Protein Dephosphorylation Research Articles

PPM1J regulates meat quality feature and glycerophospholipids composition in broiler by modulating protein dephosphorylation

The quality of broiler meat affects consumers’ purchasing decisions. Numerous studies have shown that phosphorylation of proteins in muscle can affect muscle quality. Here, metabolomics and transcriptomics were used to systematically identify the genetic regulation of differences in meat flavor among different broiler. By constructing the meat flavor-related metabolite-gene networks, we identified that protein phosphatase magnesium/manganese-dependent 1J (PPM1J), which is known to regulate a range of biological processes by modulating reversible protein phosphorylation, was a differentially expressed gene with the highest connectivity to meat flavor-related metabolites. Gain- and loss-of-function analysis revealed that PPM1J induced muscular atrophy, improved meat quality and regulated the composition of glycerophospholipids. More importantly, phosphoproteome and metabolome results found that PPM1J participates in the regulation of meat quality feature and glycerophospholipids composition by catalyzing protein dephosphorylation. Our study provides a basis for further understanding the molecular mechanism of meat quality feature and glycerophospholipids composition in broiler.

Interaction of SARS-CoV-2 Spike protein with ACE2 induces cortical actin modulation, including dephosphorylation of ERM proteins and reduction of cortical stiffness.

Cell surface cortical actin is a regulatory target for viral infection. We aimed to investigate the effect of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection on host cell cortical stiffness, an indicator of cortical actin structure. The receptor-binding domain (RBD) of SARS-CoV-2 Spike (S) protein induced a reduction in cortical stiffness in ACE2-expressing cells. The interaction of RBD with ACE2 caused the inactivation of Ezrin/Radixin/Moesin (ERM) proteins. We further investigated the effects of the RBD of SARS-CoV-2 Omicron variants, BA.1 and BA.5. These RBDs influenced cortical stiffness depending on their affinity for ACE2. Our study provides the first evidence that the interaction of the SARS-CoV-2 S protein with ACE2 induces mechanobiological signals and attenuates the cortical actin.

Bayesian mapping of protein kinases to vasopressin-regulated phosphorylation sites in renal collecting duct.

Vasopressin controls water permeability in the renal collecting duct by regulating the water channel protein, aquaporin-2 (AQP2). Phosphoproteomic studies have identified multiple proteins that undergo phosphorylation changes in response to vasopressin. The kinases responsible for the phosphorylation of most of these sites have not been identified. Here, we use large-scale Bayesian data integration to predict the responsible kinases for 51 phosphoproteomically identified vasopressin-regulated phosphorylation sites in the renal collecting duct. To do this, we applied Bayes' rule to rank the 515 known mammalian protein kinases for each site. Bayes' rule was applied recursively to integrate each of the seven independent datasets, each time using the posterior probability vector of a given step as the prior probability vector of the next step. In total, 30 of the 33 phosphorylation sites that increase with vasopressin were predicted to be phosphorylated by protein kinase A (PKA) catalytic subunit-α, consistent with prior studies implicating PKA in vasopressin signaling. Eighteen of the vasopressin-regulated phosphorylation sites were decreased in response to vasopressin and all but three of these sites were predicted to be targets of extracellular signal-regulated kinases, ERK1 and ERK2. This result implies that ERK1 and ERK2 are inhibited in response to vasopressin V2 receptor occupation, secondary to PKA activation. The six phosphorylation sites not predicted to be phosphorylated by PKA or ERK1/2 are potential targets of other protein kinases previously implicated in aquaporin-2 regulation, including cyclin-dependent kinase 18 (CDK18), calmodulin-dependent kinase 2δ (CAMK2D), AMP-activated kinase catalytic subunit-α-1 (PRKAA1) and CDC42 binding protein kinase β (CDC42BPB).NEW & NOTEWORTHY Vasopressin regulates water transport in the renal collecting duct in part through phosphorylation or dephosphorylation of proteins that regulate aquaporin-2. Prior studies have identified 51 vasopressin-regulated phosphorylation sites in 45 proteins. This study uses Bayesian data integration techniques to combine information from multiple prior proteomics and transcriptomics studies to predict the protein kinases that phosphorylate the 51 sites. Most of the regulated sites were predicted to be phosphorylated by protein kinase A or ERK1/ERK2.

Subchronic administration of scopolamine reverses UCMS-induced behavior in mice via eEF2 protein dephosphorylation

BackgroundThe cholinergic system has been increasingly linked to the pathophysiology of mood disorders such as depression, with the potential involvement of nicotinic and/or muscarinic receptors. Conventional antidepressants usually require weeks of daily dosing to achieve a full antidepressant response. In contrast, clinical studies have shown that scopolamine, a nonselective muscarinic acetylcholine receptor antagonist, can induce potent and rapid antidepressant effects, requiring only a few days of treatment. This study aimed to examine the suitability of the unpredictable chronic mild stress (UCMS) model of depression to reproduce the above scopolamine antidepressant activity patterns.MethodsRapid and sustained antidepressant-like effects were assessed by using the splash test, sucrose preference test (SPT), tail suspension test (TST), and forced swimming test (FST) in animals undergoing the UCMS procedure and stress-naïve C57BL/6J mice. Western Blotting was used to measure tropomyosin receptor kinase B (TrkB), mammalian target of rapamycin (mTOR), eukaryotic elongation factor (eEF2) and postsynaptic density protein 95 (PSD95) levels.ResultsScopolamine induced antidepressant-like effects in a dose-dependent manner only after subchronic, but not single, administration in the UCMS model of depression in C57BL/6J mice without affecting locomotor activity. Specifically, scopolamine administered at a dose of 0.3 mg/kg for four consecutive days significantly reversed the UCMS-induced depressive-like behavior, such as apathy, anhedonia, and behavioral despair, while scopolamine, given at the same dose but only once, did not relieve the above symptoms. Scopolamine treatment was accompanied by eEF2 protein dephosphorylation and its subsequent reactivation in the prefrontal cortex (PFC).ConclusionSubchronic administration of scopolamine is needed to ameliorate UCMS-induced depressive-like behavior. The suggested mechanism of scopolamine action covers eEF2 protein activity in the PFC.

Protein Phosphatase 2ACα Regulates ATR-Mediated Endogenous DNA Damage Response Against Microcephaly.

Protein phosphatase 2A (PP2A) is an abundant heterotrimeric holoenzyme in eukaryotic cells coordinating with specific kinases to regulate spatial-temporal protein dephosphorylation in various biological processes. However, the function of PP2A in cortical neurogenesis remains largely unknown. Here, we report that neuronal-specific deletion of Pp2acα in mice displayed microcephaly, with significantly smaller brains and defective learning and memory ability. Mechanistically, neuronal Pp2acα deficiency resulted in elevated endogenous DNA damage and activation of ATR/CHK1 signaling. It was further induced by the loss of direct interaction between PP2AC and ATR as well as the function of PP2AC to dephosphorylate ATR. Importantly, ATR/CHK1 signaling dysregulation altered both the expression and activity of several critical downstream factors including P53, P21, Bcl2, and Bax, which led to decreased proliferation of cortical progenitor cells and increased apoptosis in developing cortical neurons. Taken together, our results indicate an essential function of PP2ACα in endogenous DNA damage response-mediated ATR signaling during neurogenesis, and defective PP2ACα in neurons contributes to microcephaly.

Role of Protein Phosphatases in Tumor Angiogenesis: Assessing PP1, PP2A, PP2B and PTPs Activity.

Tumor angiogenesis, the formation of new blood vessels to support tumor growth and metastasis, is a complex process regulated by a multitude of signaling pathways. Dysregulation of signaling pathways involving protein kinases has been extensively studied, but the role of protein phosphatases in angiogenesis within the tumor microenvironment remains less explored. However, among angiogenic pathways, protein phosphatases play critical roles in modulating signaling cascades. This review provides a comprehensive overview of the involvement of protein phosphatases in tumor angiogenesis, highlighting their diverse functions and mechanisms of action. Protein phosphatases are key regulators of cellular signaling pathways by catalyzing the dephosphorylation of proteins, thereby modulating their activity and function. This review aims to assess the activity of the protein tyrosine phosphatases and serine/threonine phosphatases. These phosphatases exert their effects on angiogenic signaling pathways through various mechanisms, including direct dephosphorylation of angiogenic receptors and downstream signaling molecules. Moreover, protein phosphatases also crosstalk with other signaling pathways involved in angiogenesis, further emphasizing their significance in regulating tumor vascularization, including endothelial cell survival, sprouting, and vessel maturation. In conclusion, this review underscores the pivotal role of protein phosphatases in tumor angiogenesis and accentuate their potential as therapeutic targets for anti-angiogenic therapy in cancer.

Neuronal Excitation Induces Tau Protein Dephosphorylation via Protein Phosphatase 1 Activation to Promote Its Binding with Stable Microtubules.

The tau protein is a microtubule-associated protein that promotes microtubule stabilization. The phosphorylation of the tau protein has been linked to its dissociation from microtubules. Here, we examined the relationship between neuronal depolarization activity and tau protein phosphorylation by employing model systems in culture as well as in vivo. The KCl-evoked depolarization of cultured neurons has often been used to investigate the effects of neuronal activity. We found dephosphorylation at AT8 sites (S202, T205), T212, AT180 sites (T231, S235), and S396 in KCl-simulated cultured neurons. We also found that the KCl-induced tau protein dephosphorylation increases the level of the tau protein fractionated with stable microtubules. In an in vivo experiment, we demonstrated that the exposure of mice to a new environment activates protein phosphatase 1 in the mouse hippocampus and induces tau protein dephosphorylation. We also found an increased amount of the tau protein in a stable microtubule fraction, suggesting that the dephosphorylation of the tau protein may lead to its increased microtubule association in vivo. These results suggest that the association of microtubules with tau proteins may be regulated by the tau protein phosphorylation status affected by neuronal electrical activity.

Cardiac-Specific Suppression of Valosin-Containing Protein Induces Progressive Heart Failure and Premature Mortality Correlating with Temporal Dysregulations in mTOR Complex 2 and Protein Phosphatase 1.

Valosin-containing protein (VCP), an ATPase-associated protein, is emerging as a crucial regulator in cardiac pathologies. However, the pivotal role of VCP in the heart under physiological conditions remains undetermined. In this study, we tested a hypothesis that sufficient VCP expression is required for cardiac development and physiological cardiac function. Thus, we generated a cardiac-specific VCP knockout (KO) mouse model and assessed the consequences of VCP suppression on the heart through physiological and molecular studies at baseline. Our results reveal that homozygous KO mice are embryonically lethal, whereas heterozygous KO mice with a reduction in VCP by ~40% in the heart are viable at birth but progressively develop heart failure and succumb to mortality at the age of 10 to 12 months. The suppression of VCP induced a selective activation of the mammalian target of rapamycin complex 1 (mTORC1) but not mTORC2 at the early age of 12 weeks. The prolonged suppression of VCP increased the expression (by ~2 folds) and nuclear translocation (by >4 folds) of protein phosphatase 1 (PP1), a key mediator of protein dephosphorylation, accompanied by a remarked reduction (~80%) in AKTSer473 phosphorylation in VCP KO mouse hearts at a later age but not the early stage. These temporal molecular alterations were highly associated with the progressive decline in cardiac function. Overall, our findings shed light on the essential role of VCP in the heart under physiological conditions, providing new insights into molecular mechanisms in the development of heart failure.

Loss of protein phosphatase 2A regulatory subunit PPP2R5A is associated with increased incidence of stress-induced proarrhythmia.

Protein phosphatase 2A (PP2A) is a serine/threonine-selective holoenzyme that controls Ca2+ homeostasis and contractility of the heart via dephosphorylation of regulatory proteins. In some genetically modified mouse models with increased arrhythmogenicity, a reduced expression of the regulatory subunit B56α of PP2A was found as a concomitant effect. Whether there is a general correlation between the abundance of B56α and the promotion of cardiac arrhythmogenesis remains unclear. The aim of this study was therefore to investigate the role of PP2A-B56α in the propensity for arrhythmic activity in the heart. The experimental analysis of this question has been addressed by using a mouse model with deletion of the PP2A-B56α gene, PPP2R5A (KO), in comparison to wild-type animals (WT). Evidence for arrhythmogenicity was investigated in whole animal, isolated heart and cardiomyocytes by ECG, recording of monophasic action potential (MAP) induced by programmed electrical stimulation (PES), measurement of Ca2+ transients under increased pacing frequencies and determination of total K+ channel currents (I K). ECG measurements showed a prolongation of QT time in KO vs. WT. KO mice exhibited a higher rate of premature ventricular contractions in the ECG. MAP measurements in Langendorff-perfused KO hearts showed increased episodes of ventricular tachyarrhythmia induced by PES. However, the KO hearts showed values for MAP duration that were similar to those in WT hearts. In contrast, KO showed more myocardial cells with spontaneous arrhythmogenic Ca2+ transient events compared to WT. The whole-cell patch-clamp technique applied to ventricular cardiomyocytes revealed comparable peak potassium channel current densities between KO and WT. These findings support the assumption that a decrease or even the loss of PP2A-B56α leads to an increased propensity of triggered arrhythmias. This could be based on the increased spontaneous Ca2+ tansients observed.

Protein tyrosine phosphatases: emerging role in cancer therapy resistance.

Tyrosine phosphorylation of intracellular proteins is a post-translational modification that plays a regulatory role in signal transduction during cellular events. Dephosphorylation of signal transduction proteins caused by protein tyrosine phosphatases (PTPs) contributed their role as a convergent node to mediate cross-talk between signaling pathways. In the context of cancer, PTP-mediated pathways have been identified as signaling hubs that enabled cancer cells to mitigate stress induced by clinical therapy. This is achieved by the promotion of constitutive activation of growth-stimulatory signaling pathways or modulation of the immune-suppressive tumor microenvironment. Preclinical evidences suggested that anticancer drugs will release their greatest therapeutic potency when combined with PTP inhibitors, reversing drug resistance that was responsible for clinical failures during cancer therapy. This review aimed to elaborate recent insights that supported the involvement of PTP-mediated pathways in the development of resistance to targeted therapy and immune-checkpoint therapy. This review proposed the notion of PTP inhibition in anticancer combination therapy as a potential strategy in clinic to achieve long-term tumor regression. Ongoing clinical trials are currently underway to assess the safety and efficacy of combination therapy in advanced-stage tumors.

Retinoic acid signalling inhibits myogenesis by blocking MYOD translation in pig skeletal muscle cells

Vitamin A is an essential nutrient in animals, playing important roles in animal health. In the pig industry, proper supplementation of vitamin A in the feed can improve pork production performance, while deficiency or excessive intake can lead to growth retardation or disease. However, the specific molecular mechanisms through which vitamin A operates on pig skeletal muscle growth as well as muscle stem cell function remain unexplored. Therefore, in this study, we isolated the pig primary skeletal muscle stem cells (pMuSCs) and treated with retinoic acid (RA), the natural metabolite of vitamin A, and then examined the myogenic capacity of pMuSCs via immunostaining, real-time PCR, CCK8 and western-blot analysis. Unexpectedly, the RA caused a significant decrease in the proliferation and differentiation of pMuSCs. Mechanistically, the RA addition induced the activation of retinoic acid receptor gamma (RARγ), which inhibited the myogenesis through the blockage of protein translation of the master myogenic regulator myogenic differentiation 1 gene (MYOD). Specifically, RARγ inactivate AKT kinase (AKT) signalling and lead to dephosphorylation of eukaryotic translation initiation factor 4E binding protein 1 (eIF4EBP1), which in turn repress the eukaryotic translation initiation factor 4E (eIF4E) complex and block mRNA translation of MYOD. Inhibition of AKT could rescue the myogenic defects of RA-treated pMuSCs. Our findings revealed that retinoid acid signalling inhibits the skeletal muscle stem cell proliferation and differentiation in pigs. Therefore, the vitamin A supplement in the feedstuff should be cautiously optimized to avoid the potential adverse consequences on muscle development associated with the excessive levels of retinoic acid.

AKAP2 plays a pivotal role in renal acid-base homeostasis during metabolic acidosis

Reversible phosphorylation and dephosphorylation of kidney proteins modulate their activities and localization and play a central role in maintaining fluid and acid-base homeostasis. The proximal tubule and the intercalated cells of the nephron are main sites of acid-base balance. It was previously demonstrated that some kidney transporters involved in the pH homeostasis were regulated by the c-AMP/Protein Kinase A (PKA) signaling pathway.The A-Kinase anchoring protein 2 (AKAP2), is a scaffold protein that binds to the Protein Kinase A (PKA) allowing the phosphorylation of target proteins but is also known to interact with Protein Phosphatase 1 and consequently contributes to protein dephosphorylation. In this study, we hypothesize that AKAP2 regulates the activity and localization of renal transporters during metabolic acidosis. An inducible and nephron-specific Akap2Pax8/LC1 knockout mouse model was generated. Fluorescent immunostaining showed that AKAP2 is present in the tubules mainly at the apical plasma membrane. AKAP2 knock-out mice in normal conditions presented an acidic urine pH and polyuria compared to control mice. Ex-vivo data from micro-perfused CCDs demonstrated a higher Na+ reabsorption and K+ secretion in AKAP2 KO mice. This suggests its importance in maintaining acid-base balance even under physiological conditions. Moreover, when treated with an ammonium chloride diet, KO mice were sensitive to the acid load; showed a lower blood pH, hyperchloremia, a lower bicarbonate level and a two-fold higher blood ammonia. The sensitivity of the KO mice further supports the role of AKAP2 in acid-base homeostasis. Although we think this is due to renal acid-base transporters activity deregulation, the consideration of a potential defect in water reabsorption mechanisms contributing to acidosis is also valid. Our study provides valuable insights into the role of AKAP2 in maintaining fluid and acid-base homeostasis in the kidneys. The findings open avenues for further research into the molecular mechanisms underlying AKAP2-mediated regulation of renal transporters and its impact on acid-base balance. Swiss National Science Foundation (SNSF). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Tracking Inhibition of Human Small C-Terminal Domain Phosphatase 1 Using 193 nm Ultraviolet Photodissociation Mass Spectrometry.

Working in tandem with kinases via a dynamic interplay of phosphorylation and dephosphorylation of proteins, phosphatases regulate many cellular processes and thus represent compelling therapeutic targets. Here we leverage ultraviolet photodissociation to shed light on the binding characteristics of two covalent phosphatase inhibitors, T65 and rabeprazole, and their respective interactions with the human small C-terminal domain phosphatase 1 (SCP1) and its single-point mutant C181A, in which a nonreactive alanine replaces one key reactive cysteine. Top-down MS/MS analysis is used to localize the binding of T65 and rabeprazole on the two proteins and estimate the relative reactivities of each cysteine residue.

The Odds of Protein Translation Control Under Stress.

Physical or chemical stress is commonly known to inhibit protein translation at the cellular level. Since the process of protein translation requires catalysis by a multi-component machinery containing eukaryotic initiation factors (eIFs) and ribosomes in a sequence of reactions, how the process fails to proceed and whether certain genes can escape such blockade have provoked research efforts. Lines of evidence have demonstrated that phosphorylation of eIF4E or dephosphorylation of 4E-binding proteins (4E-BPs) prevents the formation of the eukaryotic translation initiation factor 4F (eIF4F) complex, whereas phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2α) due to activation of heme-regulated inhibitor (HRI), general control nonderepressible 2 (GCN2), protein kinase RNA-like endoplasmic reticulum kinase (PERK), or protein kinase R (PKR) by a diverse array of stressors prevents eIF2-GTP-tRNAiMet ternary complex assembly. These signal the abandonment of translation initiation via 5'-7-methylguanine (m7G) cap recognition by eIF4E. Stress can promote cleavage of tRNAs, impediment of rRNA processing, changes in the epitranscriptomic landscape, ribosome stalling or collision, activation of ribosomal surveillance systems, and assembly of the stress granules. Although these events contribute to the general inhibition of protein translation, a few proteins can bypass such negativity and become translated selectively. Such selective protein translation is primarily m7G cap independent through the integrated stress response or Internal Ribosomal Entry Site (IRES). The newly synthesized proteins often influence cell fate, facilitate cell survival, and build endogenous defense. Insights into the general inhibition of protein translation and selective translation of specific proteins will advance our understanding of the etiology or progression of human diseases involving cellular stress from viral infection or inflammation to myocardial infarction, stroke, or neurodegenerative disease. Antioxid. Redox Signal. 40, 943-947.

Significance of hepatitis B virus capsid dephosphorylation via polymerase

BackgroundIt is generally believed that hepatitis B virus (HBV) core protein (HBc) dephosphorylation (de-P) is important for viral DNA synthesis and virion secretion. HBV polymerase contains four domains for terminal protein, spacer, reverse transcriptase, and RNase H activities.MethodsHBV Polymerase mutants were transfected into HuH-7 cells and assayed for replication and HBc de-P by the Phos-tag gel analysis. Infection assay was performed by using a HepG2-NTCP-AS2 cell line.ResultsHere, we show that a novel phosphatase activity responsible for HBc de-P can be mapped to the C-terminal domain of the polymerase overlapping with the RNase H domain. Surprisingly, while HBc de-P is crucial for viral infectivity, it is essential for neither viral DNA synthesis nor virion secretion. The potential origin, significance, and mechanism of this polymerase-associated phosphatase activity are discussed in the context of an electrostatic homeostasis model. The Phos-tag gel analysis revealed an intriguing pattern of “bipolar distribution” of phosphorylated HBc and a de-P HBc doublet.ConclusionsIt remains unknown if such a polymerase-associated phosphatase activity can be found in other related biosystems. This polymerase-associated phosphatase activity could be a druggable target in clinical therapy for hepatitis B.

Unveiling the mitophagy puzzle in non-alcoholic fatty liver disease (NAFLD): Six hub genes for early diagnosis and immune modulatory roles

BackgroundNon-alcoholic fatty liver disease (NAFLD) stands as a predominant chronic liver ailment globally, yet its pathogenesis remains elusive. This study aims to identify Hub mitophagy-related genes (MRGs), and explore the underlying pathological mechanisms through which these hub genes regulate NAFLD. MethodsA total of 3 datasets were acquired from the GEO database and integrated to identify differentially expressed genes (DEGs) in NAFLD and perform Gene Set Enrichment Analysis (GSEA). By intersecting DEGs with MRGs, mitophagy-related differentially expressed genes (MRDEGs) were obtained. Then, hub MRGs with diagnostic biomarker capability for NAFLD were screened and a diagnostic prediction model was constructed and assessed using Nomogram, Decision Curve Analysis (DCA), and ROC curves. Functional enrichment analysis was conducted on the identified hub genes to explore their biological significance. Additionally, regulatory networks were constructed using databases. NAFLD was stratified into high and low-risk groups based on the Riskscore from the diagnostic prediction model. Furthermore, single-sample gene set enrichment analysis (ssGSEA) and CIBERSORT algorithms were employed to analyze immune cell infiltration patterns and the relationship between Hub MRGs and immune cells. ResultsThe integrated dataset comprised 122 NAFLD samples and 31 control samples. After screening, 18 MRDEGs were identified. Subsequently, six hub MRGs (NR4A1, PPP2R2A, P4HA1, TUBB6, DUSP1, NAMPT) with diagnostic potential were selected through WGCNA, logistic regression, SVM, RF, and LASSO models, all significantly downregulated in NAFLD samples compared to the control group. A diagnostic prediction model based on these six genes demonstrated robust predictive performance. Functional enrichment analysis of the six hub genes revealed involvement in processes such as protein phosphorylation or dephosphorylation. Correlation analysis demonstrated a significant association between hub MRGs and infiltrating immune cells. ConclusionWe identified six hub MRGs in NAFLD and constructed a diagnostic prediction model based on these six genes, applicable for early NAFLD diagnosis. These genes may participate in regulating NAFLD progression through the modulation of mitophagy and immune activation. Our findings may contribute to subsequent clinical and basic research on NAFLD.

EGFR targeting PhosTACs as a dual inhibitory approach reveals differential downstream signaling.

We recently developed a heterobifunctional approach [phosphorylation targeting chimeras (PhosTACs)] to achieve the targeted protein dephosphorylation (TPDephos). Here, we envisioned combining the inhibitory effects of receptor tyrosine kinase inhibitors (RTKIs) and the active dephosphorylation by phosphatases to achieve dual inhibition of kinases. We report an example of tyrosine phosphatase-based TPDephos and the effective epidermal growth factor receptor (EGFR) tyrosine dephosphorylation. We also used phosphoproteomic approaches to study the signaling transductions affected by PhosTAC-related molecules at the proteome-wide level. This work demonstrated the differential signaling pathways inhibited by PhosTAC compared with the TKI, gefitinib. Moreover, a covalent PhosTAC selective for mutated EGFR was developed and showed its inhibitory potential for dysregulated EGFR. Last, EGFR PhosTACs, consistent with EGFR dephosphorylation profiles, induced apoptosis and inhibited cancer cell viability during prolonged PhosTAC treatment. PhosTACs showcased their potential of modulating RTKs activity, expanding the scope of bifunctional molecule utility.

DUSP3 modulates IRES-dependent translation of mRNAs through dephosphorylation of the HNRNPC protein in cells under genotoxic stimulus.

The dual-specificity phosphatase 3 (DUSP3) regulates cell cycle progression, proliferation, senescence, and DNA repair pathways under genotoxic stress. This phosphatase interacts with HNRNPC protein suggesting an involvement in the regulation of HNRNPC-ribonucleoprotein complex stability. In this work, we investigate the impact of DUSP3 depletion on functions of HNRNPC aiming to suggest new roles for this enzyme. The DUSP3 knockdown results in the tyrosine hyperphosphorylation state of HNRNPC increasing its RNA binding ability. HNRNPC is present in the cytoplasm where it interacts with IRES trans-acting factors (ITAF) complex, which recruits the 40S ribosome on mRNA during protein synthesis, thus facilitating the translation of mRNAs containing IRES sequence in response to specific stimuli. In accordance with that, we found that DUSP3 is present in the 40S, monosomes and polysomes interacting with HNRNPC, just like other previously identified DUSP3 substrates/interacting partners such as PABP and NCL proteins. By downregulating DUSP3, Tyr-phosphorylated HNRNPC preferentially binds to IRES-containing mRNAs within ITAF complexes preferentially in synchronized or stressed cells, as evidenced by the higher levels of proteins such as c-MYC and XIAP, but not their mRNAs such as measured by qPCR. Under DUSP3 absence, this increased phosphorylated-HNRNPC/RNA interaction reduces HNRNPC-p53 binding in presence of RNAs releasing p53 for specialized cellular responses. Similarly, to HNRNPC, PABP physically interacts with DUSP3 in an RNA-dependent manner. Overall, DUSP3 can modulate cellular responses to genotoxic stimuli at the translational level by maintaining the stability of HNRNPC-ITAF complexes and regulating the intensity and specificity of RNA interactions with RRM-domain proteins.

The PP2A-like phosphatase Ppg1 mediates assembly of the Far complex to balance gluconeogenic outputs and enables adaptation to glucose depletion.

To sustain growth in changing nutrient conditions, cells reorganize outputs of metabolic networks and appropriately reallocate resources. Signaling by reversible protein phosphorylation can control such metabolic adaptations. In contrast to kinases, the functions of phosphatases that enable metabolic adaptation as glucose depletes are poorly studied. Using a Saccharomyces cerevisiae deletion screen, we identified the PP2A-like phosphatase Ppg1 as required for appropriate carbon allocations towards gluconeogenic outputs-trehalose, glycogen, UDP-glucose, UDP-GlcNAc-after glucose depletion. This Ppg1 function is mediated via regulation of the assembly of the Far complex-a multi-subunit complex that tethers to the ER and mitochondrial outer membranes forming localized signaling hubs. The Far complex assembly is Ppg1 catalytic activity-dependent. Ppg1 regulates the phosphorylation status of multiple ser/thr residues on Far11 to enable the proper assembly of the Far complex. The assembled Far complex is required to maintain gluconeogenic outputs after glucose depletion. Glucose in turn regulates Far complex amounts. This Ppg1-mediated Far complex assembly, and Ppg1-Far complex dependent control of gluconeogenic outputs enables adaptive growth under glucose depletion. Our study illustrates how protein dephosphorylation is required for the assembly of a multi-protein scaffold present in localized cytosolic pools, to thereby alter gluconeogenic flux and enable cells to metabolically adapt to nutrient fluctuations.

PPP2R1B abolishes colorectal cancer liver metastasis and sensitizes Oxaliplatin by inhibiting MAPK/ERK signaling pathway

BackgroundPatients with colorectal cancer (CRC) with liver metastasis or drug resistance have a poor prognosis. Previous research has demonstrated that PPP2R1B inactivation results in the development of CRC. However, the role of PPP2R1B in CRC metastasis and drug resistance is unclear.MethodsVenny 2.1 was used to determine the intersection between survival-related differentially expressed genes (DEGs) and liver metastasis-related DEGs according to RNA-seq data from The Cancer Genome Atlas (TCGA) and the GEO database (GSE179979). LC‒MS/MS and coimmunoprecipitation were performed to predict and verify the substrate protein of PPP2R1B. Gene Set Variation Analysis (GSVA) was subsequently utilized to assess pathway enrichment levels. The predictive performance of PPP2R1B was assessed by regression analysis, Kaplan–Meier (KM) survival analysis and drug sensitivity analysis. Immunohistochemistry (IHC), qRT-PCR and western blotting were performed to measure the expression levels of related mRNAs or proteins. Biological features were evaluated by wound healing, cell migration and invasion assays and CCK-8 assays. A mouse spleen infection liver metastasis model was generated to confirm the role of PPP2R1B in the progression of liver metastasis in vivo.ResultsAccording to bioinformatics analysis, PPP2R1B is significantly associated with liver metastasis and survival in CRC patients, and these findings were further verified via immunohistochemistry (IHC), qPCR and Western blotting. Pathway enrichment and LC‒MS/MS analysis revealed that PPP2R1B is negatively associated with the MAPK/ERK signalling pathway. Additionally, PD98059, a MAPK/ERK pathway inhibitor, inhibited EMT in vitro by reversing the changes in key proteins involved in EMT signalling (ZEB1, E-cadherin and Snail) and ERK/MAPK signalling (p-ERK) mediated by PPP2R1B. Oxaliplatin sensitivity prediction and validation revealed that PPP2R1B silencing decreased Oxaliplatin chemosensitivity, and these effects were reversed by PD98059 treatment. Moreover, PPP2R1B was coimmunoprecipitated with p-ERK in vitro. A negative correlation between PPP2R1B and p-ERK expression was also observed in clinical CRC samples, and the low PPP2R1B/high p-ERK coexpression pattern indicated a poor prognosis in CRC patients. In vivo, PPP2R1B silencing significantly promoted liver metastasis.ConclusionsThis study revealed that PPP2R1B induces dephosphorylation of the p-ERK protein, inhibits liver metastasis and increases Oxaliplatin sensitivity in CRC patients and could be a potential candidate for therapeutic application in CRC.