Regulation Of Protein Phosphatases Research Articles

Sex-Dependent Cardiac Growth Responses to Adrenergic Stimulation in Mice Lacking the Protein Phosphatase Regulator B55α

Sex-Dependent Cardiac Growth Responses to Adrenergic Stimulation in Mice Lacking the Protein Phosphatase Regulator B55α

The PP2A regulator IER5L supports prostate cancer progression

Prostate cancer exhibits high prevalence and accounts for a high number of cancer-related deaths. The discovery and characterization of molecular determinants of aggressive prostate cancer represents an active area of research. The Immediate Early Response (IER) family of genes, which regulate Protein Phosphatase 2A (PP2A) activity, has emerged among the factors that influence cancer biology. Here, we show that the less studied member of this family, Immediate Early Response 5 like (IER5L), is upregulated in aggressive prostate cancer. Interestingly, the upregulation of IER5L expression exhibits a robust association with metastatic disease in prostate and is recapitulated in other cancer types. In line with this observation, IER5L silencing reduces foci formation, migration and invasion ability in a variety of human and murine prostate cancer cell lines. In vivo, using zebrafish and immunocompromised mouse models, we demonstrate that IER5L-silencing reduces prostate cancer tumor growth, dissemination, and metastasis. Mechanistically, we characterize the transcriptomic and proteomic landscapes of IER5L-silenced cells. This approach allowed us to identify DNA replication and monomeric G protein regulators as downstream programs of IER5L through a pathway that is consistent with the regulation of PP2A. In sum, we report the alteration of IER5L in prostate cancer and beyond and provide biological and molecular evidence of its contribution to tumor aggressiveness.

Coiled-coil domain containing 159 is required for spermatid head and tail assembly in mice†.

The centrosome is critical for maintaining the sperm head-tail connection and the formation of flagellar microtubules. In this study, we found that in mouse testes, CCDC159 (coiled-coil domain-containing protein 159) is specifically localized to the head-tail coupling apparatus (HTCA) of spermatids, a structure that ensures sperm head-tail tight conjunction. CCDC159 contains a C-terminal coiled-coil domain that functions as the centrosomal localization signal. Gene knockout (KO) of Ccdc159 in mice resulted in acephalic spermatozoa, abnormal flagella, and male infertility. To explore the mechanism behind CCDC159 regulating spermatogenesis, we identified CCDC159-binding proteins using a yeast two-hybrid screen and speculated that CCDC159 participates in HTCA assembly by regulating protein phosphatase PP1 activity. Further RNA-sequencing analyses of Ccdc159 KO testes revealed numerous genes involved in male gamete generation that were downregulated. Together, our results show that CCDC159 in spermatids is a novel centrosomal protein anchoring the sperm head to the tail. Considering the limitation of KO mouse model in clarifying the biological function of CCDC159 in spermatogenesis, a gene-rescue experiment will be performed in the future.

Arginine vasopressin regulates the renal Na+-Cl- and Na+-K+-Cl- cotransporters through with-no-lysine kinase 4 and inhibitor 1 phosphorylation.

Vasopressin regulates water homeostasis via the V2 receptor in the kidney at least in part through protein kinase A (PKA) activation. Vasopressin, through an unknown pathway, upregulates the activity and phosphorylation of Na+-Cl- cotransporter (NCC) and Na+-K+-2Cl- cotransporter 2 (NKCC2) by Ste20-related proline/alanine-rich kinase (SPAK) and oxidative stress-responsive kinase 1 (OSR1), which are regulated by the with-no-lysine kinase (WNK) family. Phosphorylation of WNK4 at PKA consensus motifs may be involved. Inhibitor 1 (I1), a protein phosphatase 1 (PP1) inhibitor, may also play a role. In human embryonic kidney (HEK)-293 cells, we assessed the phosphorylation of WNK4, SPAK, NCC, or NKCC2 in response to forskolin or desmopressin. WNK4 and cotransporter phosphorylation were studied in desmopressin-infused WNK4-/- mice and in tubule suspensions. In HEK-293 cells, only wild-type WNK4 but not WNK1, WNK3, or a WNK4 mutant lacking PKA phosphorylation motifs could upregulate SPAK or cotransporter phosphorylation in response to forskolin or desmopressin. I1 transfection maximized SPAK phosphorylation in response to forskolin in the presence of WNK4 but not of mutant WNK4 lacking PP1 regulation. We observed direct PP1 regulation of NKCC2 dephosphorylation but not of NCC or SPAK in the absence of WNK4. WNK4-/- mice with desmopressin treatment did not increase SPAK/OSR1, NCC, or NKCC2 phosphorylation. In stimulated tubule suspensions from WNK4-/- mice, upregulation of pNKCC2 was reduced, whereas upregulation of SPAK phosphorylation was absent. These findings suggest that WNK4 is a central node in which kinase and phosphatase signaling converge to connect cAMP signaling to the SPAK/OSR1-NCC/NKCC2 pathway.NEW & NOTEWORTHY With-no-lysine kinases regulate the phosphorylation and activity of the Na+-Cl- and Na+-K+-2Cl- cotransporters. This pathway is modulated by arginine vasopressin (AVP). However, the link between AVP and WNK signaling remains unknown. Here, we show that AVP activates WNK4 through increased phosphorylation at putative protein kinase A-regulated sites and decreases its dephosphorylation by protein phosphatase 1. This work increases our understanding of the signaling pathways mediating AVP actions in the kidney.

The SDS22:PP1:I3 complex: SDS22 binding to PP1 loosens the active site metal to prime metal exchange

SDS22 and Inhibitor-3 (I3) are two ancient regulators of protein phosphatase 1 (PP1) that regulate multiple essential biological processes. Both SDS22 and I3 form stable dimeric complexes with PP1; however, and atypically for PP1 regulators, they also form a triple complex, where both proteins bind to PP1 simultaneously (SPI complex). Here we report the crystal structure of the SPI complex. While both regulators bind PP1 in conformations identical to those observed in their individual PP1 complexes, PP1 adopts the SDS22-bound conformation, which lacks its M1 metal. Unexpectedly, surface plasmon resonance (SPR) revealed that the affinity of I3 for the SDS22:PP1 complex is ∼10-fold lower than PP1 alone. We show that this change in binding affinity is solely due to the interaction of I3 with the PP1 active site, specifically PP1’s M2 metal, demonstrating that SDS22 likely allows for PP1 M2 metal exchange and thus PP1 biogenesis.

PPP1R12C Promotes Atrial Hypocontractility in Atrial Fibrillation.

Atrial fibrillation (AF)-the most common sustained cardiac arrhythmia-increases thromboembolic stroke risk 5-fold. Although atrial hypocontractility contributes to stroke risk in AF, the molecular mechanisms reducing myofilament contractile function remain unknown. We tested the hypothesis that increased expression of PPP1R12C (protein phosphatase 1 regulatory subunit 12C)-the PP1 (protein phosphatase 1) regulatory subunit targeting MLC2a (atrial myosin light chain 2)-causes hypophosphorylation of MLC2a and results in atrial hypocontractility. Right atrial appendage tissues were isolated from human patients with AF versus sinus rhythm controls. Western blots, coimmunoprecipitation, and phosphorylation studies were performed to examine how the PP1c (PP1 catalytic subunit)-PPP1R12C interaction causes MLC2a dephosphorylation. In vitro studies of pharmacological MRCK (myotonic dystrophy kinase-related Cdc42-binding kinase) inhibitor (BDP5290) in atrial HL-1 cells were performed to evaluate PP1 holoenzyme activity on MLC2a. Cardiac-specific lentiviral PPP1R12C overexpression was performed in mice to evaluate atrial remodeling with atrial cell shortening assays, echocardiography, and AF inducibility with electrophysiology studies. In human patients with AF, PPP1R12C expression was increased 2-fold versus sinus rhythm controls (P=2.0×10-2; n=12 and 12 in each group) with >40% reduction in MLC2a phosphorylation (P=1.4×10-6; n=12 and 12 in each group). PPP1R12C-PP1c binding and PPP1R12C-MLC2a binding were significantly increased in AF (P=2.9×10-2 and 6.7×10-3, respectively; n=8 and 8 in each group). In vitro studies utilizing drug BDP5290, which inhibits T560-PPP1R12C phosphorylation, demonstrated increased PPP1R12C binding with both PP1c and MLC2a and dephosphorylation of MLC2a. Mice treated with lentiviral PPP1R12C vector demonstrated a 150% increase in left atrial size versus controls (P=5.0×10-6; n=12, 8, and 12), with reduced atrial strain and atrial ejection fraction. Pacing-induced AF in mice treated with lentiviral PPP1R12C vector was significantly higher than in controls (P=1.8×10-2 and 4.1×10-2, respectively; n=6, 6, and 5). Patients with AF exhibit increased levels of PPP1R12C protein compared with controls. PPP1R12C overexpression in mice increases PP1c targeting to MLC2a and causes MLC2a dephosphorylation, which reduces atrial contractility and increases AF inducibility. These findings suggest that PP1 regulation of sarcomere function at MLC2a is a key determinant of atrial contractility in AF.

LINC-PINT suppresses breast cancer cell proliferation and migration via MEIS2/PPP3CC/NF-κB pathway by sponging miR-576-5p

BackgroundBreast cancer (BCa) is the most frequent malignant tumor in women. Long non-coding RNAs (lncRNAs) have been acknowledged to exert critical regulating functions in various cancers. Long intergenic non-protein coding RNA, p53 induced transcript (LINC-PINT) has been reported to be a chemosensitizer and a tumor suppressor in BCa. However, its downstream molecular mechanism contributing to its tumor-suppressing role remains to be explored in BCa. MethodsLINC-PINT expression in BCa tissues and cells was measured using quantitative real-time polymerase chain reaction (RT-qPCR). The proliferation of transfected BCa cells was examined by counting kit-8 (CCK-8) and EdU assay. The migrating ability of indicate BCa cells was assessed by wound healing assays. Bioinformatics analysis and mechanism experiments such as RNA immunoprecipitation (RIP), RNA pull down assay, and luciferase reporter assay, were applied to demonstrate the downstream targets of LINC-PINT. ResultsLINC-PINT was downregulated in BCa tissues and cell lines. Overexpression of LINC-PINT suppressed BCa cell proliferation and migration. LINC-PINT could interact with miR-576-5p to upregulate Meis homeobox 2 (MEIS2) that positively regulated protein phosphatase 3 catalytic subunit gamma (PPP3CC) by inactivating the nuclear factor-κB (NF-κB) pathway. ConclusionsThese findings elucidated the anti-tumor role of LINC-PINT in BCa via the miR-576-5p/MEIS2/PPP3CC/NF-κB axis, which suggested that LINC-PINT might serve as a potential therapeutic target for BCa.

ALA induces stomatal opening through regulation among PTPA, PP2AC, and SnRK2.6.

5-Aminolevulinic acid (ALA), as a new natural plant growth regulator, has been proved to regulate protein phosphatase 2A (PP2A) activity to promote stomatal opening in apple (Malus domestica) leaves. However, the molecular mechanisms underlying remain unclear. Here, we cloned and transformed MdPTPA, MdPP2AC, and MdSnRK2.6 of apple into tobaccos (Nicotiana tabacum) and found that over-expression (OE)-MdPTPA or OE-MdPP2AC promoted stomatal aperture while OE-MdSnRK2.6 induced stomatal closure under normal or drought condition. The Ca2+ and H2O2 levels in the guard cells of OE-MdPTPA and OE-MdPP2AC was decreased but flavonols increased, and the results in OE-SnRK2.6 was contrary. Exogenous ALA stimulated PP2A activity but depressed SnRK2.6 activity in transgenic tobaccos, leading to less Ca2+, H2O2 and more flavonols in guard cells, and consequently stomatal opening. OE-MdPTPA improved stomatal opening and plant growth but impaired drought tolerance, while OE-MdSnRK2.6 improved drought tolerance but depressed the leaf P n. Only OE-MdPP2AC improved stomatal opening, leaf P n, plant growth, as well as drought tolerance. These suggest that the three genes involved in ALA-regulating stomatal movement have their respective unique biological functions. Yeast two-hybrid (Y2H) assays showed that MdPP2AC interacted with MdPTPA or MdSnRK2.6, respectively, but no interaction of MdPTPA with MdSnRK2.6 was found. Yeast three-hybrid (Y3H) assay showed that MdPTPA promoted the interactions between MdPP2AC and MdSnRK2.6. Therefore, we propose a regulatory module of PTPA-PP2AC-SnRK2.6 that may be involved in mediating the ALA-inducing stomatal aperture in green plants.

Characterization of GEXP15 as a Potential Regulator of Protein Phosphatase 1 in Plasmodium falciparum.

The Protein Phosphatase type 1 catalytic subunit (PP1c) (PF3D7_1414400) operates in combination with various regulatory proteins to specifically direct and control its phosphatase activity. However, there is little information about this phosphatase and its regulators in the human malaria parasite, Plasmodium falciparum. To address this knowledge gap, we conducted a comprehensive investigation into the structural and functional characteristics of a conserved Plasmodium-specific regulator called Gametocyte EXported Protein 15, GEXP15 (PF3D7_1031600). Through in silico analysis, we identified three significant regions of interest in GEXP15: an N-terminal region housing a PP1-interacting RVxF motif, a conserved domain whose function is unknown, and a GYF-like domain that potentially facilitates specific protein-protein interactions. To further elucidate the role of GEXP15, we conducted in vitro interaction studies that demonstrated a direct interaction between GEXP15 and PP1 via the RVxF-binding motif. This interaction was found to enhance the phosphatase activity of PP1. Additionally, utilizing a transgenic GEXP15-tagged line and live microscopy, we observed high expression of GEXP15 in late asexual stages of the parasite, with localization predominantly in the nucleus. Immunoprecipitation assays followed by mass spectrometry analyses revealed the interaction of GEXP15 with ribosomal- and RNA-binding proteins. Furthermore, through pull-down analyses of recombinant functional domains of His-tagged GEXP15, we confirmed its binding to the ribosomal complex via the GYF domain. Collectively, our study sheds light on the PfGEXP15-PP1-ribosome interaction, which plays a crucial role in protein translation. These findings suggest that PfGEXP15 could serve as a potential target for the development of malaria drugs.

Hemizygous variants in protein phosphatase 1 regulatory subunit 3F (PPP1R3F) are associated with a neurodevelopmental disorder characterized by developmental delay, intellectual disability and autistic features.

Protein phosphatase 1 regulatory subunit 3F (PPP1R3F) is a member of the glycogen targeting subunits (GTSs), which belong to the large group of regulatory subunits of protein phosphatase 1 (PP1), a major eukaryotic serine/threonine protein phosphatase that regulates diverse cellular processes. Here, we describe the identification of hemizygous variants in PPP1R3F associated with a novel X-linked recessive neurodevelopmental disorder in 13 unrelated individuals. This disorder is characterized by developmental delay, mild intellectual disability, neurobehavioral issues such as autism spectrum disorder, seizures and other neurological findings including tone, gait and cerebellar abnormalities. PPP1R3F variants segregated with disease in affected hemizygous males that inherited the variants from their heterozygous carrier mothers. We show that PPP1R3F is predominantly expressed in brain astrocytes and localizes to the endoplasmic reticulum in cells. Glycogen content in PPP1R3F knockout astrocytoma cells appears to be more sensitive to fluxes in extracellular glucose levels than in wild-type cells, suggesting that PPP1R3F functions in maintaining steady brain glycogen levels under changing glucose conditions. We performed functional studies on nine of the identified variants and observed defects in PP1 binding, protein stability, subcellular localization and regulation of glycogen metabolism in most of them. Collectively, the genetic and molecular data indicate that deleterious variants in PPP1R3F are associated with a new X-linked disorder of glycogen metabolism, highlighting the critical role of GTSs in neurological development. This research expands our understanding of neurodevelopmental disorders and the role of PP1 in brain development and proper function.

Serine/Threonine Protein Phosphatases 1 and 2A in Lung Endothelial Barrier Regulation.

Vascular barrier dysfunction is characterized by increased permeability and inflammation of endothelial cells (ECs), which are prominent features of acute lung injury (ALI), acute respiratory distress syndrome (ARDS), and sepsis, and a major complication of the SARS-CoV-2 infection and COVID-19. Functional impairment of the EC barrier and accompanying inflammation arises due to microbial toxins and from white blood cells of the lung as part of a defensive action against pathogens, ischemia-reperfusion or blood product transfusions, and aspiration syndromes-based injury. A loss of barrier function results in the excessive movement of fluid and macromolecules from the vasculature into the interstitium and alveolae resulting in pulmonary edema and collapse of the architecture and function of the lungs, and eventually culminates in respiratory failure. Therefore, EC barrier integrity, which is heavily dependent on cytoskeletal elements (mainly actin filaments, microtubules (MTs), cell-matrix focal adhesions, and intercellular junctions) to maintain cellular contacts, is a critical requirement for the preservation of lung function. EC cytoskeletal remodeling is regulated, at least in part, by Ser/Thr phosphorylation/dephosphorylation of key cytoskeletal proteins. While a large body of literature describes the role of phosphorylation of cytoskeletal proteins on Ser/Thr residues in the context of EC barrier regulation, the role of Ser/Thr dephosphorylation catalyzed by Ser/Thr protein phosphatases (PPases) in EC barrier regulation is less documented. Ser/Thr PPases have been proposed to act as a counter-regulatory mechanism that preserves the EC barrier and opposes EC contraction. Despite the importance of PPases, our knowledge of the catalytic and regulatory subunits involved, as well as their cellular targets, is limited and under-appreciated. Therefore, the goal of this review is to discuss the role of Ser/Thr PPases in the regulation of lung EC cytoskeleton and permeability with special emphasis on the role of protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) as major mammalian Ser/Thr PPases. Importantly, we integrate the role of PPases with the structural dynamics of the cytoskeleton and signaling cascades that regulate endothelial cell permeability and inflammation.

Targeting protein phosphatases in cancer immunotherapy and autoimmune disorders.

Protein phosphatases act as key regulators of multiple important cellular processes and are attractive therapeutic targets for various diseases. Although extensive effort has been dedicated to phosphatase-targeted drug discovery, early expeditions for competitive phosphatase inhibitors were plagued by druggability issues, leading to the stigmatization of phosphatases as difficult targets. Despite challenges, persistent efforts have led to the identification of several drug-like, non-competitive modulators of some of these enzymes - including SH2 domain-containing protein tyrosine phosphatase 2, protein tyrosine phosphatase 1B, vascular endothelial protein tyrosine phosphatase and protein phosphatase 1 - reigniting interest in therapeutic targeting of phosphatases. Here, we discuss recent progress in phosphatase drug discovery, with emphasis on the development of selective modulators that exhibit biological activity. The roles and regulation of protein phosphatases in immune cells and their potential as powerful targets for immuno-oncology and autoimmunity indications are assessed.

Regulation of PP1 interaction with I-2, neurabin, and F-actin

Reversible phosphorylation is a fundamental regulatory mechanism required for many biological processes and is coordinated by the opposing actions of protein kinases and phosphatases. Protein phosphatase 1 (PP1) is a major protein phosphatase that plays an important role in many fundamental physiological processes including synaptic transmission and memory formation. Here we investigate the regulation of PP1 by prominent signaling proteins and synaptic scaffolds including GSK3β, inhibitor-2 (I-2), neurabin (Nrb), and actin. While GSK3β is known to regulate PP1 via phosphorylation of the PP1-binding protein I-2, we found that GSK3β directly regulates PP1 via inhibitory phosphorylation in neurons. Additionally, using bioluminescence resonance energy transfer (BRET), we found that GSK3β alters PP1-I-2 interaction in living cells. The effect of GSK3β on PP1-I-2 interaction is independent of the PP1 C-terminal tail, contrary to predictions based on previous findings from purified proteins. I-2 has been shown to form a trimeric complex with PP1 and Nrb, a major synaptic scaffold for promoting PP1 localization to the actin cytoskeleton. Utilizing BRET, we found that Nrb promotes PP1-actin interaction, however no BRET was detected between I-2 and F-actin. Finally, we found that stabilizing F-actin promotes Nrb-PP1 binding and may also lead to conformational changes between Nrb-I-2 and Nrb-F-actin complexes. Overall, our findings elaborate the dynamic regulation of PP1 complexes by GSK3β, targeting proteins, and actin polymerization.

Protein phosphatase-1 inhibitor-2 promotes PP1γ positive regulation of synaptic transmission.

Inhibitor-2 (I-2) is a prototypic inhibitor of protein phosphatase-1 (PP1), a major serine-threonine phosphatase that regulates synaptic plasticity and learning and memory. Although I-2 is a potent inhibitor of PP1 in vitro, our previous work has elucidated that, in vivo, I-2 may act as a positive regulator of PP1. Here we show that I-2 and PP1γ, but not PP1α, positively regulate synaptic transmission in hippocampal neurons. Moreover, we demonstrated that I-2 enhanced PP1γ interaction with its major synaptic scaffold, neurabin, by Förster resonance energy transfer (FRET)/Fluorescence lifetime imaging microscopy (FLIM) studies, while having a limited effect on PP1 auto-inhibitory phosphorylation. Furthermore, our study indicates that the effect of I-2 on PP1 activity in vivo is dictated by I-2 threonine-72 phosphorylation. Our work thus demonstrates a molecular mechanism by which I-2 positively regulates PP1 function in synaptic transmission.

The Caenorhabditis elegans ASPP homolog APE-1 is a junctional protein phosphatase 1 modulator.

How serine/threonine phosphatases are spatially and temporally tuned by regulatory subunits is a fundamental question in cell biology. Ankyrin repeat, SH3 domain, proline-rich-region-containing proteins are protein phosphatase 1 catalytic subunit binding partners associated with cardiocutaneous diseases. Ankyrin repeat, SH3 domain, proline-rich-region-containing proteins localize protein phosphatase 1 catalytic subunit to cell-cell junctions, but how ankyrin repeat, SH3 domain, proline-rich-region-containing proteins localize and whether they regulate protein phosphatase 1 catalytic subunit activity in vivo is unclear. Through a Caenorhabditis elegans genetic screen, we find that loss of the ankyrin repeat, SH3 domain, proline-rich-region-containing protein homolog, APE-1, suppresses a pathology called "jowls," providing us with an in vivo assay for APE-1 activity. Using immunoprecipitations and mass spectrometry, we find that APE-1 binds the protein phosphatase 1 catalytic subunit called GSP-2. Through structure-function analysis, we discover that APE-1's N-terminal half directs the APE-1-GSP-2 complex to intercellular junctions. Additionally, we isolated mutations in highly conserved residues of APE-1's ankyrin repeats that suppress jowls yet do not preclude GSP-2 binding, implying APE-1 does more than simply localize GSP-2. Indeed, in vivo reconstitution of APE-1 suggests the ankyrin repeats modulate phosphatase output, a function we find to be conserved among vertebrate homologs.

Increased CDCA2 Level Was Related to Poor Prognosis in Hepatocellular Carcinoma and Associated With Up-Regulation of Immune Checkpoints.

BackgroundCell division cycle-associated protein 2 (CDCA2) is a member of cell cycle-related proteins. CDCA2 plays a role in the regulation of protein phosphatase 1(PP1) γ-dependent DNA damage response (DDR) and H3 phosphorylation. CDCA2 promotes the tumorigenesis and development of several types of cancers by promoting the proliferation of tumor cells. However, the relationship between CDCA2 expression and the clinicopathological characteristics of hepatocellular carcinoma (HCC) is unknown.MethodsGene expression information and clinical data were downloaded from The Cancer Genome Atlas (TCGA) database. The expression of CDCA2 and its correlation to clinical characteristics in HCC were analyzed. The expression level of CDCA2 was validated in HCC cell lines. The relationship between CDCA2 expression and the survival of patients with HCC was analyzed by using Kaplan–Meier method. The prognostic value of CDCA2 in HCC was estimated by Cox regression analysis. The expression difference of CDCA2 between HCC and normal tissues and its correlation to survival were verified in independent datasets. Gene set enrichment analysis (GSEA) was used to screen the CDCA2-related signaling pathways.ResultsCell division cycle-associated protein 2 expression was upregulated in HCC tissues (p < 0.001) and increased CDCA2 was correlated to increased T stage, pathologic stage, histologic grade, and alpha-fetoprotein (AFP) level (p < 0.001). In addition, CDCA2 was overexpressed in HCC cell lines HepG2 and LM3. High CDCA2 expression level was associated with poor overall survival [hazard ratio (HR) = 1.69; 95% CI, 1.20–1.40, p = 0.003], disease specific survival (HR = 1.73; 95% CI, 1.11–2.71, p = 0.016), and progress free interval (HR = 1.74; 95% CI, 1.30–2.34, p < 0.001). Overexpression of CDCA2 and its correlation to poor survival in HCC were verified in Gene Expression Omnibus (GEO) datasets and Kaplan–Meier plotter database. Increased CDCA2 expression was associated with upregulation of PD-L1 (Spearman's coefficient = 0.207, p < 0.001), PD-L2 (Spearman coefficient's = 0.118, p < 0.05), and CTLA4 (Spearman's coefficient = 0.355, p < 0.001). GSEA showed that homologous recombination pathway, insulin signaling pathway, mitogen-activated protein kinase (MAPK) pathway, mismatch repair pathway, mechanistic target of rapamycin (mTOR) pathway, Notch pathway, T cell receptor pathway, toll like receptor pathway, and WNT pathway were enriched in CDCA2 high expression phenotype.ConclusionCell division cycle-associated protein 2 may serve as an independent biomarker for poor prognosis in HCC and increased CDCA2 expression was associated with upregulation of immune checkpoints.

AMPA Receptor Antagonists Facilitate NEDD4-2-Mediated GRIA1 Ubiquitination by Regulating PP2B-ERK1/2-SGK1 Pathway in Chronic Epilepsy Rats.

The neural precursor cell expressed by developmentally downregulated gene 4-2 (NEDD4-2) is a ubiquitin E3 ligase that has a high affinity toward binding and ubiquitinating glutamate ionotropic receptor α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) type subunit 1 (GRIA1, also referred to GluR1 or GluA1). Since dysregulation of GRIA1 surface expression is relevant to the responsiveness to AMPA receptor (AMPAR) antagonists (perampanel and GYKI 52466) in chronic epilepsy rats, it is likely that NEDD4-2 may be involved in the pathogenesis of intractable epilepsy. However, the role of NEDD4-2-mediated GRIA1 ubiquitination in refractory seizures to AMPAR antagonists is still unknown. In the present study, both AMPAR antagonists recovered the impaired GRIA1 ubiquitination by regulating protein phosphatase 2B (PP2B)-extracellular signal-regulated kinase 1/2 (ERK1/2)-serum and glucocorticoid-regulated kinase 1 (SGK1)-NEDD4-2 signaling pathway in responders (whose seizure activities are responsive to AMPAR), but not non-responders (whose seizure activities were uncontrolled by AMPAR antagonists). In addition, cyclosporin A (CsA, a PP2B inhibitor) co-treatment improved the effects of AMPAR antagonists in non-responders, independent of AKT signaling pathway. Therefore, our findings suggest that dysregulation of PP2B-ERK1/2-SGK1-NEDD4-2-mediated GRIA1 ubiquitination may be responsible for refractory seizures and that this pathway may be a potential therapeutic target for improving the treatment of intractable epilepsy in response to AMPAR antagonists.

Deletion or inhibition of prolyl oligopeptidase blocks lithium-induced phosphorylation of GSK3b and Akt by activation of protein phosphatase 2A.

Alterations in prolyl oligopeptidase (PREP) activity have been connected, for example, with bipolar and major depressive disorder, and several studies have reported that lack or inhibition of PREP blocks the effects of lithium on inositol 1,4,5-triphosphate (IP3 ) levels. However, the impact of PREP modulation on other intracellular targets of lithium, such as glycogen synthase kinase 3 beta (GSK3b) or protein kinase B (Akt), has not been studied. We recently found that PREP regulates protein phosphatase 2A (PP2A), and because GSK3b and Akt are PP2A substrates, we studied if PREP-related lithium insensitivity is dependent on PP2A. To assess this, HEK-293 and SH-SY5Y cells with PREP deletion or PREP inhibition (KYP-2047) were exposed to lithium, and thereafter, the phosphorylation levels of GSK3b and Akt were measured by Western blot. As expected, PREP deletion and inhibition blocked the lithium-induced phosphorylation on GSK3b and Akt in both cell lines. When lithium exposure was combined with okadaic acid, a PP2A inhibitor, KYP-2047 did not have effect on lithium-induced GSK3b and Akt phosphorylation. Therefore, we conclude that PREP deletion or inhibition blocks the intracellular effects of lithium on GSK3b and Akt via PP2A activation.

Protein phosphatase 1: life‐course regulation by SDS22 and Inhibitor‐3

Protein phosphatase 1 (PP1) is expressed in all eukaryotic cells and catalyzes a sizable fraction of protein Ser/Thr dephosphorylation events. It is tightly regulated in space and time through association with a wide array of regulatory interactors of protein phosphatase one (RIPPOs). Suppressor-of-Dis2-number 2 (SDS22) and Inhibitor-3 (I3), which form a ternary complex with PP1, are the first two evolved and most widely expressed RIPPOs. Their deletion causes mitotic-arrest phenotypes and is lethal in some organisms. The role of SDS22 and I3 in PP1 regulation has been a mystery for decades as they were independently identified as both activators and inhibitors of PP1. This conundrum has largely been solved by recent reports showing that SDS22 and I3 control multiple steps of the life course of PP1. Indeed, they contribute to (a) the stabilization and activation of newly translated PP1, (b) the translocation of PP1 to the nucleus, and (c) the storage of PP1 as a reserve for holoenzyme assembly. Preliminary evidence suggests that SDS22 and I3 may also function as scavengers of released or aged PP1 for re-use in holoenzyme assembly or proteolytical degradation, respectively. Hence, SDS22 and I3 are emerging as master regulators of the life course of PP1.

Regulation of Synaptic Transmission and Plasticity by Protein Phosphatase 1.

Protein phosphatases, by counteracting protein kinases, regulate the reversible phosphorylation of many substrates involved in synaptic plasticity, a cellular model for learning and memory. A prominent phosphatase regulating synaptic plasticity and neurologic disorders is the serine/threonine protein phosphatase 1 (PP1). PP1 has three isoforms (α, β, and γ, encoded by three different genes), which are regulated by a vast number of interacting subunits that define their enzymatic substrate specificity. In this review, we discuss evidence showing that PP1 regulates synaptic transmission and plasticity, as well as presenting novel models of PP1 regulation suggested by recent experimental evidence. We also outline the required targeting of PP1 by neurabin and spinophilin to achieve substrate specificity at the synapse to regulate AMPAR and NMDAR function. We then highlight the role of inhibitor-2 in regulating PP1 function in plasticity, including its positive regulation of PP1 function in vivo in memory formation. We also discuss the distinct function of the three PP1 isoforms in synaptic plasticity and brain function, as well as briefly discuss the role of inhibitory phosphorylation of PP1, which has received recent emphasis in the regulation of PP1 activity in neurons.