Protein Phosphatase Research Articles

The cellular protein phosphatase 2A is a crucial host factor for Marburg virus transcription.

Our study elucidates the crucial role of host protein phosphatase 2A (PP2A) in Marburg virus (MARV) transcription. The regulatory subunit B56 of PP2A facilitates VP30 dephosphorylation, and hence transcription activation, via binding to NP. Our results, together with previous data, reveal a conserved mechanism of filovirus VP30 dephosphorylation by host factor PP2A at the NP interface and provide novel insights into potential pan-filovirus therapies.

C-TERMINAL DOMAIN PHOSPHATASE-LIKE 1 promotes flowering with TAF15b by repressing the floral repressor gene FLOWERING LOCUS C

Arabidopsis TBP-ASSOCIATED FACTOR15b (TAF15b) is a plant-specific component of the transcription factor IID (TFIID) complex. TAF15b is involved in the autonomous pathway for flowering and represses the transcription of FLOWERING LOCUS C (FLC), a major floral repressor in Arabidopsis. While components of the autonomous flowering pathway have been extensively studied, scant attention has been directed towards elucidating the direct transcriptional regulators responsible for repressing FLC transcription. Here, we demonstrate that C-TERMINAL DOMAIN PHOSPHATASE-LIKE 1 (CPL1) is a physical and functional partner of TAF15b, playing a role for FLC repression. CPL1 is a protein phosphatase that dephosphorylates the C-terminal domain (CTD) of RNA polymerase II (Pol II). Through the immunoprecipitation and mass spectrometry technique, we identified CPL1 as an interacting partner of TAF15b. Similar to taf15b, the cpl1 mutant showed a late-flowering phenotype caused by an increase in FLC levels. Additionally, the increase in cpl1 was correlated with the enrichment of phosphorylated Pol II in the FLC chromatin, as expected. We also discovered that CPL1 and TAF15b share additional common target genes through transcriptome analysis. These results suggest that TAF15b and CPL1 cooperatively repress transcription through the dephosphorylation of Pol II, especially at the FLC locus.

Spatiotemporal regulation of MELK during mitosis.

Maternal Embryonic Leucine Zipper Kinase (MELK) has been studied intensively in recent years due to its overexpression in multiple cancers. However, the cell biology of MELK remains less characterized despite its well-documented association with mitosis. Here we report a distinctive pattern of human MELK that translocates from the cytoplasm to cell cortex within 3min of anaphase onset. The cortex association lasts about 30min till telophase. The spatiotemporal specific localization of MELK depends on the interaction between its Threonine-Proline (TP) rich domain and kinase associated 1 (KA1) domain, which is regulated by CDK1 kinase and PP4 protein phosphatase. KA1 domains are known to regulate kinase activities through various intramolecular interactions. Our results revealed a new role for KA1 domain to control subcellular localization of a protein kinase.

Abstract B092: PP2A activation alters macropinosome processing in pancreatic cancer cells leading to metabolic stress and cancer cell death

Abstract The majority of pancreatic ductal adenocarcinoma (PDAC) patients present with late-stage, metastatic disease that is often resistant to therapeutics, resulting in the lowest survival rate of all major cancers. Metabolic rewiring in response to oncogenic signals plays a critical role in PDAC cell survival, tumor growth, and metastasis. In contrast to normal epithelial cells, these metabolic alterations make PDAC tumors dependent on glutamine and glucose for survival, highlighting a unique metabolic vulnerability that can be therapeutically exploited. However, during times of nutrient stress, PDAC cells can circumvent this vulnerability by engulfing extracellular fluids to replenish amino acids, including glutamine, in a process called, macropinocytosis. Macropinocytosis occurs downstream of oncogenic KRAS, a small GTPase that is almost universally mutated in PDAC patients. The inhibition of macropinocytosis in vivo reduces PDAC tumor growth, underscoring the importance of this pathway to cancer cell survival. However, the signaling mechanisms that regulate this process and the contribution of macropinocytic signaling to aggressive cancer phenotypes remains poorly understood. Protein phosphatase 2A (PP2A) is a heterotrimeric complex that reguates a wide variety of cell signaling pathways, inlcuding KRAS, and is commonly deregulated in human PDAC tumors. Recently, PP2A has been implicated as an important regulator of macropinocytosis, but the mechanism by which this occurs is unknown. We demosntrate that suppression of PP2A accelerates the formation of KRAS-driven precursor lesions in an in vivo mouse model of PDAC, implicating PP2A as a critical regulator of mutant KRAS phenotypes. Here, we show that acute PP2A activation prevents the fusion of macropinosomes with the lysosome, robbing PDAC cells of critical metabolic nutrients and driving cell death. Furthermore, we find PP2A associates with the lipid kinase, PIKfyve, a key regulator of macropinosome-lysosome fusion. Finally, we determined that PP2A activating compounds can function synergistically with metabolic inhibitors, support a new therapeutic strategy in this aggressive and deadly cancer. Together, our results implicate PP2A as a critical suppressor of PDAC metabolic plasticity and highlight the use of PP2A activating compounds to prevent PDAC nutrient scavenging. Citation Format: Garima Baral, Claire M Pfeffer, Indiraa Doraivel, Sara N Filippelli, Brittany N Heil, Brittany L Allen-Petersen. PP2A activation alters macropinosome processing in pancreatic cancer cells leading to metabolic stress and cancer cell death [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B092.

PPP2R5E: New gene potentially involved in specific learning disorders and myopathy

Protein phosphatase 2A (PP2A) is a family of multifunctional enzymatic complexes crucial for cellular signalling, playing a pivotal role in brain function and development. Mutations in specific genes encoding PP2A complexes have been associated with neurodevelopmental disorders with hypotonia and high risk of seizures. In the current work, we present an individual with specific learning problems, motor coordination disorders, hypotonia and behavioural issues. Although whole exome sequencing (WES) did not unveil pathogenic variants in known genes related to these symptoms, a de novo heterozygous variant Glu191Lys was identified within PPP2R5E, encoding the PP2A regulatory subunit B56ε. The novel variant was not observed in the four healthy brothers and was not detected as parental somatic mosaicism. The mutation predicted a change of charge of the mutated amino acid within a conserved LFDSEDPRER motif common to all PPP2R5 B-subunits. Biochemical assays demonstrated a decreased interaction with the PP2A A and C subunits, leading to disturbances in holoenzyme formation, and thus likely, function. For the first time, we report a potential causal link between the observed variant within the PPP2R5E gene and the symptoms manifested in the subject, spanning specific learning problems and motor coordination disorders potentially associated with myopathy.

The ameliorative potential of metformin against aluminum-induced neurotoxicity: Insights from in vitro studies.

Alzheimer's disease (AD) is increasingly recognized as a metabolic disorder, often referred to as type 3 diabetes, due to its strong association with insulin resistance. Chronic exposure to aluminum, a known neurotoxin, has been identified as a significant risk factor in the development and progression of AD. This study explores the potential of metformin, a common anti-diabetic drug, to mitigate aluminum-induced neurotoxicity in an in vitro model of AD. Our findings reveal that metformin significantly reduces oxidative stress markers such as malonaldehyde, carbonyl groups, and reactive oxygen species while enhancing antioxidant defenses. Metformin modulates critical signaling pathways, including glycogen synthase kinase 3 beta (GSK3-β)/RAC-alpha serine/threonine protein kinase (RAC-alpha serine/threonine protein kinase (Akt1)/protein phosphatase 2A (PP2A) and Wnt/β-catenin, decreasing Tau protein levels and promoting neurogenesis. These results suggest that metformin may offer a novel therapeutic approach for AD, particularly in cases where aluminum exposure is a contributing factor.

101 5-HT4 receptor-mediated vasorelaxation occurs through a cyclic mononucleotide-dependent mechanism in the isolated bovine lateral saphenous vein

Abstract Serotonin (5-HT)-mediated vasorelaxation of the isolated bovine lateral saphenous vein occurs primarily through 5-HT4 receptor activation. Vasorelaxation responses mediated through 5-HT4 have only been documented to occur in cattle and sheep and thus, may represent a novel vasorelaxation mechanism that is specific to ruminants. In tissues from other species, the 5-HT4 receptor and its associated G protein couple with adenylate cyclase. However, the mechanisms contributing to 5-HT4-mediated vasorelaxation in vascular tissue remain undefined. To better understand the mechanisms of 5-HT4-mediated vasorelaxation, lateral saphenous veins from cattle (n = 4) were collected and assessed for vasoactivity in response to increasing concentrations of a selective 5-HT4 receptor agonist (BIMU-8) in the absence and presence of selective inhibitors of downstream proteins involved with cyclic mononucleotide-mediated signaling. Isolated vessel segments were suspended in chambers of a multi-myograph containing 5 mL of continuously oxygenated Krebs-Henseleit buffer and equilibrated to 1 g tension for 90 min. After the equilibration period, vessels were exposed to dimethyl sulfoxide (DMSO; vehicle control) or selective inhibitors of downstream protein targets dissolved in DMSO (1×10-5 M) for 5 min. Vessels were pre-contracted with 1×10-4 M phenylephrine for 10 min to achieve a sustained increase in tension. After the 10-min pre-contraction period, vessels were exposed to increasing concentrations (1×10-9 to 1×10-4) of BIMU-8 non-cumulatively. Vasoactive response data were normalized as a percentage of the maximum contractile response induced by the phenylephrine pre-contraction. In the absence of selective inhibitors (control), BIMU-8 induced (P < 0.01) dose-dependent relaxations (mean -log EC50 = 6.30 M) with a 112% increase in maximal vasorelaxation activity occurring at 1×10-4 M. In the presence of the selective inhibitors for adenylate cyclase (NKY80) or guanylate cyclase (NS2028), 66% and 86% of the vasorelaxation responses to BIMU-8 were attenuated (P < 0.01), respectively. In the presence of the selective inhibitors for protein kinase A [protein kinase A inhibitor fragment (6-22) amide] or protein kinase G (Rp-8-bromo-PET-Cyclic GMPS), 66% and 97% of the maximal vasorelaxation responses to BIMU-8 were attenuated (P < 0.01), respectively. In the presence of non-selective inhibitors for Ca2+ channels and K+ channels, vasorelaxation responses to BIMU-8 were attenuated (P < 0.01). In the presence of selective inhibitors for the protein phosphatase 1 and 2A subunits of myosin light chain (calyculin A) and heat shock protein synthesis (quercetin), vasorelaxation responses to BIMU-8 were attenuated (P < 0.01). The findings of the current study demonstrate that 5-HT4-receptor mediated vasorelaxation occurs through a cyclic mononucleotide-dependent mechanism involving cross-activation of protein kinase A and G signaling. Downstream proteins involved in the signaling cascade leading to vasorelaxation include Ca2+ channels, K+ channels, myosin light chain phosphatase, and heat shock proteins. More research is needed to fully describe the cellular and molecular mechanisms responsible for 5-HT4 receptor-mediated vasorelaxation.

CAVPENET Peptide Inhibits Prostate Cancer Cells Proliferation and Migration through PP1γ-Dependent Inhibition of AKT Signaling.

Protein phosphatase 1 (PP1) complexes have emerged as promising targets for anticancer therapies. The ability of peptides to mimic PP1-docking motifs, and so modulate interactions with regulatory factors, has enabled the creation of highly selective modulators of PP1-dependent cellular processes that promote tumor growth. The major objective of this study was to develop a novel bioactive cell-penetrating peptide (bioportide), which, by mimicking the PP1-binding motif of caveolin-1 (CAV1), would regulate PP1 activity, to hinder prostate cancer (PCa) progression. The designed bioportide, herein designated CAVPENET, and a scrambled homologue, were synthesized using microwave-assisted solid-phase methodologies and evaluated using PCa cell lines. Our findings indicate that CAVPENET successfully entered PCa cells to influence both viability and migration. This tumor suppressor activity of CAVPENET was attributed to inhibition of AKT signaling, a consequence of increased PP1γ activity. This led to the suppression of glycolytic metabolism and alteration in lipid metabolism, collectively representing the primary mechanism responsible for the anticancer properties of CAVPENET. Our results underscore the potential of the designed peptide as a novel therapy for PCa patients, setting the stage for further testing in more advanced models to fully realize its therapeutic promise.

Nuclear lamin A/C phosphorylation by loss of androgen receptor leads to cancer-associated fibroblast activation

Alterations in nuclear structure and function are hallmarks of cancer cells. Little is known about these changes in Cancer-Associated Fibroblasts (CAFs), crucial components of the tumor microenvironment. Loss of the androgen receptor (AR) in human dermal fibroblasts (HDFs), which triggers early steps of CAF activation, leads to nuclear membrane changes and micronuclei formation, independent of cellular senescence. Similar changes occur in established CAFs and are reversed by restoring AR activity. AR associates with nuclear lamin A/C, and its loss causes lamin A/C nucleoplasmic redistribution. AR serves as a bridge between lamin A/C and the protein phosphatase PPP1. Loss of AR decreases lamin-PPP1 association and increases lamin A/C phosphorylation at Ser 301, a characteristic of CAFs. Phosphorylated lamin A/C at Ser 301 binds to the regulatory region of CAF effector genes of the myofibroblast subtype. Expression of a lamin A/C Ser301 phosphomimetic mutant alone can transform normal fibroblasts into tumor-promoting CAFs.

Development of luminescent probes for real-time detection of the CDK/PP2A balance during the cell cycle.

From a biochemical viewpoint, the cell cycle is controlled by the phosphorylation of cyclin-dependent kinase (CDK) substrates, and the phosphorylation level is determined by the enzymatic balance between CDK and protein phosphatase 2A (PP2A). However, the conventional techniques for analyzing protein phosphorylation using radioisotopes and antibodies involve many operational steps and take days before obtaining results, making them difficult to apply to high-throughput screening and real-time observations. In this study, we developed luminescent probes with a light intensity that changes depending on its phosphorylation state. We modified the Nano-lantern probe (Renilla luciferase-based Ca2+ probe) by introducing a CDK-substrate peptide and a phosphopeptide-binding domain into the luciferase. Our initial trial resulted in new probes that could report the CDK/PP2A balance in a purified system. Further modifications of these probes (replacing the phospho-Ser with phospho-Thr and randomly replacing its surrounding amino acids) improved the dynamic range by up to four-fold, making them practical for use in the Xenopus egg extracts system, where many physiological events can be reproduced. Taken together, our new probes enabled the monitoring of the CDK/PP2A balance in real time, and are applicable to high-throughput systems; the new probes thus appear promising for use in substrate and drug screening.

Variants in LRRC7 lead to intellectual disability, autism, aggression and abnormal eating behaviors

Members of the leucine rich repeat (LRR) and PDZ domain (LAP) protein family are essential for animal development and histogenesis. Densin-180, encoded by LRRC7, is the only LAP protein selectively expressed in neurons. Densin-180 is a postsynaptic scaffold at glutamatergic synapses, linking cytoskeletal elements with signalling proteins such as the α-subunit of Ca2+/calmodulin-dependent protein kinase II. We have previously observed an association between high impact variants in LRRC7 and Intellectual Disability; also three individual cases with variants in LRRC7 had been described. We identify here 33 individuals (one of them previously described) with a dominant neurodevelopmental disorder due to heterozygous missense or loss-of-function variants in LRRC7. The clinical spectrum involves intellectual disability, autism, ADHD, aggression and, in several cases, hyperphagia-associated obesity. A PDZ domain variant interferes with synaptic targeting of Densin-180 in primary cultured neurons. Using in vitro systems (two hybrid, BioID, coimmunoprecipitation of tagged proteins from 293T cells) we identified new candidate interaction partners for the LRR domain, including protein phosphatase 1 (PP1), and observed that variants in the LRR reduced binding to these proteins. We conclude that LRRC7 encodes a major determinant of intellectual development and behaviour.

Protein phosphatase 2Cm-regulated branched-chain amino acid catabolic defect in dorsal root ganglion neurons drives pain sensitization

Maladaptive changes of metabolic patterns in the lumbar dorsal root ganglion (DRG) are critical for nociceptive hypersensitivity genesis. The accumulation of branched-chain amino acids (BCAAs) in DRG has been implicated in mechanical allodynia and thermal hyperalgesia, but the exact mechanism is not fully understood. This study aimed to explore how BCAA catabolism in DRG modulates pain sensitization. Wildtype male mice were fed a high-fat diet (HFD) for 8 weeks. Adult PP2Cmfl/fl mice of both sexes were intrathecally injected with pAAV9-hSyn-Cre to delete the mitochondrial targeted 2 C-type serine/threonine protein phosphatase (PP2Cm) in DRG neurons. Here, we reported that BCAA catabolism was impaired in the lumbar 4–5 (L4-L5) DRGs of mice fed a high-fat diet (HFD). Conditional deletion of PP2Cm in DRG neurons led to mechanical allodynia, heat and cold hyperalgesia. Mechanistically, the genetic knockout of PP2Cm resulted in the upregulation of C-C chemokine ligand 5/C-C chemokine receptor 5 (CCL5/CCR5) axis and an increase in transient receptor potential ankyrin 1 (TRPA1) expression. Blocking the CCL5/CCR5 signaling or TRPA1 alleviated pain behaviors induced by PP2Cm deletion. Thus, targeting BCAA catabolism in DRG neurons may be a potential management strategy for pain sensitization.

Establishment and biological characteristics of a human buccal mucosa squamous cell carcinoma cell line SCC117

Objective: To establish a human buccal mucosa squamous cell carcinoma (BMSCC) cell line SCC117 in China, analyze and identify its basic biological characteristics. Methods: A 59-year-old Chinese male patient with BMSCC in the Department of Oral and Maxillofacial Surgery, Peking University School and Hospital of Stomatology in January 2011 was included in this study, his surgical specimens were primary cultured in vitro by improved tissue block culture method. The BMSCC cell line SCC117 was established after continuous passage and stable growth. The morphological characteristics of the cells were observed by light and electron microscope, and their basic biological characteristics were analyzed by growth curve, chromosome karyotype and xenotransplantation tumorigenicity in nude mice experiment. The expressions of cytokeratin (CK14), tumor-related proteins retinoblastoma tumor suppressor protein (RB), P53, E-cadherin, P21, phosphatase and tensin homolog deleted on chromosome ten (PTEN) were detected by immunohistochemical and human papilloma virus (HPV) were tested by PCR. SCC117 was identified by short tandem repeat (STR) analysis of genomic DNA. Results: SCC117, a human BMSCC cell line, had been continuously subcultured in vitro for more than 150 generations. The cells grew in polygonal mosaic and lost contact inhibition, the typical desmosomes and tensional fibrils were observed by electron microscope, and CK14 was positive by immunohistochemistry. The doubling time was 40.16 h, the chromosome mode of the cell line was concentrated between 67 and 69, hypo-triploid. All 4 nude mice inoculated with SCC117 cells developed tumors, indicating that the SCC117 cell line had the ability of xenogeneic tumorigenesis. The histopathological type of the transplanted tumor in nude mice was consistent with that of the primary tumor tissue, both of which were squamous cell carcinoma. The immunohistochemical results showed that in both human primary tumor and the transplanted tumor tissue in nude mice, RB, P53, and E-cadherin were all positive, P21 was weakly positive, while PTEN was negative. SCC117 was tested negative for the presence of HPV. STR sequence analysis showed that SCC117 cell line originated from primary tumor tissue and was not cross-contaminated by other cell lines. Conclusions: The human BMSCC cell line SCC117 was successfully established in China, which could provide a new experimental model for the study of oral SCC without HPV infection, especially BMSCC.

Reactive Oxygen species dependent increase in H3K27 acetylation by intermittent hypoxia is regulated by H3S28 phosphorylation.

Histones play a crucial role in regulating gene expression through post -translational modifications (PTMS) which include acetylation, methylation and phosphorylation. We have previously identified histone 3 acetylation (H3Kac) and methylation (H3Kme) as an early epigenetic mechanism associated with intermittent hypoxia (IH), a hallmark feature of sleep apnea. The goal of the present study was to determine the molecular mechanisms underlying IH increased H3 acetylation. IH-induced H3 acetylation was blocked by an antioxidant. Conversely, reactive oxygen species (ROS) mimetics, increased H3 acetylated protein expression similar to IH, suggesting a role for ROS. Trichostatin A (TSA), an HDAC (histone deacetylase) inhibitor mimicked IH-induced H3 acetylation under normoxic conditions, while pharmacological blockade of p300/CBP (HAT, histone acetylase) with CTK7A abolished IH-induced H3 acetylation. These results suggest that interplay between HATs and HDACs regulate ROS-dependent H3 acetylation by IH. Lysine 27 (H3K27) on H3 was one of the lysines specifically acetylated by IH and this acetylation was associated with dephsophorylation of H3 at serine 28 (H3S28). Inhibition of S28 dephosphorylation by protein phosphatase inhibitors (PIC or Calyculin A), prevented H3K27 acetylation by IH. Conversely, inhibiting K27 acetylation with CTK7A, increased S28 phosphorylation in IH-exposed cells. These findings highlight the intricate balance between H3 acetylation and phosphorylation in response to IH, shedding light on epigenetic mechanism regulating gene expression. (Supported by NIH-PO1-HL90554).

Protein phosphatase 4 maintains the survival of primordial follicles by regulating autophagy in oocytes

In mammalian ovary, the primordial follicle pool serves as the source of developing follicles and fertilizable ova. To maintain the normal length of female reproductive life, the primordial follicles must have adequate number and be kept in a quiescent state before menopause. However, the molecular mechanisms underlying primordial follicle survival are poorly understood. Here, we provide genetic evidence showing that lacking protein phosphatase 4 (PPP4) in oocytes, a member of PP2A-like subfamily, results in infertility in female mice. A large quantity of primordial follicles has been depleted around the primordial follicle pool formation phase and the ovarian reserve is exhausted at about 7 months old. Further investigation demonstrates that depletion of PPP4 causes the abnormal activation of mTOR, which suppresses autophagy in primordial follicle oocytes. The abnormal primordial follicle oocytes are eventually erased by pregranulosa cells in the manner of lysosome invading. These results show that autophagy prevents primordial follicles over loss and PPP4-mTOR pathway governs autophagy during the primordial follicle formation and dormant period.

Spontaneous and chaperone-assisted metal loading in the active site of protein phosphatase-1.

Protein phosphatase PP1 has two active-site metals (Zn2+/Fe2+) that are essential for catalysis. However, when expressed in bacteria, PP1 has two Mn2+-ions in its active site, indicating that the incorporation of Zn2+/Fe2+ depends on additional eukaryotic component(s). Here, we used purified, metal-deficient PP1 to study metal incorporation. Fe2+ was incorporated spontaneously, but Zn2+ was not. Mn2+-incorporation at physiological pH depended on the co-expression of PP1 with PPP1R2 (Inhibitor-2) or PPP1R11 (Inhibitor-3), or a pre-incubation of PP1 at pH 4. We also demonstrate that PPP1R2 and PPP1R11 are Zn2+-binding proteins but are, by themselves, not able to load PP1 with Zn2+. Our data suggest that PPP1R2 and PPP1R11 function as metal chaperones for PP1 but depend on co-chaperone(s) and/or specific modification(s) for the transfer of associated Zn2+ to PP1.

Cytosolic Factors Controlling PASTA Kinase-Dependent ReoM Phosphorylation.

Bacteria adapt the biosynthesis of their envelopes to specific growth conditions and prevailing stress factors. Peptidoglycan (PG) is the major component of the cell wall in Gram-positive bacteria, where PASTA kinases play a central role in PG biosynthesis regulation. Despite their importance for growth, cell division and antibiotic resistance, the mechanisms of PASTA kinase activation are not fully understood. ReoM, a recently discovered cytosolic phosphoprotein, is one of the main substrates of the PASTA kinase PrkA in the Gram-positive human pathogen Listeria monocytogenes. Depending on its phosphorylation, ReoM controls proteolytic stability of MurA, the first enzyme in the PG biosynthesis pathway. The late cell division protein GpsB has been implicated in PASTA kinase signalling. Consistently, we show that L. monocytogenes prkA and gpsB mutants phenocopied each other. Analysis of invivo ReoM phosphorylation confirmed GpsB as an activator of PrkA leading to the description of structural features in GpsB that are important for kinase activation. We further show that ReoM phosphorylation is growth phase-dependent and that this kinetic is reliant on the protein phosphatase PrpC. ReoM phosphorylation was inhibited in mutants with defects in MurA degradation, leading to the discovery that MurA overexpression prevented ReoM phosphorylation. Overexpressed MurA must be able to bind its substrates and interact with ReoM to exert this effect, but the extracellular PASTA domains of PrkA or MurJ flippases were not required. Our results indicate that intracellular signals control ReoM phosphorylation and extend current models describing the mechanisms of PASTA kinase activation.

PPM1D activity promotes cellular transformation by preventing senescence and cell death

Cell cycle checkpoints, oncogene-induced senescence and programmed cell death represent intrinsic barriers to tumorigenesis. Protein phosphatase magnesium-dependent 1 (PPM1D) is a negative regulator of the tumour suppressor p53 and has been implicated in termination of the DNA damage response. Here, we addressed the consequences of increased PPM1D activity resulting from the gain-of-function truncating mutations in exon 6 of the PPM1D. We show that while control cells permanently exit the cell cycle and reside in senescence in the presence of DNA damage caused by ionising radiation or replication stress induced by the active RAS oncogene, RPE1-hTERT and BJ-hTERT cells carrying the truncated PPM1D continue proliferation in the presence of DNA damage, form micronuclei and accumulate genomic rearrangements revealed by karyotyping. Further, we show that increased PPM1D activity promotes cell growth in the soft agar and formation of tumours in xenograft models. Finally, expression profiling of the transformed clones revealed dysregulation of several oncogenic and tumour suppressor pathways. Our data support the oncogenic potential of PPM1D in the context of exposure to ionising radiation and oncogene-induced replication stress.

Mechanisms underlying age-associated exacerbation of pulmonary veno-occlusive disease.

Pulmonary veno-occlusive disease (PVOD) is a rare but severe form of pulmonary hypertension characterized by the obstruction of pulmonary arteries and veins, causing increased pulmonary artery pressure and leading to right ventricular (RV) heart failure. PVOD is often resistant to conventional pulmonary arterial hypertension (PAH) treatments and has a poor prognosis, with a median survival time of 2-3 years after diagnosis. We previously showed that the administration of a chemotherapy agent mitomycin C (MMC) in rats mediates PVOD through the activation of the eukaryotic initiation factor 2 (eIF2) kinase protein kinase R (PKR) and the integrated stress response (ISR), resulting in the impairment of vascular endothelial junctional structure and barrier function. Here, we demonstrate that aged rats over 1 year exhibit more severe vascular remodeling and RV hypertrophy than young adult rats following MMC treatment. This is attributed to an age-associated elevation of basal ISR activity and depletion of protein phosphatase 1, leading to prolonged eIF2 phosphorylation and sustained ISR activation. Pharmacological blockade of PKR or ISR mitigates PVOD phenotypes in both age groups, suggesting that targeting the PKR/ISR axis could be a potential therapeutic strategy for PVOD.

Phosphorylated transcription factor PuHB40 mediates ROS-dependent anthocyanin biosynthesis in pear exposed to high light.

Plants are increasingly vulnerable to environmental stresses because of global warming and climate change. Stress-induced reactive oxygen species (ROS) accumulation results in plant cell damage, even cell death. Anthocyanins are important antioxidants that scavenge ROS to maintain ROS homeostasis. However, the mechanism underlying ROS-induced anthocyanin accumulation is unclear. In this study, we determined that the HD-Zip I family member transcription factor PuHB40 mediates ROS-dependent anthocyanin biosynthesis under high-light stress in pear (Pyrus ussuriensis). Specifically, PuHB40 induces the PuMYB123-like-PubHLH3 transcription factor complex for anthocyanin biosynthesis. The PuHB40-mediated transcriptional activation depends on its phosphorylation level, which is regulated by protein phosphatase PP2A. Elevated ROS content maintains high PuHB40 phosphorylation levels while also enhancing the PuHB40-induced PuMYB123-like transcription by decreasing the PuPP2AA2 expression, ultimately leading to increased anthocyanin biosynthesis. Our study reveals a pathway regulating the ROS-induced anthocyanin biosynthesis in pears, further clarifying the mechanism underlying the abiotic stress-induced anthocyanin biosynthesis, which may have implications for improving plant stress tolerance.