proteins to the nucleus to regulate transcription. Here we investigated the possibility that the PHLPP2 isoform can regulate GRK5-induced cardiomyocyte hypertrophy in neonatal rat ventricular myocytes (NRVMs). We show that removal of PHLPP2 by siRNA induces hypertrophic growth of NRVMs as measured by cell size changes at baseline, potentiated PE-induced cell size changes, and re-expression of fetal genes atrial natriuretic factor and brain natriuretic peptide. Endogenous GRK5 and PHLPP2 were found to interact in NRVMs, and PE-induced nuclear accumulation of GRK5 was enhanced upon down-regulation of PHLPP2. Conversely, overexpression of PHLPP2 blocked PE-induced hypertrophic growth, re-expression of fetal genes, and nuclear accumulation of GRK5, which depended on its phosphatase activity. Finally, using siRNA against GRK5, we found that GRK5 was necessary for the hypertrophic response induced by PHLPP2 knockdown. Our findings demonstrate for the first time a novel regulation of GRK5 by the phosphatase PHLPP2, which modulates hypertrophic growth. Understanding the signaling pathways affected by PHLPP2 has potential for new therapeutic targets in the treatment of cardiac hypertrophy and failure.

Berberine induced modulation of PHLPP2-Akt-MST1 kinase signaling is coupled with mitochondrial impairment and hepatoma cell death

Pleckstrin homology domain leucine-rich repeat protein phosphatase 2 (PHLPP2) has been known to exert tumor suppressive activity for long without much knowledge about its regulation and implications. Protein kinase B (Akt), Protein kinase C (PKC) and Ribosomal protein S6 Kinase (S6K) are known downtargets of PHLPP2, regulating a plethora of life processes viz. cell growth, survival and evasion from apoptosis. Present study decoded the crucial role of PHLPP2 in inducing apoptosis by its interaction with the newly found binding partner Mammalian sterile 20-like kinase 1 (Mst1) in berberine (BBR)-treated human hepatoma cells. HepG2 cells were exposed to (50 μM, 100 μM) berberine for different time intervals (18 h, 24 h). The results showed enhanced expression of PHLPP2 at transcriptional (2.13 fold, P < 0.01) and translational level (4 fold, P < 0.001), but not of PHLPP1, in berberine-treated HepG2 cells. Elevated expression of PHLPP2 was reported to inactivate Akt by dephosphorylating it on Ser473 (P < 0.001). As Akt is known to inhibit apoptotic effect of Mst1, we found that PHLPP2 mediated inactivation of Akt releases its repression from Mst1 leading to heightened phosphorylation of Mst1 on its activating site Thr183 (1.5 fold, P < 0.001). Consequently, coordination between PHLPP2, Akt and Mst1 stimulated downstream targets c-jun N-terminal kinase (JNK), Bim and Bak which are direct activators of pro-apoptotic proteins leading to cell death. Further, PHLPP2/Mst1 knock-down efficiently curtailed anti-proliferative effect of berberine by restoring the basal level of downstream anti-apoptotic proteins. In addition, pre-treatment of NAC (5 mM) showed that ROS generation was a primitive event to initiate activation of stress kinases. Thus, our findings suggest that PHLPP2, Akt and Mst1 constitute an autoinhibitory triangle which may be partly responsible for antiproliferative effect of berberine.

The Protein Phosphatase PHLPP1 Suppresses Insulin Signaling and Inflammation in Mouse Model

The PH domain Leucine Rich Repeat Protein Phosphatase 1 (PHLPP1) directly dephosphorylates and inactivates Akt2, the Akt isozyme that is a key transducer of insulin signaling. Elevated levels of PHLPP1 protein have been associated with obesity and insulin resistance in humans, suggesting PHLPP1 may be a novel therapeutic target to combat insulin resistance in diabetes and metabolic syndrome. Here we examine how loss of PHLPP1 affects insulin signaling using the Phlpp1−/− mice. Insulin tolerance tests of mice on a normal chow diet reveal a modest but statistically‐significant increase in the insulin sensitivity of mice lacking PHLPP1 compared to littermate Phlpp1+/+ controls. Analyses of liver from these mice reveal elevated Akt signaling in the mice lacking PHLPP1, as assessed by increased phosphorylation of Akt itself and downstream substrates such as GSK‐3 and MDM2. In addition, consistent with previous studies, the steady state levels of EGF receptor are elevated. We also note that there was an approximately 15% increase in the steady state levels of PHLPP2. Consistent with in vivo results, deletion of PHLPP1 in HepG2 liver cells also increases insulin‐dependent signaling, assessed by increased phosphorylation of the insulin receptor and Akt. Thus, both in vivo and in vitro studies are consistent with PHLPP1 suppressing insulin signaling. In contrast to results from normal chow diet, Phlpp1−/− and Phlpp1+/+ mice subjected to a high fat diet had the same insulin sensitivity. Furthermore, biochemical analysis of liver from these mice revealed a striking decrease in phosphorylation of Akt and downstream substrates such as S6kinase, and a decrease in PKCα. PHLPP2 levels remained elevated. Additionally, we also found that in livers, high fat diet promotes an up‐regulation of pro‐inflammatory genes such as Il6 and Tnfa, which is significantly enhanced in PHLPP1 deficient mice. Our data are consistent with a model in which loss of PHLPP1 enhances both insulin sensitivity and inflammation. The pro‐inflammatory effects of high fat diet may be exacerbated in the Phlpp1−/− cells, masking an insulin sensitivity phenotype.Support or Funding InformationThis work was funded by NIH P01DK054441This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

MiR-214-Dependent Increase of PHLPP2 Levels Mediates the Impairment of Insulin-Stimulated Akt Activation in Mouse Aortic Endothelial Cells Exposed to Methylglyoxal.

Evidence has been provided linking microRNAs (miRNAs) and diabetic complications, by the regulation of molecular pathways, including insulin-signaling, involved in the pathophysiology of vascular dysfunction. Methylglyoxal (MGO) accumulates in diabetes and is associated with cardiovascular complications. This study aims to analyze the contribution of miRNAs in the MGO-induced damaging effect on insulin responsiveness in mouse aortic endothelial cells (MAECs). miRNA modulation was performed by transfection of specific miRNA mimics and inhibitors in MAECs, treated or not with MGO. miRNA-target protein levels were evaluated by Western blot. PH domain leucine-rich repeat protein phosphatase 2 (PHLPP2) regulation by miR-214 was tested by luciferase assays and by the use of a target protector specific for miR-214 on PHLPP2-3′UTR. This study reveals a 4-fold increase of PHLPP2 in MGO-treated MAECs. PHLPP2 levels inversely correlate with miR-214 modulation. Moreover, miR-214 overexpression is able to reduce PHLPP2 levels in MGO-treated MAECs. Interestingly, a direct regulation of PHLPP2 is proved to be dependent by miR-214. Finally, the inhibition of miR-214 impairs the insulin-dependent Akt activation, while its overexpression rescues the insulin effect on Akt activation in MGO-treated MAECs. In conclusion, this study shows that PHLPP2 is a target of miR-214 in MAECs, and identifies miR-214 downregulation as a contributing factor to MGO-induced endothelial insulin-resistance.

Transmembrane and Coiled-Coil Domain 1 Impairs the AKT Signaling Pathway in Urinary Bladder Urothelial Carcinoma: A Characterization of a Tumor Suppressor.

Purpose: Urinary bladder urothelial carcinoma (UBUC) is a common malignant disease in developed countries. Cell-cycle dysregulation resulting in uncontrolled cell proliferation has been associated with UBUC development. This study aimed to explore the roles of TMCO1 in UBUCs.Experimental Design: Data mining, branched DNA assay, immunohistochemistry, xenograft, cell culture, quantitative RT-PCR, immunoblotting, stable and transient transfection, lentivirus production and stable knockdown, cell-cycle, cell viability and proliferation, soft-agar, wound-healing, transwell migration and invasion, coimmunoprecipitation, immunocytochemistry, and AKT serine/threonine kinase (AKT) activity assays and site-directed mutagenesis were used to study TMCO1 involvement in vivo and in vitroResults: Data mining identified that the TMCO1 transcript was downregulated during the progression of UBUCs. In distinct UBUC-derived cell lines, changes in TMCO1 levels altered the cell-cycle distribution, cell viability, cell proliferation, and colony formation and modulated the AKT pathway. TMCO1 recruited the PH domain and leucine-rich repeat protein phosphatase 2 (PHLPP2) to dephosphorylate pAKT1(serine 473) (S473). Mutagenesis at S60 of the TMCO1 protein released TMCO1-induced cell-cycle arrest and restored the AKT pathway in BFTC905 cells. Stable TMCO1 (wild-type) overexpression suppressed, whereas T33A and S60A mutants recovered, tumor size in xenograft mice.Conclusions: Clinical associations, xenograft mice, and in vitro indications provide solid evidence that the TMCO1 gene is a novel tumor suppressor in UBUCs. TMCO1 dysregulates cell-cycle progression via suppression of the AKT pathway, and S60 of the TMCO1 protein is crucial for its tumor-suppressor roles. Clin Cancer Res; 23(24); 7650-63. ©2017 AACR.

Tubastatin ameliorates pulmonary fibrosis by targeting the TGFβ-PI3K-Akt pathway.

BackgroundIdiopathic pulmonary fibrosis (IPF) is a chronic, progressive and fatal disease. Histone deacetylase 6 (HDAC6) alters function and fate of various proteins via deacetylation of lysine residues, and is implicated in TGF-β1-induced EMT (epithelial-mesenchymal transition). However, the role of HDAC6 in pulmonary fibrosis is unknown.MethodsHDAC6 expression in IPF and control lungs was assessed by quantitative real-time PCR (qRT-PCR) and immunoblots. Lung fibroblasts were treated with TGF-β1 ± HDAC6 inhibitors (Tubacin, Tubastatin, ACY1215, or MC1568), and fibrotic markers such as type I collagen were assessed using qRT-PCR and immunoblots. Mice were treated with bleomycin (oropharyngeal aspiration; single dose) ± Tubastatin (intraperitoneally injection; daily for 21 days), and lung collagen expression was gauged using immunoblots and trichrome staining. In a separate experiment, HDAC6 wild-type (WT) and knockout (KO) mice were administered bleomycin, and lungs were evaluated in the same manner.ResultsHDAC6 expression was deregulated in IPF lungs. Among the HDAC6 inhibitors tested, only Tubastatin significantly repressed TGF-β1-induced expression of type-1 collagen in lung fibroblasts, and this finding was coupled with decreased Akt phosphorylation and increased Akt-PHLPP (PH domain and Leucine rich repeat Protein Phosphatase) association. Tubastatin repressed TGF-β1-induced S6K phosphorylation, HIF-1α expression, and VEGF expression. Tubastatin also repressed TGF-β1-induced inhibition of LC3B-II (a marker of autophagosome formation). In bleomycin-treated mouse lungs, HDAC6 expression was increased, and Tubastatin repressed type-1 collagen expression. However, in HDAC6 KO mice, bleomycin-induced type-1 collagen expression was not repressed compared to WT mice. Knockdown of HDAC6, as well as HDAC10, another potential Tubastatin target, did not inhibit TGF-β1-induced collagen expression in lung fibroblasts.ConclusionsHDAC6 expression is altered during lung fibrogenesis. Tubastatin represses TGF-β1-induced collagen expression, by diminishing Akt phosphorylation and regulating downstream targets such as HIF-1α-VEGF axis and autophagy. Tubastatin-treated WT mice are protected against bleomycin-induced fibrosis, but HDAC6 KO mice are not. Our data suggest that Tubastatin ameliorates pulmonary fibrosis, by targeting the TGFβ-PI3K-Akt pathway, likely via an HDAC6-independent mechanism.

Degradation of PHLPP2 by KCTD17, via a Glucagon-Dependent Pathway, Promotes Hepatic Steatosis

Obesity-induced nonalcoholic fatty liver disease (NAFLD) develops, in part, via excess insulin-stimulated hepatic de novo lipogenesis, which increases, paradoxically, in patients with obesity-induced insulin resistance. Pleckstrin homology domain leucine-rich repeat protein phosphatase 2 (PHLPP2) terminates insulin signaling by dephosphorylating Akt; levels of PHLPP2 are reduced in livers from obese mice. We investigated whether loss of hepatic PHLPP2 is sufficient to induce fatty liver in mice, mechanisms of PHLPP2 degradation in fatty liver, and expression of genes that regulate PHLPP2 in livers of patients with NAFLD. C57BL/6J mice (controls), obese db/db mice, and mice with liver-specific deletion of PHLPP2 (L-PHLPP2) fed either normal chow or high-fat diet (HFD) were analyzed for metabolic phenotypes, including glucose tolerance and hepatic steatosis. PHLPP2-deficient primary hepatocytes or CRISPR/Cas9-mediated PHLPP2-knockout hepatoma cells were analyzed for insulin signaling and gene expression. We performed mass spectrometry analyses of liver tissues from C57BL/6J mice transduced with Ad-HA-Flag-PHLPP2 to identify posttranslational modifications to PHLPP2 and proteins that interact with PHLPP2. We measured levels of mRNAs by quantitative reverse transcription polymerase chain reaction in liver biopsies from patients with varying degrees of hepatic steatosis. PHLPP2-knockout hepatoma cells and hepatocytes from L-PHLPP2 mice showed normal initiation ofinsulin signaling, but prolonged insulin action. Chow-fed L-PHLPP2 mice had normal glucose tolerance but hepatic steatosis. In HFD-fed C57BL/6J or db/db obese mice, endogenous PHLPP2 was degraded by glucagon and PKA-dependent phosphorylation of PHLPP2 (at Ser1119 and Ser1210), which led to PHLPP2 binding to potassium channel tetramerization domain containing 17 (KCTD17), a substrate-adaptor for Cul3-RING ubiquitin ligases. Levels of KCTD17 mRNA were increased in livers of HFD-fed C57BL/6J or db/db obese mice and in liver biopsies patients with NAFLD, compared with liver tissues from healthy control mice or patients without steatosis. Knockdown of KCTD17 with small hairpin RNA in primary hepatocytes increased PHLPP2 protein but not Phlpp2 mRNA, indicating that KCTD17 mediates PHLPP2 degradation. KCTD17 knockdown in obese mice prevented PHLPP2 degradation and decreased expression of lipogenic genes. In mouse models of obesity, we found that PHLPP2 degradation induced lipogenesis without affecting gluconeogenesis. KCTD17, which is up-regulated in liver tissues of obese mice and patients with NAFLD, binds to phosphorylated PHLPP2 to target it for ubiquitin-mediated degradation; this increases expression of genes that regulate lipogenesis to promote hepatic steatosis. Inhibitors of this pathway might be developed for treatment of patients with NAFLD.

Interplay between the phosphatase PHLPP1 and E3 ligase RNF41 stimulates proper kinetochore assembly via the outer-kinetochore protein SGT1

Kinetochores link chromosomes to spindle microtubules and are essential for accurate chromosome segregation during cell division. Kinetochores assemble at the centromeric region of chromosomes as a multiprotein complex. However, the molecular mechanisms of kinetochore assembly have not yet been fully elucidated. In this study, we identified pleckstrin homology domain and leucine-rich repeat protein phosphatase 1 (PHLPP1) as a regulatory phosphatase that facilitates proper kinetochore assembly. We found that PHLPP1 interacted with the essential outer-kinetochore protein SGT1 and stabilized its protein levels. Loss of PHLPP1 from cells led to SGT1 degradation and thereby caused defective kinetochore assembly. We also found that the ring finger protein 41 (RNF41) as an E3 ligase ubiquitinated and degraded SGT1 in a phosphorylation-dependent manner. PHLPP1 dephosphorylated SGT1 at four conserved residues (Ser-17, Ser-249, Ser-289, and Thr-233) and thereby prevented SGT1 from associating with RNF41, in turn, countering SGT1 degradation. Importantly, depletion of RNF41 or expression of a non-phosphorylatable SGT1 mutant rescued the kinetochore defects caused by the loss of PHLPP1. Taken together, our results suggest that PHLPP1 plays an important role in the assembly of kinetochores by counteracting RNF41-mediated SGT1 degradation.

Abstract 4488: Tumor suppressor phosphatase PHLPP1 regulates transcription factor activity and gene expression in inflammation

Abstract Epigenetic mechanisms play a key role in cancer and are a major contributor in driving the aberrantly high levels of oncogenic receptor tyrosine kinases (RTK) in cancer. Understanding mechanisms leading to altered transcription of oncogenes and tumor suppressors has the potential to provide new therapeutic targets. Here we show that the PH domain leucine-rich repeat protein phosphatase 1 (PHLPP1), a tumor suppressor that directly dephosphorylates and inactivates Akt, has an additional function of regulating approximately 10% of the transcriptome. Specifically, we used non-biased high-throughput RNA-sequencing and chromatin immunoprecipitation-sequencing techniques to analyze genome-wide mRNA levels and acetylation patterns of genes in cells from wild-type and PHLPP1 knockout mice. De novo motif analysis of the promoter for the genes regulated by PHLPP1 identified enrichment in the recognition motifs for a number of transcription factors, including ones involved in inflammatory signaling. Biochemical analysis revealed that PHLPP1 regulates the phosphorylation and activation of these transcription factors, such that loss of PHLPP1 leads to enhanced inflammatory signaling. Our data support a model in which PHLPP1 dephosphorylates specific transcription factors to act as the brakes to inflammatory signaling, a hallmark of cancer. Citation Format: Ksenya Cohen Katsenelson, Joshua D. Stender, Christopher K. Glass, Alexandra C. Newton. Tumor suppressor phosphatase PHLPP1 regulates transcription factor activity and gene expression in inflammation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4488. doi:10.1158/1538-7445.AM2017-4488

Abstract 3463: Mitotic regulation of the tumor suppressor phosphatase PHLPP1

Abstract The PH domain Leucine-rich repeat Protein Phosphatases (PHLPP) are tumor suppressors originally discovered for their ability to directly dephosphorylate and inactivate the pro-survival kinase Akt, a key transducer of growth factor signaling. In the decade since this discovery, a number of other PHLPP targets have been elucidated, but still little is known about the molecular mechanisms governing the function and regulation of PHLPP itself. Here we report that PHLPP1 is hyperphosphorylated during mitosis and that, additionally, PHLPP1 suppresses the phosphorylation of the mitotic kinase CDK1. Specifically, we show that PHLPP1 undergoes an electrophoretic mobility shift in mitotic cells that is lost when lysates are treated in vitro with lambda phosphatase, suggesting that PHLPP1 is phosphorylated during mitosis. In addition to a cell cycle-dependent change in its post-translational modifications, PHLPP1 suppresses the phosphorylation of the main mitotic cyclin dependent kinase, CDK1, at Tyr15, an inhibitory phosphorylation that must be removed in order for cells to progress through mitosis. First, Tyr15 phosphorylation is increased in phlpp1-/- mouse embryonic fibroblasts (MEFs) compared to wild-type MEFs. Second, pharmacological inhibition of PHLPP1 in wild-type MEFs results in an increase in Tyr15 phosphorylation. Activation of key mitotic kinases, such as CDK1, and inactivation of phosphatases is required for the large increase in protein phosphorylation that occurs during mitosis. As such, reversible protein phosphorylation plays a role in regulating proper progression through mitosis, which is essential as errors in mitosis can result in aneuploidy, a hallmark of cancer. Our data support a previously undescribed role for PHLPP1 in regulating mitotic progression, potentially providing novel insight into how PHLPP suppresses tumorigenesis. Citation Format: Agnieszka T. Grzechnik, Alexandra C. Newton. Mitotic regulation of the tumor suppressor phosphatase PHLPP1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3463. doi:10.1158/1538-7445.AM2017-3463

Analysis of the function of microRNA-375 in humans using bioinformatics.

MicroRNA-375 (miR-375) is expressed at low levels in many types of solid tumor, particularly in gastrointestinal tumors. It is considered to be important in the development of cancer and certain diseases. Thus, more detailed knowledge is required on the particular functions of miR-375. miRs function by regulating target genes. Therefore, in the current study, miRWalk (which includes the data from 10 prediction software programs) was used to predict the target genes of miR-375. The genes, which were co-predicted using five different software programs were further analyzed using Database for Annotation, Visualization and Integrated Discovery online software [including gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis]. Subsequently, the online tool, Search Tool for the Retrieval of Interacting Genes, was used to analyze the protein-protein interaction and construct modules using Cytoscape. The result demonstrated 6,574 predicted genes, 1,325 of which were co-predicted. The GO analysis result indicated that, in biological processes, the co-predicted genes were significantly enriched in the regulation of nervous system development and cell differentiation, and the highest enrichment of molecular function was ion binding. In KEGG analysis, the genes were enriched in the Hippo signaling pathway, glutamatergic synapse, circadian entrainment and the phosphoinositide 3-kinase (PI3K)-Akt signaling pathway. The top 10 hub proteins were mechanistic target of rapamycin, PH domain and leucine rich repeat protein phosphatase 1, ubiquitously transcribed tetratricopeptide repeat containing, Y-linked, histone deacetylase 2, F-box and leucine rich repeat protein 19, KIT proto-oncogene receptor tyrosine kinase, angiotensinogen, Janus kinase 2, fibroblast growth factor 2 and RNA polymerase II subunit A. These proteins predominantly regulate the development and progression of cancer, hypertension, essential thrombocythemia and inflammation. The genes in the top seven modules selected were identified to be primarily enriched in chemokines, extracellular matrix-receptor interaction, focal adhesion, the PI3K-Akt signaling pathway, amoebiasis and protein processing signaling pathway. Thus, the target genes and hub proteins that were predicted in the current study were identified to be important in regulating the development and progression of cancer and certain diseases. Furthermore, they present potential novel biomarkers for tumor diagnosis and candidate targets for treatment, and indicate that further research is required to establish the functions of miR-375.

MicroRNA-27a contributes to the malignant behavior of gastric cancer cells by directly targeting PH domain and leucine-rich repeat protein phosphatase 2

BackgroundAccumulating evidence indicates that microRNA-27a (miR-27a) is involved in carcinogenesis and tumor progression. However, the exact function and molecular mechanism of miR-27a in gastric cancer remain unclear.MethodsQuantitative real-time PCR (qRT-PCR) was used to quantify the expression of miR-27a and its target gene. The function of miR-27a in gastric cancer was investigated through in vitro and in vivo assays (MTT assay, colony formation assay, flow cytometry assay, wound healing assay, migration and invasion assay, immunohistochemistry (IHC), immunofluorescence (IF) and Western blot). A luciferase reporter assay was conducted to confirm the target gene of miR-27a.ResultsWe found that miR-27a was commonly overexpressed in gastric cancer and high expression of miR-27a was associated with distant metastasis, lymph node metastasis, advanced T stage and advanced clinical stage. Functional assays demonstrated that overexpression of miR-27a in AGS cells accelerated cell proliferation, migration and invasion and suppressed apoptosis. Meanwhile, opposite results were observed in SGC-7901 cells when miR-27a was suppressed. Consistently, down-regulation of miR-27a inhibited the growth and metastasis of engrafted tumors in vivo. Furthermore, we found PH domain and leucine-rich repeat protein phosphatase 2 (PHLPP2) to be a new target of miR-27a, and downregulation of PHLPP2 could rescue the effect of anti-miR-27a in gastric cancer cells. In addition, miR-27a-mediated suppression of PHLPP2 led to stimulation of the AKT/GSK3β pathway.ConclusionsOur data suggest that miR-27a functions as a crucial oncogenic miRNA in gastric cancer. It can promote proliferation and metastasis of tumor cells by suppressing PHLPP2 and activating the AKT/GSK3β pathway. Therefore, miR-27a is a potential novel therapeutic target in gastric cancer treatment.

Expression of PH Domain Leucine-rich Repeat Protein Phosphatase, Forkhead Homeobox Type O 3a and RAD51, and their Relationships with Clinicopathologic Features and Prognosis in Ovarian Serous Adenocarcinoma.

Background:Ovarian serous adenocarcinoma can be divided into low- and high-grade tumors, which exhibit substantial differences in pathogenesis, clinicopathology, and prognosis. This study aimed to investigate the differences in the PH domain leucine-rich repeat protein phosphatase (PHLPP), forkhead homeobox type O 3a (FoxO3a), and RAD51 protein expressions, and their associations with prognosis in patients with low- and high-grade ovarian serous adenocarcinomas.Methods:The PHLPP, FoxO3a, and RAD51 protein expressions were examined in 94 high- and 26 low-grade ovarian serous adenocarcinomas by immunohistochemistry. The differences in expression and their relationships with pathological features and prognosis were analyzed.Results:In high-grade serous adenocarcinomas, the positive rates of PHLPP and FoxO3a were 24.5% and 26.6%, while in low-grade tumors, they were 23.1% and 26.9%, respectively (P < 0.05 vs. the control specimens; low- vs. high-grade: P > 0.05). The positive rates of RAD51 were 70.2% and 65.4% in high- and low-grade serous adenocarcinomas, respectively (P < 0.05 vs. the control specimens; low- vs. high-grade: P > 0.05). Meanwhile, in high-grade tumors, Stage III/IV tumors and lymph node and omental metastases were significantly associated with lower PHLPP and FoxO3a and higher RAD51 expression. The 5-year survival rates of patients with PHLPP- and FoxO3a-positive high-grade tumors (43.5% and 36.0%) were significantly higher than in patients with PHLPP-negative tumors (5.6% and 7.2%, respectively; P < 0.05). Similarly, the 5-year survival rate of RAD51-positive patients (3.0%) was significantly lower than in negative patients (42.9%; P < 0.05). In low-grade tumors, the PHLPP, FoxO3a, and RAD51 expressions were not significantly correlated with lymph node metastasis, omental metastasis, Federation of Gynecology and Obstetrics stage, or prognosis.Conclusions:Abnormal PHLPP, FoxO3a, and RAD51 protein expressions may be involved in the development of high- and low-grade ovarian serous adenocarcinomas, suggesting common molecular pathways. Decreased PHLPP and FoxO3a and increased RAD51 protein expression may be important molecular markers for poor prognosis, and RAD51 may be an independent prognosis factor, of high-grade, but not low-grade, ovarian serous adenocarcinomas.

PHLPP regulates hexokinase 2-dependent glucose metabolism in colon cancer cells

Increased glucose metabolism is considered as one of the most important metabolic alterations adapted by cancer cells in order to generate energy as well as high levels of glycolytic intermediates to support rapid proliferation. PH domain leucine-rich repeat protein phosphatase (PHLPP) belongs to a novel family of Ser/Thr protein phosphatases that function as tumor suppressors in various types of human cancer. Here we determined the role of PHLPP in regulating glucose metabolism in colon cancer cells. Knockdown of PHLPP increased the rate of glucose consumption and lactate production, whereas overexpression of PHLPP had the opposite effect. Bioenergetic analysis using Seahorse Extracelluar Flux Analyzer revealed that silencing PHLPP expression induced a glycolytic shift in colon cancer cells. Mechanistically, we found that PHLPP formed a complex with Akt and hexokinase 2 (HK2) in the mitochondrial fraction of colon cancer cells and knockdown of PHLPP enhanced Akt-mediated phosphorylation and mitochondrial localization of HK2. Depletion of HK2 expression or treating cells with Akt and HK2 inhibitors reversed PHLPP loss-induced increase in glycolysis. Furthermore, PHLPP knockdown cells became addicted to glucose as a major energy source in that glucose starvation significantly decreased cancer cell survival. As HK2 is the key enzyme that determines the direction and magnitude of glucose flux, our study identified PHLPP as a novel regulator of glucose metabolism by controlling HK2 activity in colon cancer cells.

PHLPPing through history: a decade in the life of PHLPP phosphatases.

In the decade since their discovery, the PH domain leucine-rich repeat protein phosphatases (PHLPP) have emerged as critical regulators of cellular homeostasis, and their dysregulation is associated with various pathophysiologies, ranging from cancer to degenerative diseases, such as diabetes and heart disease. The two PHLPP isozymes, PHLPP1 and PHLPP2, were identified in a search for phosphatases that dephosphorylate Akt, and thus suppress growth factor signaling. However, given that there are over 200 000 phosphorylated residues in a single cell, and fewer than 50 Ser/Thr protein phosphatases, it is not surprising that PHLPP has many other cellular functions yet to be discovered, including a recently identified role in regulating the epigenome. Both PHLPP1 and PHLPP2 are commonly deleted in human cancers, supporting a tumor suppressive role. Conversely, the levels of one isozyme, PHLPP1, are elevated in diabetes. Thus, mechanisms to correctly control PHLPP activity in cells are critical for normal cellular homeostasis. This review summarizes the known functions of PHLPP and its role in disease.

Pleckstrin Homology (PH) Domain Leucine-rich Repeat Protein Phosphatase Controls Cell Polarity by Negatively Regulating the Activity of Atypical Protein Kinase C

The proper establishment of epithelial polarity allows cells to sense and respond to signals that arise from the microenvironment in a spatiotemporally controlled manner. Atypical PKCs (aPKCs) are implicated as key regulators of epithelial polarity. However, the molecular mechanism underlying the negative regulation of aPKCs remains largely unknown. In this study, we demonstrated that PH domain leucine-rich repeat protein phosphatase (PHLPP), a novel family of Ser/Thr protein phosphatases, plays an important role in regulating epithelial polarity by controlling the phosphorylation of both aPKC isoforms. Altered expression of PHLPP1 or PHLPP2 disrupted polarization of Caco2 cells grown in 3D cell cultures as indicated by the formation of aberrant multi-lumen structures. Overexpression of PHLPP resulted in a decrease in aPKC phosphorylation at both the activation loop and the turn motif sites; conversely, knockdown of PHLPP increased aPKC phosphorylation. Moreover, in vitro dephosphorylation experiments revealed that both aPKC isoforms were substrates of PHLPP. Interestingly, knockdown of PKCζ, but not PKCι, led to similar disruption of the polarized lumen structure, suggesting that PKCζ likely controls the polarization process of Caco2 cells. Furthermore, knockdown of PHLPP altered the apical membrane localization of aPKCs and reduced the formation of aPKC-Par3 complex. Taken together, our results identify a novel role of PHLPP in regulating aPKC and cell polarity.

Cheliensisin A (Chel A) induces apoptosis in human bladder cancer cells by promoting PHLPP2 protein degradation.

Cheliensisin A (Chel A), a styryl-lactone compound extracted from Goniothalamus cheliensis, is reported to have significant anti-cancer effects in various cancer cells. Here we demonstrated that Chel A treatment resulted in apoptosis and an inhibition of anchorage-independent growth in human bladder cancer T24, T24T and U5637 cells. Mechanistic studies showed that such effect is mediated by PH domain and Leucine rich repeat Protein Phosphatases (PHLPP2) protein. Chel A treatment led to PHLPP2 degradation and subsequently increased in c-Jun phosphorylation. Moreover PHLPP2 degradation could be attenuated by inhibition of autophagy, which was mediated by Beclin 1. Collectively, we discover that Chel A treatment induces Beclin-dependent autophagy, consequently mediates PHLPP2 degradation and JNK/C-Jun phosphorylation and activation, further in turn contributing to apoptosis in human bladder cancer cells. Current studies provide a significant insight into understanding of anticancer effect of Chel A in treatment of human bladder cancer.

Immune dysregulation in patients with PTEN hamartoma tumor syndrome: Analysis of FOXP3 regulatory T cells

BackgroundPatients with heterozygous germline mutations in phosphatase and tensin homolog deleted on chromosome 10 (PTEN) experience autoimmunity and lymphoid hyperplasia.ObjectivesBecause regulation of the phosphoinositide 3-kinase (PI3K) pathway is critical for maintaining regulatory T (Treg) cell functions, we investigate Treg cells in patients with heterozygous germline PTEN mutations (PTEN hamartoma tumor syndrome [PHTS]).MethodsPatients with PHTS were assessed for immunologic conditions, lymphocyte subsets, forkhead box P3 (FOXP3)+ Treg cell levels, and phenotype. To determine the functional importance of phosphatases that control the PI3K pathway, we assessed Treg cell induction in vitro, mitochondrial depolarization, and recruitment of PTEN to the immunologic synapse.ResultsAutoimmunity and peripheral lymphoid hyperplasia were found in 43% of 79 patients with PHTS. Immune dysregulation in patients with PHTS included lymphopenia, CD4+ T-cell reduction, and changes in T- and B-cell subsets. Although total CD4+FOXP3+ Treg cell numbers are reduced, frequencies are maintained in the blood and intestine. Despite pathogenic PTEN mutations, the FOXP3+ T cells are phenotypically normal. We show that the phosphatase PH domain leucine-rich repeat protein phosphatase (PHLPP) downstream of PTEN is highly expressed in normal human Treg cells and provides complementary phosphatase activity. PHLPP is indispensable for the differentiation of induced Treg cells in vitro and Treg cell mitochondrial fitness. PTEN and PHLPP form a phosphatase network that is polarized at the immunologic synapse.ConclusionHeterozygous loss of function of PTEN in human subjects has a significant effect on T- and B-cell immunity. Assembly of the PTEN-PHLPP phosphatase network allows coordinated phosphatase activities at the site of T-cell receptor activation, which is important for limiting PI3K hyperactivation in Treg cells despite PTEN haploinsufficiency.

Defects in the CAPN1 Gene Result in Alterations in Cerebellar Development and Cerebellar Ataxia in Mice and Humans.

A CAPN1 missense mutation in Parson Russell Terrier dogs is associated with spinocerebellar ataxia. We now report that homozygous or heterozygous CAPN1-null mutations in humans result in cerebellar ataxia and limb spasticity in four independent pedigrees. Calpain-1 knockout (KO) mice also exhibit a mild form of ataxia due to abnormal cerebellar development, including enhanced neuronal apoptosis, decreased number of cerebellar granule cells, and altered synaptic transmission. Enhanced apoptosis is due to absence of calpain-1-mediated cleavage of PH domain and leucine-rich repeat protein phosphatase 1 (PHLPP1), which results in inhibition of the Akt pro-survival pathway in developing granule cells. Injection of neonatal mice with the indirect Akt activator, bisperoxovanadium, or crossing calpain-1 KO mice with PHLPP1 KO mice prevented increased postnatal cerebellar granule cell apoptosis and restored granule cell density and motor coordination in adult mice. Thus, mutations in CAPN1 are an additional cause of ataxia in mammals, including humans.

Calpain-1 and calpain-2 play opposite roles in retinal ganglion cell degeneration induced by retinal ischemia/reperfusion injury

Calpain has been shown to be involved in neurodegeneration, and in particular in retinal ganglion cell (RGC) death resulting from increased intraocular pressure (IOP) and ischemia. However, the specific roles of the two major calpain isoforms, calpain-1 and calpain-2, in RGC death have not been investigated. Here, we show that calpain-1 and calpain-2 were sequentially activated in RGC dendrites after acute IOP elevation. By combining the use of a selective calpain-2 inhibitor (C2I) and calpain-1 KO mice, we demonstrated that calpain-1 activity supported survival, while calpain-2 activity promoted cell death of RGCs after IOP elevation. Calpain-1 activation cleaved PH domain and leucine-rich repeat protein phosphatase 1 (PHLPP1) and activated the Akt pro-survival pathway, while calpain-2 activation cleaved striatal-enriched protein tyrosine phosphatase (STEP) and activated STEP-mediated pro-death pathway in RGCs after IOP elevation. Systemic or intravitreal C2I injection to wild-type mice 2h after IOP elevation promoted RGC survival and improved visual function. Our data indicate that calpain-1 and calpain-2 play opposite roles in high IOP-induced ischemic injury and that a selective calpain-2 inhibitor could prevent acute glaucoma-induced RGC death and blindness.