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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 -
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 -
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 -
Cell Research Jul 2014Mitophagy, or mitochondria autophagy, plays a critical role in selective removal of damaged or unwanted mitochondria. Several protein receptors, including Atg32 in... (Review)
Review
Mitophagy, or mitochondria autophagy, plays a critical role in selective removal of damaged or unwanted mitochondria. Several protein receptors, including Atg32 in yeast, NIX/BNIP3L, BNIP3 and FUNDC1 in mammalian systems, directly act in mitophagy. Atg32 interacts with Atg8 and Atg11 on the surface of mitochondria, promoting core Atg protein assembly for mitophagy. NIX/BNIP3L, BNIP3 and FUNDC1 also have a classic motif to directly bind LC3 (Atg8 homolog in mammals) for activation of mitophagy. Recent studies have shown that receptor-mediated mitophagy is regulated by reversible protein phosphorylation. Casein kinase 2 (CK2) phosphorylates Atg32 and activates mitophagy in yeast. In contrast, in mammalian cells Src kinase and CK2 phosphorylate FUNDC1 to prevent mitophagy. Notably, in response to hypoxia and FCCP treatment, the mitochondrial phosphatase PGAM5 dephosphorylates FUNDC1 to activate mitophagy. Here, we mainly focus on recent advances in our understanding of the molecular mechanisms underlying the activation of receptor-mediated mitophagy and the implications of this catabolic process in health and disease.
Topics: Animals; Apoptosis; Autophagy-Related Protein 8 Family; Autophagy-Related Proteins; Casein Kinase II; Cell Hypoxia; Humans; Membrane Proteins; Microtubule-Associated Proteins; Mitochondria; Mitochondrial Dynamics; Mitochondrial Proteins; Mitophagy; Phosphorylation; Proto-Oncogene Proteins; Receptors, Cytoplasmic and Nuclear; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Tumor Suppressor Proteins; Vesicular Transport Proteins; src-Family Kinases
PubMed: 24903109
DOI: 10.1038/cr.2014.75 -
The FEBS Journal Jul 2016Glucan phosphatases are a recently discovered class of enzymes that dephosphorylate starch and glycogen, thereby regulating energy metabolism. Plant genomes encode two... (Review)
Review
Glucan phosphatases are a recently discovered class of enzymes that dephosphorylate starch and glycogen, thereby regulating energy metabolism. Plant genomes encode two glucan phosphatases, called Starch EXcess4 (SEX4) and Like Sex Four2 (LSF2), that regulate starch metabolism by selectively dephosphorylating glucose moieties within starch glucan chains. Recently, the structures of both SEX4 and LSF2 were determined, with and without phosphoglucan products bound, revealing the mechanism for their unique activities. This review explores the structural and enzymatic features of the plant glucan phosphatases, and outlines how they are uniquely adapted to perform their cellular functions. We outline the physical mechanisms used by SEX4 and LSF2 to interact with starch glucans: SEX4 binds glucan chains via a continuous glucan-binding platform comprising its dual-specificity phosphatase domain and carbohydrate-binding module, while LSF2 utilizes surface binding sites. SEX4 and LSF2 both contain a unique network of aromatic residues in their catalytic dual-specificity phosphatase domains that serve as glucan engagement platforms and are unique to the glucan phosphatases. We also discuss the phosphoglucan substrate specificities inherent to SEX4 and LSF2, and outline structural features within the active site that govern glucan orientation. This review defines the structural mechanism of the plant glucan phosphatases with respect to phosphatases, starch metabolism and protein-glucan interaction, thereby providing a framework for their application in both agricultural and industrial settings.
Topics: Arabidopsis Proteins; Dual-Specificity Phosphatases; Glucans; Plant Proteins; Protein Binding; Starch
PubMed: 26934589
DOI: 10.1111/febs.13703 -
ACS Chemical Neuroscience Feb 2023Amyloid fibers of the protein α-synuclein, found in Lewy body deposits, are hallmarks of Parkinson's disease. We here show that α-synuclein amyloids catalyze...
Amyloid fibers of the protein α-synuclein, found in Lewy body deposits, are hallmarks of Parkinson's disease. We here show that α-synuclein amyloids catalyze biologically relevant chemical reactions in vitro. Amyloid fibers, but not monomers, of α-synuclein catalyzed hydrolysis of the model ester -nitrophenyl acetate and dephosphorylation of the model phosphoester -nitrophenyl-orthophosphate. When His50 was replaced with Ala in α-synuclein, dephosphorylation but not esterase activity of amyloids was diminished. Truncation of the protein's C-terminus had no effect on fiber catalytic efficiency. Catalytic activity of α-synuclein fibers may be a new gain-of-function that plays a role in Parkinson's disease.
Topics: Humans; alpha-Synuclein; Parkinson Disease; Lewy Bodies; Amyloid
PubMed: 36745416
DOI: 10.1021/acschemneuro.2c00799 -
The Journal of Cell Biology Sep 2020The tumor suppressor PTEN is essential for early development. Its lipid phosphatase activity converts PIP3 to PIP2 and antagonizes the PI3K-Akt pathway. In this study,...
The tumor suppressor PTEN is essential for early development. Its lipid phosphatase activity converts PIP3 to PIP2 and antagonizes the PI3K-Akt pathway. In this study, we demonstrate that PTEN's protein phosphatase activity is required for epiblast epithelial differentiation and polarization. This is accomplished by reconstitution of PTEN-null embryoid bodies with PTEN mutants that lack only PTEN's lipid phosphatase activity or both PTEN's lipid and protein phosphatase activities. Phosphotyrosine antibody immunoprecipitation and mass spectrometry were used to identify Abi1, a core component of the WASP-family verprolin homologous protein (WAVE) regulatory complex (WRC), as a new PTEN substrate. We demonstrate that PTEN dephosphorylation of Abi1 at Y213 and S216 results in Abi1 degradation through the calpain pathway. This leads to down-regulation of the WRC and reorganization of the actin cytoskeleton. The latter is critical to the transformation of nonpolar pluripotent stem cells into the polarized epiblast epithelium. Our findings establish a link between PTEN and WAVE-Arp2/3-regulated actin cytoskeletal dynamics in epithelial morphogenesis.
Topics: Actin Cytoskeleton; Adaptor Proteins, Signal Transducing; Animals; Calpain; Cell Differentiation; Cell Line, Tumor; Cytoskeletal Proteins; Down-Regulation; Epithelial Cells; Epithelium; Female; Germ Layers; Humans; Mice; Mice, Inbred C57BL; Morphogenesis; PTEN Phosphohydrolase; Pregnancy; Signal Transduction
PubMed: 32673396
DOI: 10.1083/jcb.201910041 -
Scientific Reports Feb 2018Dishevelled (Dvl) family proteins are key mediators of Wnt signalling and function in both canonical and noncanonical branches. Dvl2, the most studied Dvl protein, is...
Dishevelled (Dvl) family proteins are key mediators of Wnt signalling and function in both canonical and noncanonical branches. Dvl2, the most studied Dvl protein, is extensively regulated by phosphorylation. Several kinases were found to be critical for Dvl2 localisation, stability control and functional segregation. For example, S143-phosphorylated Dvl2 was detected, together with CK1δ/ε, at the centrosome and basal body of primary cilia and plays pivotal roles during ciliogenesis. However, relatively less is known about Dvl dephosphorylation and the phosphatases involved. Here, we identified PP5 (PPP5C) as a phosphatase of Dvl2. PP5 interacts with and can directly dephosphorylate Dvl2. Knockdown of PP5 caused elevated Dvl2 phosphorylation both at the basal level and upon Wnt stimulation. In the Dvl2 protein, S143, the 10B5 cluster and other sites were dephosphorylated by PP5. Interestingly, comparison of PP5 with PP2A, another known Dvl2 phosphatase, revealed that PP5 and PP2A are not fully redundant in the regulation of Dvl2 phosphorylation status. In hTERT-RPE1 cells, PP5 was found at the basal body of cilia, where S143-phosphorylated Dvl2 also resides. Functional assays revealed modest effects on ciliogenesis after PP5 depletion or over-expression. Taken together, our results provided evidence to suggest PP5 as a new phosphatase for Dvl2.
Topics: Cells, Cultured; Cilia; Dishevelled Proteins; HEK293 Cells; HeLa Cells; Humans; MCF-7 Cells; Nuclear Proteins; Phosphoprotein Phosphatases; Phosphorylation; Proteolysis; Retinal Pigment Epithelium; Signal Transduction; Wnt Proteins
PubMed: 29426949
DOI: 10.1038/s41598-018-21124-3 -
PLoS Pathogens Jul 2020Hepatitis B virus (HBV) replicates its genomic DNA via viral DNA polymerase self-primed reverse transcription of a RNA pre-genome in the nucleocapsid assembled by 120...
Hepatitis B virus (HBV) replicates its genomic DNA via viral DNA polymerase self-primed reverse transcription of a RNA pre-genome in the nucleocapsid assembled by 120 core protein (Cp) dimers. The arginine-rich carboxyl-terminal domain (CTD) of Cp plays an important role in the selective packaging of viral DNA polymerase-pregenomic (pg) RNA complex into nucleocapsid. Previous studies suggested that the CTD is initially phosphorylated at multiple sites to facilitate viral RNA packaging and subsequently dephosphorylated in association with viral DNA synthesis and secretion of DNA-containing virions. However, our recent studies suggested that Cp is hyper-phosphorylated as free dimers and its dephosphorylation is associated with pgRNA encapsidation. Herein, we provide further genetic and biochemical evidence supporting that extensive Cp dephosphorylation does take place during the assembly of pgRNA-containing nucleocapsids, but not empty capsids. Moreover, we found that cellular protein phosphatase 1 (PP1) is required for Cp dephosphorylation and pgRNA packaging. Interestingly, the PP1 catalytic subunits α and β were packaged into pgRNA-containing nucleocapsids, but not empty capsids, and treatment of HBV replicating cells with core protein allosteric modulators (CpAMs) promoted empty capsid assembly and abrogated the encapsidation of PP1 α and β. Our study thus identified PP1 as a host cellular factor that is co-packaged into HBV nucleocapsids, and plays an essential role in selective packaging of the viral DNA-polymerase-pgRNA complex through catalyzing Cp dephosphorylation.
Topics: Cell Line; Hepatitis B; Hepatitis B virus; Humans; Nucleocapsid; Peptide Fragments; Phosphorylation; Protein Phosphatase 1; RNA, Viral; Viral Core Proteins; Virus Assembly
PubMed: 32702076
DOI: 10.1371/journal.ppat.1008669 -
Cell Death & Disease Sep 2022Mixed lineage leukemia 1 (MLL1) is a histone H3 lysine 4 (H3K4) methyltransferase that interacts with WD repeat domain 5 (WDR5) to regulate cell survival, proliferation,...
Mixed lineage leukemia 1 (MLL1) is a histone H3 lysine 4 (H3K4) methyltransferase that interacts with WD repeat domain 5 (WDR5) to regulate cell survival, proliferation, and senescence. The role of MLL1 in the pathogenesis of acute kidney injury (AKI) is unknown. In this study, we demonstrate that MLL1, WDR5, and trimethylated H3K4 (H3K4me3) were upregulated in renal tubular cells of cisplatin-induced AKI in mice, along with increased phosphorylation of p53 and decreased expression of E-cadherin. Administration of MM102, a selective MLL1/WDR5 complex inhibitor, improved renal function and attenuated tubular injury and apoptosis, while repressing MLL1, WDR5, and H3K4me3, dephosphorylating p53 and preserving E-cadherin. In cultured mouse renal proximal tubular cells (RPTCs) exposed to cisplatin, treatment with MM102 or transfection with siRNAs for either MLL1 or WDR5 also inhibited apoptosis and p53 phosphorylation while preserving E-cadherin expression; p53 inhibition with Pifithrin-α lowered cisplatin-induced apoptosis without affecting expression of MLL1, WDR5, and H3K4me3. Interestingly, silencing of E-cadherin offset MM102's cytoprotective effects, but had no effect on p53 phosphorylation. These findings suggest that MLL1/WDR5 activates p53, which, in turn, represses E-cadherin, leading to apoptosis during cisplatin-induced AKI. Further studies showed that MM102 effectively inhibited cisplatin-triggered DNA damage response (DDR), as indicated by dephosphorylation of ataxia telangiectasia mutated (ATM) and ATM and Rad-3 related (ATR) proteins, dephosphorylation of checkpoint kinase 1 and 2 (Chk1 and Chk2); depression of γ-H2AX; and restrained cell cycle arrest, as evidenced by decreased expression of p21 and phospho-histone H3 at serine 10 in vitro and in vivo. Overall, we identify MLL1 as a novel DDR regulator that drives cisplatin-induced RPTC apoptosis and AKI by modulating the MLL1/WDR5-/ATR/ATM-Chk-p53-E-cadherin axis. Targeting the MLL1/WDR5 complex may have a therapeutic potential for the treatment of AKI.
Topics: Acute Kidney Injury; Animals; Apoptosis; Cadherins; Cisplatin; Histone Methyltransferases; Histones; Kidney; Leukemia; Mice; Myeloid-Lymphoid Leukemia Protein; Tumor Suppressor Protein p53
PubMed: 36068197
DOI: 10.1038/s41419-022-05104-0