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International Journal of Biological... 2023Numerous mitochondrial abnormalities are reported to result from excessive inflammation during endotoxemia. Prohibitin 2 (PHB2) and phosphoglycerate mutase 5 (Pgam5)...
Numerous mitochondrial abnormalities are reported to result from excessive inflammation during endotoxemia. Prohibitin 2 (PHB2) and phosphoglycerate mutase 5 (Pgam5) have been associated with altered mitochondrial homeostasis in several cardiovascular diseases; however, their role in endotoxemia-related myocardial dysfunction has not been explored. Our experiments were aimed to evaluate the potential contribution of Pgam5 and PHB2 to endotoxemia-induced mitochondrial dysfunction in cardiomyocytes, with a focus on two endogenous protective programs that sustain mitochondrial integrity, namely mitophagy and the mitochondrial unfolded protein response (UPR). We found that PHB2 transgenic mice are resistant to endotoxemia-mediated myocardial depression and mitochondrial damage. Our assays indicated that PHB2 overexpression activates mitophagy and the UPR, which maintains mitochondrial metabolism, prevents oxidative stress injury, and enhances cardiomyocyte viability. Molecular analyses further showed that Pgam5 binds to and dephosphorylates PHB2, resulting in cytosolic translocation of mitochondrial PHB2. Silencing of Pgam5 or transfection of a phosphorylated PHB2 mutant in mouse HL-1 cardiomyocytes prevented the loss of mitochondrially-localized PHB2 and activated mitophagy and UPR in the presence of LPS. Notably, cardiomyocyte-specific deletion of Pgam5 attenuated LPS-mediated myocardial dysfunction and preserved cardiomyocyte viability. These findings suggest that Pgam5/PHB2 signaling and mitophagy/UPR are potential targets for the treatment of endotoxemia-related cardiac dysfunction.
Topics: Animals; Mice; Endotoxemia; Lipopolysaccharides; Mitophagy; Phosphoprotein Phosphatases; Prohibitins; Unfolded Protein Response
PubMed: 37781037
DOI: 10.7150/ijbs.85767 -
Acta Pharmacologica Sinica Jan 2024Acute kidney injury (AKI) is a worldwide public health problem characterized by the massive loss of tubular cells. However, the precise mechanism for initiating tubular...
Acute kidney injury (AKI) is a worldwide public health problem characterized by the massive loss of tubular cells. However, the precise mechanism for initiating tubular cell death has not been fully elucidated. Here, we reported that phosphoglycerate mutase 5 (PGAM5) was upregulated in renal tubular epithelial cells during ischaemia/reperfusion or cisplatin-induced AKI in mice. PGAM5 knockout significantly alleviated the activation of the mitochondria-dependent apoptosis pathway and tubular apoptosis. Apoptosis inhibitors alleviated the activation of the mitochondria-dependent apoptosis pathway. Mechanistically, as a protein phosphatase, PGAM5 could dephosphorylate Bax and facilitate Bax translocation to the mitochondrial membrane. The translocation of Bax to mitochondria increased membrane permeability, decreased mitochondrial membrane potential and facilitated the release of mitochondrial cytochrome c (Cyt c) into the cytoplasm. Knockdown of Bax attenuated PGAM5 overexpression-induced Cyt c release and tubular cell apoptosis. Our results demonstrated that the increase in PGAM5-mediated Bax dephosphorylation and mitochondrial translocation was implicated in the development of AKI by initiating mitochondrial Cyt c release and activating the mitochondria-dependent apoptosis pathway. Targeting this axis might be beneficial for alleviating AKI.
Topics: Mice; Animals; Cytochromes c; Phosphoglycerate Mutase; bcl-2-Associated X Protein; Apoptosis; Mitochondria; Acute Kidney Injury; Carrier Proteins; Phosphoprotein Phosphatases
PubMed: 37684381
DOI: 10.1038/s41401-023-01151-1 -
Advances in Cancer Research 2021Protein Tyrosine Phosphatases reverse cellular signals initiated by growth factors receptors and other tyrosine kinases by dephosphorylating phosphotyrosine on target... (Review)
Review
Protein Tyrosine Phosphatases reverse cellular signals initiated by growth factors receptors and other tyrosine kinases by dephosphorylating phosphotyrosine on target proteins. The activity of these enzymes is crucial for maintaining cell homeostasis, yet these enzymes have been often dismissed as humble house-keeping proteins. Understandably, mutations and changes in expression patterns of Protein Tyrosine Phosphatases are implicated in tumorigenesis and various carcinomas. The conserved nature of their catalytic domains makes drug discovery a challenging pursuit. In this review, we focus on describing the various classes of Protein Tyrosine Phosphatases and their catalytic domains. We also summarize their role in cancer and neurodegenerative diseases using specific members as the model system. Finally, we explain the dichotomy in the biological role of catalytically active vs the pseudoenzyme forms of Protein Tyrosine Phosphatases in the context of their membrane bound receptor forms. This chapter aims to provide a current understanding of these proteins, in the background of their foundational past research.
Topics: Humans; Neoplasms; Phosphorylation; Protein Tyrosine Phosphatases; Protein-Tyrosine Kinases; Signal Transduction
PubMed: 34353440
DOI: 10.1016/bs.acr.2021.06.001 -
The Journal of Biological Chemistry Apr 2024FOXO1 is a transcription factor and potential tumor suppressor that is negatively regulated downstream of PI3K-PKB/AKT signaling. Paradoxically, FOXO also promotes tumor...
FOXO1 is a transcription factor and potential tumor suppressor that is negatively regulated downstream of PI3K-PKB/AKT signaling. Paradoxically, FOXO also promotes tumor growth, but the detailed mechanisms behind this role of FOXO are not fully understood. In this study, we revealed a molecular cascade by which the Thr24 residue of FOXO1 is phosphorylated by AKT and is dephosphorylated by calcineurin, which is a Ca-dependent protein phosphatase. Curiously, single nucleotide somatic mutations of FOXO1 in cancer occur frequently at and near Thr24. Using a calcineurin inhibitor and shRNA directed against calcineurin, we revealed that calcineurin-mediated dephosphorylation of Thr24 regulates FOXO1 protein stability. We also found that FOXO1 binds to the promoter region of MDM2 and activates transcription, which in turn promotes MDM2-mediated ubiquitination and degradation of p53. FOXO3a and FOXO4 are shown to control p53 activity; however, the significance of FOXO1 in p53 regulation remains largely unknown. Supporting this notion, FOXO1 depletion increased p53 and p21 protein levels in association with the inhibition of cell proliferation. Taken together, these results indicate that FOXO1 is stabilized by calcineurin-mediated dephosphorylation and that FOXO1 supports cancer cell proliferation by promoting MDM2 transcription and subsequent p53 degradation.
Topics: Proto-Oncogene Proteins c-mdm2; Humans; Tumor Suppressor Protein p53; Forkhead Box Protein O1; Calcineurin; Proteolysis; Phosphorylation; Cell Proliferation; Ubiquitination; Cell Line, Tumor; Neoplasms; Forkhead Transcription Factors; Proto-Oncogene Proteins c-akt; Protein Stability
PubMed: 38519029
DOI: 10.1016/j.jbc.2024.107209 -
Advances in Biological Regulation May 2023Cellular functions are regulated by signal transduction pathway networks consisting of protein-modifying enzymes that control the activity of many downstream proteins.... (Review)
Review
Cellular functions are regulated by signal transduction pathway networks consisting of protein-modifying enzymes that control the activity of many downstream proteins. Protein kinases and phosphatases regulate gene expression by reversible phosphorylation of transcriptional factors, which are their direct substrates. Casein kinase II (CK2) is a serine/threonine kinase that phosphorylates a large number of proteins that have critical roles in cellular proliferation, metabolism and survival. Altered function of CK2 has been associated with malignant transformation, immunological disorders and other types of diseases. Protein phosphatase 1 (PP1) is a serine/threonine phosphatase, which regulates the phosphorylation status of many proteins that are essential for cellular functions. IKAROS is a DNA-binding protein, which functions as a regulator of gene transcription in hematopoietic cells. CK2 directly phosphorylates IKAROS at multiple phosphosites which determines IKAROS activity as a regulator of gene expression. PP1 binds to IKAROS via the PP1-consensus recognition site and dephosphorylates serine/threonine residues that are phosphorylated by CK2. Thus, the interplay between CK2 and PP1 signaling pathways have opposing effects on the phosphorylation status of their mutual substrate - IKAROS. This review summarizes the effects of CK2 and PP1 on IKAROS role in regulation of gene expression and its function as a tumor suppressor in leukemia.
Topics: Humans; Signal Transduction; Casein Kinase II; Genes, Tumor Suppressor; Leukemia; Phosphorylation; Gene Expression Regulation
PubMed: 36621151
DOI: 10.1016/j.jbior.2022.100942 -
Molecular Cell Dec 2023Transcription termination by RNA polymerase II (RNA Pol II) is linked to RNA 3' end processing by the cleavage and polyadenylation factor (CPF or CPSF). CPF contains...
Transcription termination by RNA polymerase II (RNA Pol II) is linked to RNA 3' end processing by the cleavage and polyadenylation factor (CPF or CPSF). CPF contains endonuclease, poly(A) polymerase, and protein phosphatase activities, which cleave and polyadenylate pre-mRNAs and dephosphorylate RNA Pol II to control transcription. Exactly how the RNA 3' end processing machinery is coupled to transcription remains unclear. Here, we combine in vitro reconstitution, structural studies, and genome-wide analyses to show that yeast CPF physically and functionally interacts with RNA Pol II. Surprisingly, CPF-mediated dephosphorylation promotes the formation of an RNA Pol II stalk-to-stalk homodimer in vitro. This dimer is compatible with transcription but not with the binding of transcription elongation factors. Disruption of the dimerization interface in cells causes transcription defects, including altered RNA Pol II abundance on protein-coding genes, tRNA genes, and intergenic regions. We hypothesize that RNA Pol II dimerization may provide a mechanistic basis for the allosteric model of transcription termination.
Topics: RNA Polymerase II; Saccharomyces cerevisiae Proteins; Genome-Wide Association Study; Transcription, Genetic; Saccharomyces cerevisiae; RNA 3' End Processing
PubMed: 38029752
DOI: 10.1016/j.molcel.2023.11.004 -
Cells Feb 2024Post-translational modifications (PTMs) are crucial mechanisms that underlie the intricacies of biological systems and disease mechanisms. This review focuses on the... (Review)
Review
Post-translational modifications (PTMs) are crucial mechanisms that underlie the intricacies of biological systems and disease mechanisms. This review focuses on the latest advancements in the design of heterobifunctional small molecules that hijack PTM machineries for target-specific modifications in living systems. A key innovation in this field is the development of proteolysis-targeting chimeras (PROTACs), which promote the ubiquitination of target proteins for proteasomal degradation. The past decade has seen several adaptations of the PROTAC concept to facilitate targeted (de)phosphorylation and acetylation. Protein fusion tags have been particularly vital in these proof-of-concept studies, aiding in the investigation of the functional roles of post-translationally modified proteins linked to diseases. This overview delves into protein-tagging strategies that enable the targeted modulation of ubiquitination, phosphorylation, and acetylation, emphasizing the synergies and challenges of integrating heterobifunctional molecules with protein tags in PTM research. Despite significant progress, many PTMs remain to be explored, and protein tag-assisted PTM-inducing chimeras will continue to play an important role in understanding the fundamental roles of protein PTMs and in exploring the therapeutic potential of manipulating protein modifications, particularly for targets not yet addressed by existing drugs.
Topics: Ubiquitination; Protein Processing, Post-Translational; Phosphorylation; Proteins
PubMed: 38474390
DOI: 10.3390/cells13050426 -
Cell Death & Disease Jan 2024Yes-associated protein (YAP) and WW domain-containing transcription regulator protein 1 (WWTR1; also known as TAZ) are the main effectors of the Hippo pathway and their...
Yes-associated protein (YAP) and WW domain-containing transcription regulator protein 1 (WWTR1; also known as TAZ) are the main effectors of the Hippo pathway and their dysregulation contributes to diseases in tissues including the liver. Although mitochondria are capable of transmitting signals to change transcriptomic landscape of diseased hepatocytes, such retrograde signaling and the related nuclear machinery are largely unknown. Here, we show that increased YAP activity is associated with mitochondrial stress during liver injury; and this is required for secondary inflammation, promoting hepatocyte death. Mitochondrial stress inducers robustly promoted YAP/TAZ dephosphorylation, nuclear accumulation, and target gene transcription. RNA sequencing revealed that the majority of mitochondrial stress transcripts required YAP/TAZ. Mechanistically, direct oxidation of RhoA by mitochondrial superoxide was responsible for PP2A-mediated YAP/TAZ dephosphorylation providing a novel physiological input for the Hippo pathway. Hepatocyte-specific Yap/Taz ablation suppressed acetaminophen-induced liver injury and blunted transcriptomic changes associated with the pathology. Our observations uncover unappreciated pathway of mitochondrial stress signaling and reveal YAP/TAZ activation as the mechanistic basis for liver injury progression.
Topics: Adaptor Proteins, Signal Transducing; YAP-Signaling Proteins; Liver; Signal Transduction; Intracellular Signaling Peptides and Proteins
PubMed: 38225223
DOI: 10.1038/s41419-024-06448-5 -
American Journal of Physiology. Renal... May 2021Phosphorylation of the thiazide-sensitive NaCl cotransporter (NCC) in the distal convoluted tubule (DCT) is altered rapidly in response to changes in extracellular K...
Phosphorylation of the thiazide-sensitive NaCl cotransporter (NCC) in the distal convoluted tubule (DCT) is altered rapidly in response to changes in extracellular K concentration ([K]). High extracellular [K] is believed to activate specific phosphatases to dephosphorylate NCC, thereby reducing its activity. This process is defective in the human disease familial hyperkalemic hypertension, in which extracellular [K] fails to dephosphorylate NCC, suggesting an interplay between NCC-activating and NCC-inactivating switches. Here, we explored the role of STE20/SPS1-related proline-alanine-rich protein kinase (SPAK) and intracellular Cl concentration in the rapid effects of extracellular K on NCC phosphorylation. SPAK was found to be rapidly dephosphorylated in vitro in human embryonic kidney cells and ex vivo in kidney slices by high [K]. Acute high-K challenge resulted in DCT1-specific SPAK dephosphorylation in vivo and dissolution of SPAK puncta. In line with the postulate of interplay between activating and inactivating switches, we found that the "on" switch, represented by with no lysine kinase 4 (WNK4)-SPAK, must be turned off for rapid NCC dephosphorylation by high [K]. Longer-term WNK-SPAK-mediated stimulation, however, altered the sensitivity of the system, as it attenuated rapid NCC dephosphorylation due to acute K loading. Although blockade of protein phosphatase (PP)1 increased NCC phosphorylation at baseline, neither PP1 nor PP3, singly or in combination, was essential for NCC dephosphorylation. Overall, our data suggest that NCC phosphorylation is regulated by a dynamic equilibrium between activating kinases and inactivating phosphatases, with kinase inactivation playing a key role in the rapid NCC dephosphorylation by high extracellular K. Although a great deal is known about mechanisms by which thiazide-sensitive NaCl cotransporter is phosphorylated and activated, much less is known about dephosphorylation. Here, we show that rapid dephosphorylation by high K depends on the Cl sensitivity of with no lysine kinase 4 and the rapid dephosphorylation of STE20/SPS1-related proline-alanine-rich protein kinase, primarily along the early distal convoluted tubule.
Topics: Animals; Chlorides; HEK293 Cells; Humans; Kidney Tubules, Distal; Kinetics; Male; Mice, Inbred C57BL; Mice, Knockout; Phosphorylation; Potassium, Dietary; Protein Serine-Threonine Kinases; Protein Transport; Solute Carrier Family 12, Member 3; Mice
PubMed: 33719576
DOI: 10.1152/ajprenal.00459.2020 -
Nature Structural & Molecular Biology Oct 2022SHOC2 acts as a strong synthetic lethal interactor with MEK inhibitors in multiple KRAS cancer cell lines. SHOC2 forms a heterotrimeric complex with MRAS and PP1C that...
SHOC2 acts as a strong synthetic lethal interactor with MEK inhibitors in multiple KRAS cancer cell lines. SHOC2 forms a heterotrimeric complex with MRAS and PP1C that is essential for regulating RAF and MAPK-pathway activation by dephosphorylating a specific phosphoserine on RAF kinases. Here we present the high-resolution crystal structure of the SHOC2-MRAS-PP1C (SMP) complex and apo-SHOC2. Our structures reveal that SHOC2, MRAS, and PP1C form a stable ternary complex in which all three proteins synergistically interact with each other. Our results show that dephosphorylation of RAF substrates by PP1C is enhanced upon interacting with SHOC2 and MRAS. The SMP complex forms only when MRAS is in an active state and is dependent on SHOC2 functioning as a scaffolding protein in the complex by bringing PP1C and MRAS together. Our results provide structural insights into the role of the SMP complex in RAF activation and how mutations found in Noonan syndrome enhance complex formation, and reveal new avenues for therapeutic interventions.
Topics: Humans; Intracellular Signaling Peptides and Proteins; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase Kinases; Noonan Syndrome; Phosphoserine; Protein Phosphatase 1; Proto-Oncogene Proteins p21(ras); raf Kinases; ras Proteins
PubMed: 36175670
DOI: 10.1038/s41594-022-00841-4