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Advances in Biological Regulation May 2022The PAH1-encoded phosphatidate phosphatase, which catalyzes the dephosphorylation of phosphatidate to produce diacylglycerol, controls the divergence of phosphatidate... (Review)
Review
The PAH1-encoded phosphatidate phosphatase, which catalyzes the dephosphorylation of phosphatidate to produce diacylglycerol, controls the divergence of phosphatidate into triacylglycerol synthesis and phospholipid synthesis. Pah1 is inactive in the cytosol as a phosphorylated form and becomes active on the nuclear/endoplasmic reticulum membrane as a dephosphorylated form by the Nem1-Spo7 protein phosphatase complex. The phosphorylation of Pah1 by protein kinases, which include casein kinases I and II, Pho85-Pho80, Cdc28-cyclin B, and protein kinases A and C, controls its cellular location, catalytic activity, and susceptibility to proteasomal degradation. Nem1 (catalytic subunit) and Spo7 (regulatory subunit), which form a protein phosphatase complex catalyzing the dephosphorylation of Pah1 for its activation, are phosphorylated by protein kinases A and C. In this review, we discuss the functions and interrelationships of the protein kinases in the control of the Nem1-Spo7/Pah1 phosphatase cascade and lipid synthesis.
Topics: Lipids; Membrane Proteins; Nuclear Proteins; Phosphatidate Phosphatase; Phosphorylation; Protein Kinases; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 35231723
DOI: 10.1016/j.jbior.2022.100889 -
Molecular Cell Oct 2022Neuronal activity induces topoisomerase IIβ (Top2B) to generate DNA double-strand breaks (DSBs) within the promoters of neuronal early response genes (ERGs) and...
Neuronal activity induces topoisomerase IIβ (Top2B) to generate DNA double-strand breaks (DSBs) within the promoters of neuronal early response genes (ERGs) and facilitate their transcription, and yet, the mechanisms that control Top2B-mediated DSB formation are unknown. Here, we report that stimulus-dependent calcium influx through NMDA receptors activates the phosphatase calcineurin to dephosphorylate Top2B at residues S1509 and S1511, which stimulates its DNA cleavage activity and induces it to form DSBs. Exposing mice to a fear conditioning paradigm also triggers Top2B dephosphorylation at S1509 and S1511 in the hippocampus, indicating that calcineurin also regulates Top2B-mediated DSB formation following physiological neuronal activity. Furthermore, calcineurin-Top2B interactions following neuronal activity and sites that incur activity-induced DSBs are preferentially localized at the nuclear periphery in neurons. Together, these results reveal how radial gene positioning and the compartmentalization of activity-dependent signaling govern the position and timing of activity-induced DSBs and regulate gene expression patterns in neurons.
Topics: Animals; Mice; Calcineurin; Calcium; DNA; DNA Breaks, Double-Stranded; DNA Topoisomerases, Type II; DNA-Binding Proteins; Neurons; Receptors, N-Methyl-D-Aspartate
PubMed: 36206766
DOI: 10.1016/j.molcel.2022.09.012 -
The FEBS Journal Feb 2021Protein phosphorylation is a major reversible post-translational modification. Protein phosphatases function as 'critical regulators' in signaling networks through... (Review)
Review
Protein phosphorylation is a major reversible post-translational modification. Protein phosphatases function as 'critical regulators' in signaling networks through dephosphorylation of proteins, which have been phosphorylated by protein kinases. A large understanding of their working has been sourced from animal systems rather than the plant or the prokaryotic systems. The eukaryotic protein phosphatases include phosphoprotein phosphatases (PPP), metallo-dependent protein phosphatases (PPM), protein tyrosine (Tyr) phosphatases (PTP), and aspartate (Asp)-dependent phosphatases. The PPP and PPM families are serine(Ser)/threonine(Thr)-specific phosphatases (STPs), while PTP family is Tyr specific. Dual-specificity phosphatases (DsPTPs/DSPs) dephosphorylate Ser, Thr, and Tyr residues. PTPs lack sequence homology with STPs, indicating a difference in catalytic mechanisms, while the PPP and PPM families share a similar structural fold indicating a common catalytic mechanism. The catalytic cysteine (Cys) residue in the conserved HCX R active site motif of the PTPs acts as a nucleophile during hydrolysis. The PPP members require metal ions, which coordinate the phosphate group of the substrate, followed by a nucleophilic attack by a water molecule and hydrolysis. The variable holoenzyme assembly of protein phosphatase(s) and the overlap with other post-translational modifications like acetylation and ubiquitination add to their complexity. Though their functional characterization is extensively reported in plants, the mechanistic nature of their action is still being explored by researchers. In this review, we exclusively overview the plant protein phosphatases with an emphasis on their mechanistic action as well as structural characteristics.
Topics: Biocatalysis; Catalytic Domain; Models, Molecular; Phosphoprotein Phosphatases; Phosphorylation; Plant Proteins; Protein Domains; Protein Subunits; Signal Transduction; Substrate Specificity
PubMed: 32542989
DOI: 10.1111/febs.15454 -
Cell Reports Aug 2023Mitochondrial morphology is regulated by the post-translational modifications of the dynamin family GTPase proteins including mitofusin 1 (MFN1), MFN2, and...
Mitochondrial morphology is regulated by the post-translational modifications of the dynamin family GTPase proteins including mitofusin 1 (MFN1), MFN2, and dynamin-related protein 1 (DRP1). Mitochondrial phosphatase phosphoglycerate mutase 5 (PGAM5) is emerging as a regulator of these post-translational modifications; however, its precise role in the regulation of mitochondrial morphology is unknown. We show that PGAM5 interacts with MFN2 and DRP1 in a stress-sensitive manner. PGAM5 regulates MFN2 phosphorylation and consequently protects it from ubiquitination and degradation. Further, phosphorylation and dephosphorylation modification of MFN2 regulates its fusion ability. Phosphorylation enhances fission and degradation, whereas dephosphorylation enhances fusion. PGAM5 dephosphorylates MFN2 to promote mitochondrial network formation. Further, using a Drosophila genetic model, we demonstrate that the MFN2 homolog Marf and dPGAM5 are in the same biological pathway. Our results identify MFN2 dephosphorylation as a regulator of mitochondrial fusion and PGAM5 as an MFN2 phosphatase.
Topics: GTP Phosphohydrolases; Phosphoric Monoester Hydrolases; Phosphoglycerate Mutase; Mitochondrial Dynamics; Mitochondrial Proteins; Dynamins
PubMed: 37498743
DOI: 10.1016/j.celrep.2023.112895 -
The Journal of Clinical Investigation Nov 2023Consumption of low dietary potassium, common with ultraprocessed foods, activates the thiazide-sensitive sodium chloride cotransporter (NCC) via the with no (K) lysine...
Consumption of low dietary potassium, common with ultraprocessed foods, activates the thiazide-sensitive sodium chloride cotransporter (NCC) via the with no (K) lysine kinase/STE20/SPS1-related proline-alanine-rich protein kinase (WNK/SPAK) pathway to induce salt retention and elevate blood pressure (BP). However, it remains unclear how high-potassium "DASH-like" diets (dietary approaches to stop hypertension) inactivate the cotransporter and whether this decreases BP. A transcriptomics screen identified Ppp1Ca, encoding PP1A, as a potassium-upregulated gene, and its negative regulator Ppp1r1a, as a potassium-suppressed gene in the kidney. PP1A directly binds to and dephosphorylates NCC when extracellular potassium is elevated. Using mice genetically engineered to constitutively activate the NCC-regulatory kinase SPAK and thereby eliminate the effects of the WNK/SPAK kinase cascade, we confirmed that PP1A dephosphorylated NCC directly in a potassium-regulated manner. Prior adaptation to a high-potassium diet was required to maximally dephosphorylate NCC and lower BP in constitutively active SPAK mice, and this was associated with potassium-dependent suppression of Ppp1r1a and dephosphorylation of its cognate protein, inhibitory subunit 1 (I1). In conclusion, potassium-dependent activation of PP1A and inhibition of I1 drove NCC dephosphorylation, providing a mechanism to explain how high dietary K+ lowers BP. Shifting signaling of PP1A in favor of activation of WNK/SPAK may provide an improved therapeutic approach for treating salt-sensitive hypertension.
Topics: Animals; Mice; Blood Pressure; Solute Carrier Family 12, Member 3; Protein Serine-Threonine Kinases; Sodium Chloride; Potassium, Dietary; Kidney; Hypertension; Potassium; Phosphorylation
PubMed: 37676724
DOI: 10.1172/JCI158498 -
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 -
The Journal of Biological Chemistry Dec 2019Endothelial cells have key functions in endothelial barrier integrity and in responses to angiogenic signals that promote cell proliferation, cell migration,...
Endothelial cells have key functions in endothelial barrier integrity and in responses to angiogenic signals that promote cell proliferation, cell migration, cytoskeletal reorganization, and formation of new blood vessels. These functions highly depend on protein-protein interactions in cell-cell junction and cell attachment complexes and on interactions with cytoskeletal proteins. Protein phosphatase 2A (PP2A) dephosphorylates several target proteins involved in cytoskeletal dynamics and cell adhesion. Our goal was to find new interacting and substrate proteins of the PP2A-B55α holoenzyme in bovine pulmonary endothelial cells. Using LC-MS/MS analysis, we identified flotillin-1 as a protein that binds recombinant GSH -transferase-tagged PP2A-B55α. Immunoprecipitation experiments, proximity ligation assays, and immunofluorescent staining confirmed the interaction between these two endogenous proteins in endothelial cells. Originally, flotillins were described as regulatory proteins for axon regeneration, but they appear to function in many cellular processes, such as membrane receptor signaling, endocytosis, and cell adhesion. Ser is a known PKC-targeted site in flotillin-1. Utilizing phosphomutants of flotillin-1 and the NanoBiT luciferase assay, we show here that phosphorylation/dephosphorylation of Ser in flotillin-1 significantly affects its interaction with PP2A-B55α and that PP2A-B55α dephosphorylates phospho-Ser Spreading, attachment, migration, and tube formation rates of S315A variant-overexpressing cells were faster than those of nontransfected or S315D-transfected cells. These results indicate that the PP2A-flotillin-1 interaction identified here affects major physiological activities of pulmonary endothelial cells.
Topics: Animals; Carbazoles; Cattle; Cell Movement; Cells, Cultured; Endothelial Cells; Holoenzymes; Membrane Proteins; Mutagenesis, Site-Directed; Neovascularization, Physiologic; Phosphorylation; Protein Interaction Domains and Motifs; Protein Kinase C; Protein Phosphatase 2; Protein Subunits; RNA Interference; RNA, Small Interfering; Up-Regulation
PubMed: 31753918
DOI: 10.1074/jbc.RA119.007980 -
Annual Review of Pharmacology and... 2014Precise control of the balance between protein phosphorylation, catalyzed by protein kinases, and protein dephosphorylation, catalyzed by protein phosphatases, is... (Review)
Review
Precise control of the balance between protein phosphorylation, catalyzed by protein kinases, and protein dephosphorylation, catalyzed by protein phosphatases, is essential for cellular homeostasis. Dysregulation of this balance leads to pathophysiological states, driving diseases such as cancer, heart disease, and diabetes. Aberrant phosphorylation of components of the pathways that control cell growth and cell survival are particularly prevalent in cancer. One of the most studied tumor suppressors in these pathways is the lipid phosphatase PTEN (phosphatase and tensin homolog deleted on chromosome ten), which dephosphorylates the lipid second messenger phosphatidylinositol 3,4,5-trisphosphate (PIP3), thus preventing activation of the oncogenic kinase AKT (v-akt murine thymoma viral oncogene homolog). In 2005, the discovery of a family of protein phosphatases whose members directly dephosphorylate and inactivate AKT introduced a new negative regulator of the phosphoinositide 3-kinase (PI3K) oncogenic pathway. Pleckstrin homology domain leucine-rich repeat protein phosphatase (PHLPP) isozymes comprise a novel tumor suppressor family whose two members, PHLPP1 and PHLPP2, are deleted as frequently as PTEN in cancers such as those of the prostate. PHLPP is thus a novel therapeutic target to suppress oncogenic pathways and is a potential candidate biomarker to stratify patients for the appropriate targeted therapeutics. This review discusses the role of PHLPP in terminating AKT signaling and how pharmacological intervention would impact this pathway.
Topics: DNA Primers; Gene Expression Regulation; Humans; Male; Molecular Targeted Therapy; Nuclear Proteins; Phosphoprotein Phosphatases; Phosphorylation; Prostatic Neoplasms; Proto-Oncogene Proteins c-akt; Signal Transduction
PubMed: 24392697
DOI: 10.1146/annurev-pharmtox-011112-140338 -
Cell Apr 2020Protein phosphatase 2A (PP2A) enzymes can suppress tumors, but they are often inactivated in human cancers overexpressing inhibitory proteins. Here, we identify a class...
Protein phosphatase 2A (PP2A) enzymes can suppress tumors, but they are often inactivated in human cancers overexpressing inhibitory proteins. Here, we identify a class of small-molecule iHAPs (improved heterocyclic activators of PP2A) that kill leukemia cells by allosterically assembling a specific heterotrimeric PP2A holoenzyme consisting of PPP2R1A (scaffold), PPP2R5E (B56ε, regulatory), and PPP2CA (catalytic) subunits. One compound, iHAP1, activates this complex but does not inhibit dopamine receptor D2, a mediator of neurologic toxicity induced by perphenazine and related neuroleptics. The PP2A complex activated by iHAP1 dephosphorylates the MYBL2 transcription factor on Ser241, causing irreversible arrest of leukemia and other cancer cells in prometaphase. In contrast, SMAPs, a separate class of compounds, activate PP2A holoenzymes containing a different regulatory subunit, do not dephosphorylate MYBL2, and arrest tumor cells in G1 phase. Our findings demonstrate that small molecules can serve as allosteric switches to activate distinct PP2A complexes with unique substrates.
Topics: Apoptosis; Cell Cycle Proteins; Cell Line, Tumor; Enzyme Activators; G1 Phase; Humans; Multiprotein Complexes; Phenothiazines; Phosphorylation; Protein Phosphatase 2; Protein Subunits; Trans-Activators; Transcription Factors
PubMed: 32315619
DOI: 10.1016/j.cell.2020.03.051 -
Cell Communication and Signaling : CCS Jan 2022G protein-coupled receptors (GPCRs) usually regulate cellular processes via activation of intracellular signaling pathways. However, we have previously shown that in...
BACKGROUND
G protein-coupled receptors (GPCRs) usually regulate cellular processes via activation of intracellular signaling pathways. However, we have previously shown that in several cell lines, GqPCRs induce immediate inactivation of the AKT pathway, which leads to JNK-dependent apoptosis. This apoptosis-inducing AKT inactivation is essential for physiological functions of several GqPCRs, including those for PGF2α and GnRH.
METHODS
Here we used kinase activity assays of PI3K and followed phosphorylation state of proteins using specific antibodies. In addition, we used coimmunoprecipitation and proximity ligation assays to follow protein-protein interactions. Apoptosis was detected by TUNEL assay and PARP1 cleavage.
RESULTS
We identified the mechanism that allows the unique stimulated inactivation of AKT and show that the main regulator of this process is the phosphatase PP2A, operating with the non-canonical regulatory subunit IGBP1. In resting cells, an IGBP1-PP2Ac dimer binds to PI3K, dephosphorylates the inhibitory pSer608-p85 of PI3K and thus maintains its high basal activity. Upon GqPCR activation, the PP2Ac-IGBP1 dimer detaches from PI3K and thus allows the inhibitory dephosphorylation. At this stage, the free PP2Ac together with IGBP1 and PP2Aa binds to AKT, causing its dephosphorylation and inactivation.
CONCLUSION
Our results show a stimulated shift of PP2Ac from PI3K to AKT termed "PP2A switch" that represses the PI3K/AKT pathway, providing a unique mechanism of GPCR-stimulated dephosphorylation. Video Abstract.
Topics: Phosphatidylinositol 3-Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; Receptors, G-Protein-Coupled; Signal Transduction
PubMed: 34998390
DOI: 10.1186/s12964-021-00805-z