Catalytic Subunit Of Protein Phosphatase Research Articles

Protein phosphatase 6 activates NF-κB to confer sensitivity to MAPK pathway inhibitors in KRAS- and BRAF-mutant cancer cells.

The Ras-mitogen-activated protein kinase (MAPK) pathway is a major target for cancer treatment. To better understand the genetic pathways that modulate cancer cell sensitivity to MAPK pathway inhibitors, we performed a CRISPR knockout screen with MAPK pathway inhibitors on a colorectal cancer (CRC) cell line carrying mutant KRAS. Genetic deletion of the catalytic subunit of protein phosphatase 6 (PP6), encoded by PPP6C, rendered KRAS- and BRAF-mutant CRC and BRAF-mutant melanoma cells more resistant to these inhibitors. In the absence of MAPK pathway inhibition, PPP6C deletion in CRC cells decreased cell proliferation in two-dimensional (2D) adherent cultures but accelerated the growth of tumor spheroids in 3D culture and tumor xenografts in vivo. PPP6C deletion enhanced the activation of nuclear factor κB (NF-κB) signaling in CRC and melanoma cells and circumvented the cell cycle arrest and decreased cyclin D1 abundance induced by MAPK pathway blockade in CRC cells. Inhibiting NF-κB activity by genetic and pharmacological means restored the sensitivity of PPP6C-deficient cells to MAPK pathway inhibition in CRC and melanoma cells in vitro and in CRC cells in vivo. Furthermore, a R264 point mutation in PPP6C conferred loss of function in CRC cells, phenocopying the enhanced NF-κB activation and resistance to MAPK pathway inhibition observed for PPP6C deletion. These findings demonstrate that PP6 constrains the growth of KRAS- and BRAF-mutant cancer cells, implicates the PP6-NF-κB axis as a modulator of MAPK pathway output, and presents a rationale for cotargeting the NF-κB pathway in PPP6C-mutant cancer cells.

Saturated fatty acids stimulate cytokine production in tanycytes via the PP2Ac-dependent signaling pathway

The hypothalamic tanycytes are crucial for free fatty acids (FFAs) detection, storage, and transport within the central nervous system. They have been shown to effectively respond to fluctuations in circulating FFAs, thereby regulating energy homeostasis. However, the precise molecular mechanisms by which tanycytes modulate lipid utilization remain unclear. Here, we report that the catalytic subunit of protein phosphatase 2 A (PP2Ac), a serine/threonine phosphatase, is expressed in tanycytes and its accumulation and activation occur in response to high-fat diet consumption. In vitro, tanycytic PP2Ac responds to palmitic acid (PA) exposure and accumulates and is activated at an early stage in an AMPK-dependent manner. Furthermore, activated PP2Ac boosts hypoxia-inducible factor-1α (HIF-1α) accumulation, resulting in upregulation of an array of cytokines. Pretreatment with a PP2Ac inhibitor, LB100, prevented the PA-induced elevation of vascular endothelial growth factor (VEGF), fibroblast growth factor 1 (FGF1), hepatocyte growth factor (HGF), and dipeptidyl peptidase IV (DPPIV or CD26). Our results disclose a mechanism of lipid metabolism in tanycytes that involves the activation of PP2Ac and highlight the physiological significance of PP2Ac in hypothalamic tanycytes in response to overnutrition and efficacious treatment of obesity.

Protein Phosphatase 6 Complex: Novel Regulator of Fetal Hemoglobin and Potential Therapeutic Target in Sickle Cell Disease

Although increasing levels of fetal hemoglobin (HbF) in sickle cell disease (SCD) significantly reduces mortality, effective pharmacologic induction of HbF has remained elusive. To identify potentially druggable molecules involved in HbF control, we carried out a domain-focused CRISPR-Cas9-based genetic screen in HUDEP2 cells targeting all 218 serine/threonine protein phosphatases with 6 independent sgRNAs each. A single phosphatase, PPP6C, emerged as a potential HbF repressor. PPP6C is the catalytic subunit of protein phosphatase 6 (PP6), a serine/threonine phosphatase complex that has been implicated in cell cycle regulation, autophagy, and innate immunity mechanisms in other cell types, but its role in erythropoiesis and HbF regulation has not previously been described. CRISPR-Cas9-based depletion of PPP6C in primary human erythroid cells elevated mRNA levels of HBG (the fetal form of β-globin) in a dose-dependent manner up to 3-4 fold, increased HbF levels 3-fold as measured by HPLC (up to 15-20% of total hemoglobin), and doubled the number of HbF-expressing cells. PPP6C depletion caused relatively few changes in the erythroid transcriptome by RNA-seq analysis, and did not measurably impair erythroid maturation. In addition, PPP6C loss in primary SCD patient-derived cells was well-tolerated, led to robust levels of HbF induction, and reduced cell sickling in vitro by up to 60%. Mechanistically, loss of PPP6C reduced the levels of the HbF repressor BCL11A by nearly 50% but left unchanged the levels of other HbF regulators, such as HRI, LRF, EKLF, NFIA/X, ZNF410, or HIC2, suggesting that PPP6C-mediated HbF regulation proceeds at least in part via loss of BCL11A. Importantly, xenotransplantation data (NBSGW) showed ~4-fold induction of HBG at 16 weeks post-transplant, suggesting that PPP6C deficiency leads to effective, sustained HbF induction in vivo. We next set out to determine whether any PP6 subunits have erythroid specificity that could be exploited therapeutically. In contrast to PPP6C, which is highly expressed in all hematopoietic lineages, the PP6 facultative subunit PPP6R1 appears to be enriched in erythroid precursors, suggesting that depletion of PPP6R1 may function as an erythroid-selective target to increase HbF levels. CRISPR-Cas9-based depletion of PPP6R1 in primary human erythroid cells showed a 2-3-fold increase in HBG and near-doubling of HbF-expressing cells, recapitulating the majority of PPP6C-mediated HbF induction. Functional studies are currently in progress to specifically determine whether additional PP6 subunits play a key role in HbF regulation. Furthermore, ongoing work utilizing dTAG-based acute depletion of PPP6C and PPP6R1 paired with phospho-proteomic studies will narrow down direct targets of the PP6 complex and provide insights into erythropoietic and HbF regulatory pathways impacted by PP6. Taken together, our data indicate that PP6, including its catalytic subunit PPP6C and erythroid-enriched regulatory subunit PPP6R1, inhibit HbF production in part via modulating BCL11A levels, and may serve as a red-cell specific therapeutic target in the treatment of SCD.

Protein phosphatase 1 regulates core PCP signaling.

Planar cell polarity (PCP) signaling polarizes epithelial cells within the plane of an epithelium. Core PCP signaling components adopt asymmetric subcellular localizations within cells to both polarize and coordinate polarity between cells. Achieving subcellular asymmetry requires additional effectors, including some mediating post-translational modifications of core components. Identification of such proteins is challenging due to pleiotropy. We used mass spectrometry-based proximity labeling proteomics to identify such regulators in the Drosophila wing. We identified the catalytic subunit of protein phosphatase1, Pp1-87B, and show that it regulates core protein polarization. Pp1-87B interacts with the core protein Van Gogh and at least one serine/threonine kinase, Dco/CKIε, that is known to regulate PCP. Pp1-87B modulates Van Gogh subcellular localization and directs its dephosphorylation in vivo. PNUTS, a Pp1 regulatory subunit, also modulates PCP. While the direct substrate(s) of Pp1-87B in control of PCP is not known, our data support the model that cycling between phosphorylated and unphosphorylated forms of one or more core PCP components may regulate acquisition of asymmetry. Finally, our screen serves as a resource for identifying additional regulators of PCP signaling.

DOK3 promotes atopic dermatitis by enabling the phosphatase PP4C to inhibit the T cell signaling mediator CARD11.

The scaffolding protein CARD11 is a critical mediator of antigen receptor signaling in lymphocytes. Hypomorphic (partial loss-of-function) mutations in CARD11 are associated with the development of severe atopic dermatitis, in which T cell receptor signaling is reduced and helper T cell differentiation is skewed to an allergy-associated type 2 phenotype. Here, we found that the docking protein DOK3 plays a key role in the pathogenesis of atopic dermatitis by suppressing CARD11 activity. DOK3 interacted with CARD11 and decreased its phosphorylation in T cells by recruiting the catalytic subunit of protein phosphatase 4, thereby dampening downstream signaling. Knocking out Dok3 enhanced the production of the cytokine IFN-γ by T cells, which conferred protection against experimental atopic dermatitis-like skin inflammation in mice. The expression of DOK3 was increased in T cells isolated from patients with atopic dermatitis and inversely correlated with IFNG expression. A subset of hypomorphic CARD11 variants found in patients with atopic dermatitis bound more strongly than wild-type CARD11 to DOK3. Our findings suggest that the strength of the interaction of DOK3 with CARD11 may predispose individuals to developing atopic dermatitis.

AKAP2-anchored protein phosphatase 1 controls prostatic neuroendocrine carcinoma cell migration and invasion

Prostate cancer (PC) is the second leading cause of cancer-related death in men. The growth of primary prostate cancer cells relies on circulating androgens and thus the standard therapy for the treatment of localized and advanced PC is the androgen deprivation therapy. Prostatic neuroendocrine carcinoma (PNEC) is an aggressive and highly metastatic subtype of prostate cancer, which displays poor prognosis and high lethality. Most of PNECs develop from prostate adenocarcinoma in response to androgen deprivation therapy, however the mechanisms involved in this transition and in the elevated biological aggressiveness of PNECs are poorly defined. Our current findings indicate that AKAP2 expression is dramatically upregulated in PNECs as compared to non-cancerous prostate tissues. Using a PNEC cell model, we could show that AKAP2 is localized both intracellularly and at the cell periphery where it colocalizes with F-actin. AKAP2 and F-actin interact directly through a newly identified actin-binding domain located on AKAP2. RNAi-mediated silencing of AKAP2 promotes the phosphorylation and deactivation of cofilin, a protein involved in actin turnover. This effect correlates with a significant reduction in cell migration and invasion. Co-immunoprecipitation experiments and proximity ligation assays revealed that AKAP2 forms a complex with the catalytic subunit of protein phosphatase 1 (PP1) in PNECs. Importantly, AKAP2-mediated anchoring of PP1 to the actin cytoskeleton regulates cofilin dephosphorylation and activation, which, in turn, enhances F-actin dynamics and favors migration and invasion. In conclusion, this study identified AKAP2 as an anchoring protein overexpressed in PNECs that controls cancer cell invasive properties by regulating cofilin phosphorylation.

Opposing Roles for the α Isoform of the Catalytic Subunit of Protein Phosphatase 1 in Inside-Out and Outside-In Integrin Signaling in Murine Platelets.

Platelet activation during hemostasis and thrombosis is facilitated by agonist-induced inside-out and integrin αIIbβ3-initiated outside-in signaling via protein kinases and phosphatases. Pharmacological inhibitor studies suggest that the serine/threonine protein phosphatase 1 (PP1) promotes platelet activation. However, since phosphatase inhibitors block all the isoforms of the catalytic subunit of PP1 (PP1c), the role of specific PP1c isoform in platelet signaling remains unclear. Here, we employed a platelet-specific PP1cα-/- mice to explore the contribution of a major PP1 isoform in platelet functions. Loss of PP1cα moderately decreased activation of integrin αIIbβ3, binding of soluble fibrinogen, and aggregation to low-dose thrombin, ADP, and collagen. In contrast, PP1cα-/- platelets displayed increased adhesion to immobilized fibrinogen, fibrin clot retraction, and thrombus formation on immobilized collagen. Mechanistically, post-fibrinogen engagement potentiated p38 mitogen-activated protein kinase (MAPK) activation in PP1cα-/- platelets and the p38 inhibitor blocked the increased integrin-mediated outside-in signaling function. Tail bleeding time and light-dye injury-induced microvascular thrombosis in the cremaster venules and arterioles were not altered in PP1cα-/- mice. Thus, PP1cα displays pleiotropic signaling in platelets as it amplifies agonist-induced signaling and attenuates integrin-mediated signaling with no impact on hemostasis and thrombosis.

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.

Identification of PP1c-PPP1R12A Substrates Using Kinase-Catalyzed Biotinylation to Identify Phosphatase Substrates.

Protein phosphatase 1 regulatory subunit 12A (PPP1R12A) interacts with the catalytic subunit of protein phosphatase 1 (PP1c) to form the myosin phosphatase complex. In addition to a well-documented role in muscle contraction, the PP1c-PPP1R12A complex is associated with cytoskeleton organization, cell migration and adhesion, and insulin signaling. Despite the variety of biological functions, only a few substrates of the PP1c-PPP1R12A complex are characterized, which limit a full understanding of PP1c-PPP1R12A activities in muscle contraction and cytoskeleton regulation. Here, the chemoproteomics method Kinase-catalyzed Biotinylation to Identify Phosphatase Substrates (K-BIPS) was used to identify substrates of the PP1c-PPP1R12A complex in L6 skeletal muscle cells. K-BIPS enriched 136 candidate substrates with 14 high confidence hits. One high confidence hit, AKT1 kinase, was validated as a novel PP1c-PPP1R12A substrate. Given the previously documented role of AKT1 in PPP1R12A phosphorylation and cytoskeleton organization, the data suggest that PP1c-PPP1R12A regulates its own phosphatase activity through an AKT1-dependent feedback mechanism to influence cytoskeletal arrangement in muscle cells.

TaTIP41 and TaTAP46 positively regulate drought tolerance in wheat by inhibiting PP2A activity.

Drought is a major environmental stress limiting global wheat (Triticum aestivum) production. Exploring drought tolerance genes is important for improving drought adaptation in this crop. Here, we cloned and characterized TaTIP41, a novel drought tolerance gene in wheat. TaTIP41 is a putative conserved component of target of rapamycin (TOR) signaling, and the TaTIP41 homoeologs were expressed in response to drought stress and abscisic acid (ABA). The overexpression of TaTIP41 enhanced drought tolerance and the ABA response, including ABA-induced stomatal closure, while its downregulation using RNA interference (RNAi) had the opposite effect. Furthermore, TaTIP41 physically interacted with TaTAP46, another conserved component of TOR signaling. Like TaTIP41, TaTAP46 positively regulated drought tolerance. Furthermore, TaTIP41 and TaTAP46 interacted with type-2A protein phosphatase (PP2A) catalytic subunits, such as TaPP2A-2, and inhibited their enzymatic activities. Silencing TaPP2A-2 improved drought tolerance in wheat. Together, our findings provide new insights into the roles of TaTIP41 and TaTAP46 in the drought tolerance and ABA response in wheat, and their potential application in improving wheat environmental adaptability. This article is protected by copyright. All rights reserved.

Phosphorylated Peptide Derived from the Myosin Phosphatase Target Subunit Is a Novel Inhibitor of Protein Phosphatase-1

Identification of specific protein phosphatase-1 (PP1) inhibitors is of special importance regarding the study of its cellular functions and may have therapeutic values in diseases coupled to signaling processes. In this study, we prove that a phosphorylated peptide of the inhibitory region of myosin phosphatase (MP) target subunit (MYPT1), R690QSRRS(pT696)QGVTL701 (P-Thr696-MYPT1690-701), interacts with and inhibits the PP1 catalytic subunit (PP1c, IC50 = 3.84 µM) and the MP holoenzyme (Flag-MYPT1-PP1c, IC50 = 3.84 µM). Saturation transfer difference NMR measurements established binding of hydrophobic and basic regions of P-Thr696-MYPT1690-701 to PP1c, suggesting interactions with the hydrophobic and acidic substrate binding grooves. P-Thr696-MYPT1690-701 was dephosphorylated by PP1c slowly (t1/2 = 81.6-87.9 min), which was further impeded (t1/2 = 103 min) in the presence of the phosphorylated 20 kDa myosin light chain (P-MLC20). In contrast, P-Thr696-MYPT1690-701 (10-500 µM) slowed down the dephosphorylation of P-MLC20 (t1/2 = 1.69 min) significantly (t1/2 = 2.49-10.06 min). These data are compatible with an unfair competition mechanism between the inhibitory phosphopeptide and the phosphosubstrate. Docking simulations of the PP1c-P-MYPT1690-701 complexes with phosphothreonine (PP1c-P-Thr696-MYPT1690-701) or phosphoserine (PP1c-P-Ser696-MYPT1690-701) suggested their distinct poses on the surface of PP1c. In addition, the arrangements and distances of the surrounding coordinating residues of PP1c around the phosphothreonine or phosphoserine at the active site were distinct, which may account for their different hydrolysis rate. It is presumed that P-Thr696-MYPT1690-701 binds tightly at the active center but the phosphoester hydrolysis is less preferable compared to P-Ser696-MYPT1690-701 or phosphoserine substrates. Moreover, the inhibitory phosphopeptide may serve as a template to synthesize cell permeable PP1-specific peptide inhibitors.

Regulation of Fetal Hemoglobin Production in Adult Erythroid Cells By Protein Phosphatase 6C (PPP6C)

Although increasing levels of fetal hemoglobin (HbF) in sickle cell disease (SCD) significantly reduces mortality, effective pharmacologic induction of HbF has remained elusive. To identify potentially druggable molecules involved in HbF control, we carried out a domain-focused CRISPR-Cas9-based genetic screen targeting all serine/threonine protein phosphatases (1308 independent sgRNAs representing 218 phosphatases). The phosphatase sgRNA library was cloned into a lentivirus scaffold and introduced into the adult-type erythroid cell line HUDEP2 stably expressing Cas9, the top and bottom 10% of HbF-expressing cells sorted, and the integrated sgRNAs sequenced. Of all protein phosphatases tested, this screen singly identified PPP6C as a novel HbF repressor. PPP6C is the catalytic subunit of protein phosphatase 6, a serine/threonine protein phosphatase that has been implicated in numerous cellular functions, including cell cycle regulation, autophagy, and innate immunity, but its role in HbF regulation has not previously been described. Depletion of PPP6C by 5 independent sgRNAs in HUDEP2 cells resulted in significant HbF enrichment and did not adversely affect cell fitness. To validate these findings, we performed CRISPR-Cas9-based depletion of PPP6C in primary human erythroid cells. Quantitation of fetal-type globin genes (gamma-globin) was determined by RT-qPCR and Western blot, and HbF protein levels were determined by flow cytometry and HPLC. Depletion of PPP6C elevated gamma-globin mRNA levels in a dose-dependent manner to 3-4 times basal levels, increased HbF levels 3-4 fold as measured by HPLC (up to 15-20% of total hemoglobin), and doubled the number of HbF-expressing cells. PPP6C depletion caused relatively few changes in the erythroid transcriptome by RNA-seq analysis and did not ostensibly impair erythroid maturation. Importantly, xenotransplantation data (NBSGW) from two independent murine transplants for a total of 10 recipient mice showed ~4-fold induction of gamma-globin at 16 weeks post-transplant, suggesting that PPP6C deficiency is well-tolerated and leads to effective, sustained HbF induction in vivo. To test whether interference with PPP6C raised HbF levels sufficiently to provide anti-sickling effects, we depleted PPP6C by CRISPR-Cas9 in SCD patient-derived cells. PPP6C depletion in SCD cells was well-tolerated, led to robust levels of HbF induction, and reduced cell sickling in vitro by greater than 60%. Mechanistically, loss of PPP6C reduced the levels of the HbF repressor BCL11A by nearly 50% but left unchanged the levels of other HbF regulators, such as HRI, LRF, EKLF, NFIA/X, ZNF410, or HIC2, suggesting that PPP6C-mediated HbF regulation may proceed at least in part via loss of BCL11A. Additional studies, including specific interrogation of potential downstream mediators of PPP6C in HbF regulation, are ongoing to further elucidate HbF regulatory pathways impacted by PPP6C. Taken together, our data indicate that PPP6C functions in a dose-dependent manner to regulate HbF in primary erythroid cells and may serve as a therapeutic target in the treatment of SCD.

Rice protein phosphatase 1 regulatory subunits OsINH2 and OsINH3 participate actively in growth and adaptive responses under abscisic acid.

Rice (Oryza sativa L.), a worldwide staple food crop, is affected by various environmental stressors that ultimately reduce yield. However, diversified physiological and molecular responses enable it to cope with adverse factors. It includes the integration of numerous signaling in which protein phosphatase 1 (PP1) plays a pivotal role. Research on PP1 has been mostly limited to the PP1 catalytic subunit in numerous cellular progressions. Therefore, we focused on the role of PP1 regulatory subunits (PP1r), OsINH2 and OsINH3, homologs of AtINH2 and AtINH3 in Arabidopsis, in rice growth and stress adaptations. Our observations revealed that these are ubiquitously expressed regulatory subunits that interacted and colocalized with their counter partners, type 1 protein phosphatase (OsTOPPs) but could not change their subcellular localization. The mutation in OsINH2 and OsINH3 reduced pollen viability, thereby affected rice fertility. They were involved in abscisic acid (ABA)-mediated inhibition of seed germination, perhaps by interacting with osmotic stress/ABA-activated protein kinases (OsSAPKs). Meanwhile, they positively participated in osmotic adjustment by proline biosynthesis, detoxifying reactive oxygen species (ROS) through peroxidases (POD), reducing malondialdehyde formation (MDA), and regulating stress-responsive genes. Moreover, their co-interaction proposed they might mediate cellular processes together or by co-regulation; however, the special behavior of two different PP1r is needed to explore. In a nutshell, this research enlightened the involvement of OsINH2 and OsINH3 in the reproductive growth of rice and adaptive strategies under stress. Hence, their genetic interaction with ABA components and deep mechanisms underlying osmotic regulation and ROS adjustment would explain their role in complex signaling. This research offers the basis for introducing stress-resistant crops.

Mapping PP1c and Its Inhibitor 2 Interactomes Reveals Conserved and Specific Networks in Asexual and Sexual Stages of Plasmodium.

Malaria parasites require multiple phosphorylation and dephosphorylation steps to drive signaling pathways for proper differentiation and transformation. Several protein phosphatases, including protein phosphatase 1 (PP1), one of the main dephosphorylation enzymes, have been shown to be indispensable for the Plasmodium life cycle. The catalytic subunit of PP1 (PP1c) participates in cellular processes via dynamic interactions with a vast number of binding partners that contribute to its diversity of action. In this study, we used Plasmodium berghei transgenic parasite strains stably expressing PP1c or its inhibitor 2 (I2) tagged with mCherry, combined with the mCherry affinity pulldown of proteins from asexual and sexual stages, followed by mass spectrometry analyses. Mapped proteins were used to identify interactomes and to cluster functionally related proteins. Our findings confirm previously known physical interactions of PP1c and reveal enrichment of common biological processes linked to cellular component assembly in both schizonts and gametocytes to biosynthetic processes/translation in schizonts and to protein transport exclusively in gametocytes. Further, our analysis of PP1c and I2 interactomes revealed that nuclear export mediator factor and peptidyl-prolyl cis-trans isomerase, suggested to be essential in P. falciparum, could be potential targets of the complex PP1c/I2 in both asexual and sexual stages. Our study emphasizes the adaptability of Plasmodium PP1 and provides a fundamental study of the protein interaction landscapes involved in a myriad of events in Plasmodium, suggesting why it is crucial to the parasite and a source for alternative therapeutic strategies.

Chebulinic Acid Suppresses Adipogenesis in 3T3-L1 Preadipocytes by Inhibiting PPP1CB Activity.

Depletion of protein phosphatase-1 catalytic subunit beta (PPP1CB), a serine/threonine protein phosphatase and potent adipogenic activator, suppresses the differentiation of 3T3-L1 preadipocytes into mature adipocytes. Therefore, PPP1CB is considered as a potential therapeutic target for obesity. We screened 1033 natural products for PPP1CB inhibitors and identified chebulinic acid, which is abundantly present in the seeds of Euphoria longana and fruits of Terminalia chebula. Chebulinic acid strongly inhibited the hydrolysis of 6,8-difluoro-4-methylumbelliferyl phosphate by PPP1CB (IC50 = 300 nM) and demonstrated potent antiadipogenic effects in 3T3-L1 preadipocytes in a concentration-dependent manner. Additional studies have demonstrated that chebulinic acid suppresses early differentiation by downregulating key transcription factors that control adipogenesis in 3T3-L1 cells. These results suggested that chebulinic acid may be a potential therapeutic agent for treating obesity by inhibiting PPP1CB activity.

Protein Phosphatase 6C (PPP6C) Loss Significantly Raises Fetal Hemoglobin Levels and Reduces Cell Sickling

Sickle cell disease (SCD) afflicts millions of people worldwide and can lead to severe complications including acute chest syndrome, stroke, avascular necrosis of bone, and nephropathy. Although increasing levels of fetal hemoglobin (HbF) significantly reduces cell sickling and SCD-related morbidity and mortality, effective HbF pharmacologic induction has remained an elusive goal. To identify additional potentially druggable molecules involved in HbF control, we carried out a domain-focused CRISPR-Cas9-based genetic screen targeting all protein phosphatases (1308 independent sgRNA representing 218 phosphatases). The phosphatase sgRNA library was cloned into a lentivirus scaffold and introduced into the erythroid cell line HUDEP2 stably expressing Cas9, and the top and bottom 10% of HbF-expressing cells were sorted and the integrated sgRNAs were sequenced. This screen identified a single protein phosphatase - PPP6C - as an HbF repressor.PPP6C is the catalytic subunit of protein phosphatase 6, a serine/threonine cytosolic protein phosphatase that is widely expressed across tissues and throughout erythroid development to broadly regulate mRNA translation. PPP6C has been implicated in numerous cellular functions, including cell cycle regulation, autophagy, and innate immunity, but its role in HbF regulation has not previously been described. Depletion of PPP6C by 5 independent sgRNAs in HUDEP2 cells resulted in significant HbF enrichment. Importantly, PPP6C depletion did not affect cellular viability or differentiation, suggesting that PPP6C may serve as a targetable HbF regulator for the treatment of SCD.To validate the findings of this genetic screen in primary human erythroid cells, we performed CRISPR-Cas9 ribonuclear protein (RNP)-based genome editing of PPP6C in a three-phase in vitro culture of adult CD34+ hematopoietic cells. HbF levels were assessed by RT-qPCR, Western blot, flow cytometry, and HPLC. We find that depletion of PPP6C protein levels by greater than 80% increases gamma-globin transcript levels in a dose-dependent manner to nearly 5 times basal levels. In addition, PPP6C loss leads to a greater than doubling in F-cell number and a 3-4-fold increase in HbF levels as measured by HPLC analysis. PPP6C depletion showed minimal effects on the erythroid transcriptome by RNA-Seq and did not significantly impair erythroid maturation. Mechanistically, loss of PPP6C leads to depletion of BCL11A protein levels by nearly 50% but unchanged levels of other key HbF regulators such as HRI and LRF, suggesting PPP6C-mediated HbF regulation may proceed at least in part via loss of BCL11A. However, additional studies are necessary to fully elucidate these underlying regulatory mechanisms. Importantly, depletion of PPP6C in SCD patient-derived cells was well tolerated, led to similar levels of HbF induction, and markedly reduced cell sickling by greater than 60%. Results from ongoing studies exploring the mechanism of PPP6C in HbF regulation will be discussed.Taken together, these data indicate that PPP6C functions in a dose-dependent manner to regulate HbF in primary erythroid cells and may serve as a therapeutic target in the treatment of SCD. DisclosuresBlobel: Fulcrum Therapeutics, Inc.: Consultancy; Pfizer: Consultancy.

Ser69 phosphorylation of TIMAP affects endothelial cell migration

Purpose/Aim: TIMAP (TGF-β-inhibited membrane-associated protein) is a regulatory subunit of protein phosphatase 1 (PP1). The N-terminal region contains a binding motif for the catalytic subunit of PP1 (PP1c) and a nuclear localization signal (NLS). Phosphorylation of TIMAP on Ser331, Ser333 and Ser337 side chains was shown to regulate the activity of the TIMAP-PP1c complex. Several studies, however, reported an additional side chain of TIMAP. Ser69 is located near to the PP1c binding motif and NLS, therefore, we hypothesized that the phosphorylation of this side chain perhaps may regulate the interaction between TIMAP and PP1c, or may affect the nuclear transport of TIMAP. Materials and Methods: To study the significance of Ser69 phosphorylation, GST-tagged or c-myc-tagged wild type, phosphomimic S69D and phosphonull S69A recombinant TIMAP proteins were expressed in bacteria or endothelial cells, respectively. Protein-protein interactions of the wild type or mutant forms of TIMAP were studied by pull-down and Western blot. Localization of TIMAP S69 mutants in pulmonary artery endothelial cells was detected by immunofluorescent staining and expression and localization of the recombinants were investigated by subcellular fractionation and Western blot. Results : Modifications of Ser69 of TIMAP had no effect on binding of PP1c, ERM or RACK1. However, S69D TIMAP showed enhanced membrane localization and an increased number of membrane protrusions were observed in the cells overexpressing this phosphomimic mutant. Furthermore, significantly faster wound healing and migration rate of the S69D mutant overexpressing cells were detected by endothelial barrier resistance measurements (ECIS). Specific interaction was shown between TIMAP and polo-like kinase 4 (PLK4), a potential kinase to phosphorylate Ser69. Conclusions: Altogether, our results indicate that Ser69 phosphorylation by PLK4 may evoke an enrichment of TIMAP in the plasma membrane region and may play an important role in endothelial cell migration without affecting the PP1c binding ability of TIMAP.

Agonist-Induced Signaling and Integrin-Mediated Function in Murine Platelets Is Distinctly Regulated By the α Isoform of the Catalytic Subunit of Protein Phosphatase 1

Platelet activation at the site of injury is promoted by agonist-induced inside-out and integrin αIIbβ3-initiated outside-in signaling. Our understanding of the agonist and integrin mediated signaling pathways has largely been driven by studies that have examined the role of protein kinases. As models of signal transduction have been refined to include protein phosphorylation and protein dephosphorylation events, the contribution of the protein phosphatases to platelet signaling and function have garnered fresh attention. This is particularly evident for the catalytic subunit of serine/threonine protein phosphatase 1 (PP1c), wherein, its role in platelet biology has been previously assessed with pharmacological agents like calyculin A and okadaic acid. Although such chemical inhibitors represent a useful tool, it lacks specificity to discriminate the different isoforms of PP1c. PP1cα represents a major platelet expressed isoform and its contribution to platelet function is not known. To explore the potential role of PP1cα isoform in platelet signaling, we generated a platelet specific PP1cα -/- mice by crossing the PP1cα floxed mice with a platelet factor 4 (PF4) cre mice. Immunoblotting studies from PP1cα -/- mice confirmed that PP1cα was lost in platelets but not in lymphocytes. A compensatory upregulation of the PP1cβ but not PP1cγ1 isoform was noticed in PP1cα -/- platelets. Loss of PP1cα decreased platelet aggregation and soluble fibrinogen binding to low dose thrombin, thromboxane analog and convulxin, suggesting that PP1cα promotes agonist-induced inside-out signaling. In contrast, PP1cα -/- platelets displayed enhanced adhesiveness to immobilized fibrinogen and increased fibrin clot retraction, processes that are dependent on integrin-mediated outside-in signaling. In a microfluidic perfusion system, whole blood from PP1cα -/- mice displayed increased thrombus formation on collagen. p38 mitogen activated protein kinase (MAPK) was activated in PP1cα -/- platelets, and p38 inhibitor blocked the increased integrin-mediated PP1cα -/- platelet functions. These observations suggest that PP1cα suppress integrin-mediated function in part by decreasing the activation of p38 MAPK. Thus, unlike the conclusion gathered from generic phosphatase inhibitors that PP1c promotes platelet function, our studies using a genetic approach indicate that PP1cα contributes to platelet signaling in a fashion that is specific to the mode of signaling. Specifically, PP1cα promotes platelet agonist-induced inside-out signaling and suppress integrin-mediated outside-in signaling. This observation suggests that chemical activators of PP1cα isoform could serve as a potential antithrombotic agent. DisclosuresNo relevant conflicts of interest to declare.

Reg1 and Snf1 regulate stress-induced relocalization of protein phosphatase-1 to cytoplasmic granules.

The compartmentalization of cellular function is achieved largely through the existence of membrane-bound organelles. However, recent work suggests a novel mechanism of compartmentalization mediated by membraneless structures that have liquid droplet-like properties and arise through phase separation. Cytoplasmic stress granules (SGs) are the best characterized and are induced by various stressors including arsenite, heat shock, and glucose deprivation. Current models suggest that SGs play an important role in protein homeostasis by mediating reversible translation attenuation. Protein phosphatase-1 (PP1) is a central cellular regulator responsible for most serine/threonine dephosphorylation. Here, we show that upon arsenite stress, PP1's catalytic subunit Glc7 relocalizes to punctate cytoplasmic granules. This altered localization requires PP1's recently described maturation pathway mediated by the multifunctional ATPase Cdc48 and PP1's regulatory subunit Ypi1. Glc7 relocalization is mediated by its regulatory subunit Reg1 and its target Snf1, the AMP-dependent protein kinase. Surprisingly, Glc7 granules are highly specific to arsenite and appear distinct from canonical SGs. Arsenite induces potent translational inhibition, and translational recovery is strongly dependent on Glc7, but independent of Glc7's well-established role in regulating eIF2α. These results suggest a novel form of stress-induced cytoplasmic granule and a new mode of translational control by Glc7.

Use of Mitotic Protein Kinase Inhibitors and Phospho-Specific Antibodies to Monitor Protein Phosphorylation During the Cell Cycle.

Reversible protein phosphorylation regulates the transitions between different phases of the cell cycle ensuring proper segregation of the duplicated genome into two daughter cells. Protein kinases and protein phosphatases establish the appropriate phosphorylation stoichiometries in diverse substrates maintaining genomic stability as a cell undergoes this complex process. Along with regulating common substrates, these opposing enzymes regulate one another by fine-tuning each other's activity both spatially and temporally throughout mitosis. Protein phosphatase catalytic subunits work together with regulatory proteins, which control their localization, activity, and specificity. Protein phosphatase 1 (PP1) recognizes its regulatory proteins via a short linear interaction motif (SLIM) called the "RVxF" motif. A subset of proteins with these "RVxF" motifs contain a phosphorylatable amino acid (S/T) at the 'x' position.Here, we describe methods to generate, affinity purify and utilize phospho-specific antibodies to monitor phosphorylation sites during the cell cycle and the appropriate use of mitotic kinase inhibitors. More specifically, we employ phospho-specific antibodies, which recognize phosphorylated RVp[S/T]F motif-containing proteins, to monitor the phosphorylation status of these motifs throughout the cell cycle. Furthermore, we use mitotic kinase inhibitors to examine the effect of kinase inhibition on the phosphorylation status of multiple RV[S/T]F motifs using these phospho-specific antibodies.