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The Journal of Neuroscience : the... Apr 2020Addictive drugs usurp the brain's intrinsic mechanism for reward, leading to compulsive and destructive behaviors. In the ventral tegmental area (VTA), the center of the...
Addictive drugs usurp the brain's intrinsic mechanism for reward, leading to compulsive and destructive behaviors. In the ventral tegmental area (VTA), the center of the brain's reward circuit, GABAergic neurons control the excitability of dopamine (DA) projection neurons and are the site of initial psychostimulant-dependent changes in signaling. Previous work established that cocaine/methamphetamine exposure increases protein phosphatase 2A (PP2A) activity, which dephosphorylates the GABAR2 subunit, promotes internalization of the GABA receptor (GABAR) and leads to smaller GABAR-activated G-protein-gated inwardly rectifying potassium (GIRK) currents in VTA GABA neurons. How the actions of PP2A become selective for a particular signaling pathway is poorly understood. Here, we demonstrate that PP2A can associate directly with a short peptide sequence in the C terminal domain of the GABAR1 subunit, and that GABARs and PP2A are in close proximity in rodent neurons (mouse/rat; mixed sexes). We show that this PP2A-GABAR interaction can be regulated by intracellular Ca Finally, a peptide that potentially reduces recruitment of PP2A to GABARs and thereby limits receptor dephosphorylation increases the magnitude of baclofen-induced GIRK currents. Thus, limiting PP2A-dependent dephosphorylation of GABARs may be a useful strategy to increase receptor signaling for treating diseases. Dysregulation of GABA receptors (GABARs) underlies altered neurotransmission in many neurological disorders. Protein phosphatase 2A (PP2A) is involved in dephosphorylating and subsequent internalization of GABARs in models of addiction and depression. Here, we provide new evidence that PP2A B55 regulatory subunit interacts directly with a small region of the C-terminal domain of the GABAR1 subunit, and that this interaction is sensitive to intracellular Ca We demonstrate that a short peptide corresponding to the PP2A interaction site on GABAR1 competes for PP2A binding, enhances phosphorylation GABAR2 S783, and affects functional signaling through GIRK channels. Our study highlights how targeting PP2A dependent dephosphorylation of GABARs may provide a specific strategy to modulate GABAR signaling in disease conditions.
Topics: Animals; Brain; Female; G Protein-Coupled Inwardly-Rectifying Potassium Channels; HEK293 Cells; Humans; Male; Mice; Mice, Inbred C57BL; Neurons; Phosphorylation; Protein Phosphatase 2; Rats; Receptors, GABA-B; Signal Transduction; Synaptic Transmission
PubMed: 32111696
DOI: 10.1523/JNEUROSCI.2654-19.2020 -
The Journal of Biological Chemistry Feb 2011The Hippo pathway regulates organ size by controlling both cell proliferation and apoptosis. TAZ functions as a transcriptional co-activator downstream of the Hippo...
The Hippo pathway regulates organ size by controlling both cell proliferation and apoptosis. TAZ functions as a transcriptional co-activator downstream of the Hippo pathway and has been implicated in human cancer development. A key step in the Hippo-TAZ pathway is phosphorylation of TAZ by LATS kinase, which leads to TAZ inhibition by both cytoplasmic retention and degradation. However, the mechanism of TAZ dephosphorylation and the responsible phosphatase are unknown. Here, we identified PP1 as a bona fide TAZ phosphatase. PP1A dephosphorylates TAZ at Ser-89 and Ser-311, promotes TAZ nuclear translocation, and stabilizes TAZ by disrupting the binding to the SCF E3 ubiquitin ligase. Furthermore, ASPP2 facilitates the interaction between TAZ and PP1 to promote TAZ dephosphorylation. As a result, PP1 and ASPP2 increase TAZ-dependent gene expression. This study demonstrates that PP1A and ASPP2 play a critical role in promoting TAZ function by antagonizing the LATS kinase through TAZ dephosphorylation.
Topics: Active Transport, Cell Nucleus; Apoptosis Regulatory Proteins; Cell Nucleus; HEK293 Cells; HeLa Cells; Humans; Intracellular Signaling Peptides and Proteins; Phosphorylation; Protein Phosphatase 1; Trans-Activators; Transcription Factors; Transcriptional Coactivator with PDZ-Binding Motif Proteins
PubMed: 21189257
DOI: 10.1074/jbc.M110.194019 -
Sub-cellular Biochemistry 2015New evidences have been reported that point to the ecto-enzyme, tissue-nonspecific alkaline phosphatase (TNAP), as a key element at the origin of two opposite phenomena,... (Review)
Review
New evidences have been reported that point to the ecto-enzyme, tissue-nonspecific alkaline phosphatase (TNAP), as a key element at the origin of two opposite phenomena, neuronal differentiation and neuronal degeneration. During brain development, TNAP plays an essential role for establishing neuronal circuits. The pro-neuritic effect induced by TNAP, which results in axonal length increase, is due to its enzymatic hydrolysis of extracellular ATP at the surrounding area of the axonal growth cone . In this way, the activation of P2X7 receptor is prevented and as a consequence there is no inhibition of axonal growth. The existence of a close functional interrelation between both purinergic elements is finally supported by the fact that both elements may control, in a reciprocal way, the expression level of the other. On the opposite stage, recent evidences indicate that TNAP plays a key role in spreading the neurotoxicity effect induced by extracellular hyperphosphorylated tau protein, the main component of intracellular neurofibrillary tangles present in the brain of Alzheimer disease patients. TNAP exhibits a broad substrate specificity and in addition to nucleotides it is able to dephosphorylate extracellular proteins, such as the hyperphosphorylated tau protein once it is released to the extracellular medium. Dephosphorylated tau protein behaves as an agonist of muscarinic M1 and M3 receptors, provoking a robust and sustained intracellular calcium increase that finally triggering neuronal death. Besides, activation of muscarinic receptors by dephosphorylated tau increases the expression of TNAP, which could explain the increase in TNAP activity and protein levels detected in Alzheimer disease.
Topics: Alkaline Phosphatase; Animals; Brain; Cell Differentiation; Embryonic Development; Humans; Neural Stem Cells; Neurodegenerative Diseases; Neurogenesis
PubMed: 26219721
DOI: 10.1007/978-94-017-7197-9_18 -
Journal of Biochemistry Jul 1996Cofilin is an actin-depolymerizing protein, whose depolymerizing activity is supposed to be regulated in part by phosphorylation and dephosphorylation. Thus, we studied...
Cofilin is an actin-depolymerizing protein, whose depolymerizing activity is supposed to be regulated in part by phosphorylation and dephosphorylation. Thus, we studied the phosphorylation states of cofilin in rat parotid acinar cells during stimulation for amylase exocytosis. Isoproterenol and carbachol induced rapid and extensive dephosphorylation of cofilin; 60-70% dephosphorylation was clearly detectable within 1 min. Membrane-permeable cyclic AMP (CPS-cAMP), phorbol ester (PMA), and Ca ionophore A23187 mimicked the effect of isoproterenol and carbachol. Protein phosphatase inhibitors (calyculin A or FK506 plus cyclosporin A) did not block the dephosphorylation in response to isoproterenol or carbachol. Furthermore, calyculin A alone strongly dephosphorylated cofilin. Although no exogenous protein phosphatases tested dephosphorylated cofilin in the homogenate, the cofilin that was isolated by immunoprecipitation was clearly dephosphorylated by protein phosphatases 1, 2A, and 2C.
Topics: Actin Depolymerizing Factors; Amylases; Animals; Calcimycin; Calcium; Carbachol; Cyclic AMP; Cyclosporine; Enzyme Inhibitors; Exocytosis; Ionophores; Isoproterenol; Marine Toxins; Microfilament Proteins; Nerve Tissue Proteins; Oxazoles; Parotid Gland; Phosphoprotein Phosphatases; Phosphorylation; Rats; Tacrolimus; Tetradecanoylphorbol Acetate
PubMed: 8864841
DOI: 10.1093/oxfordjournals.jbchem.a021390 -
Oncotarget Aug 2019Gene transcription is a highly complex and strictly regulated process. RNA polymerase II (Pol II) C-terminal domain (CTD) undergoes massive cycles of phosphorylation and...
Gene transcription is a highly complex and strictly regulated process. RNA polymerase II (Pol II) C-terminal domain (CTD) undergoes massive cycles of phosphorylation and dephosphorylation during the process of gene transcription. These post-translational modifications of CTD provide an interactive platform for various factors required for transcription initiation, elongation, termination, and co-transcriptional RNA processing. Pol II CTD kinases and phosphatases are key regulators and any deviation may cause genome-wide transcriptional dysregulation leading to various pathological conditions including cancer. PTEN, a well known tumor suppressor, is one of the most commonly somatically altered in diverse malignancies. When mutated in the germline, causes cancer predisposition. Numerous studies have demonstrated that PTEN regulates the expression of hundreds of genes, however, no mechanism is known so far. PTEN is a dual specificity phosphatase, using both lipid and protein as substrates. In the present study, we demonstrate that PTEN interacts with the RNA Pol II and that PTEN expression is inversely correlated with global phosphorylation of Pol II CTD. Furthermore, PTEN dephosphorylates Pol II CTD with a significant specificity for Ser5p. Interestingly, ChIP-seq data analysis revealed that PTEN globally binds to promoter proximal regions, and PTEN loss increases genome-wide Pol II Ser5p occupancy, suggest that PTEN is a Pol II CTD phosphatase. Our observations demonstrate an unexplored function of PTEN with the potential of global transcriptional regulation, adding a new dimension to somatic carcinogenesis and germline cancer predisposition.
PubMed: 31452836
DOI: 10.18632/oncotarget.27128 -
Cells Jul 2023Protocadherin-7 (Pcdh7) is a member of the non-clustered protocadherin δ1 subgroup of the cadherin superfamily. Pcdh7 has been revealed to control osteoclast...
Protocadherin-7 (Pcdh7) is a member of the non-clustered protocadherin δ1 subgroup of the cadherin superfamily. Pcdh7 has been revealed to control osteoclast differentiation by regulating Rho-family small GTPases, RhoA and Rac1, through its intracellular SET binding domain. However, the mechanisms by which small GTPases are regulated downstream of Pcdh7 remain unclear. Here, we demonstrate that protein phosphatase 2A (PP2A)-mediated dephosphorylation of Glycogen synthase kinase-3β (GSK3β) is required for Pcdh7-dependent activation of RhoA during osteoclast differentiation. Pcdh7-deficient (Pcdh7) cells showed impaired PP2A activity, despite their normal expression of PP2A. GSK3β, whose activity is regulated by its inhibitory phosphorylation at Ser9, was dephosphorylated during osteoclast differentiation in a Pcdh7-dependent manner. Inhibition of protein phosphatase by okadaic acid reduced dephosphorylation of GSK3β in Pcdh7 cells, while activation of PP2A by DT-061 rescued impaired dephosphorylation of GSK3β in Pcdh7 cells. Inhibition of GSK3β by AR-A014418 inhibited RANKL-induced RhoA activation and osteoclast differentiation in Pcdh7 cells. On the other hand, DT-061 treatment rescued impaired RhoA activation and RANKL-induced osteoclast differentiation in Pcdh7 cells. Taken together, these results demonstrate that PP2A dephosphorylates GSK3β and thereby activates it in a Pcdh7-dependent manner, which is required for activation of small GTPase RhoA and proper osteoclast differentiation.
Topics: Osteoclasts; Protein Phosphatase 2; Monomeric GTP-Binding Proteins; Protocadherins; Glycogen Synthase Kinase 3 beta; Cadherins
PubMed: 37566044
DOI: 10.3390/cells12151967 -
The Journal of Neuroscience : the... Mar 1989Carboxy-terminal tail domains of larger molecular mass subunits (NF-M and NF-H) of neurofilaments (NFs), which are the highly phosphorylated moieties, were observed as...
Carboxy-terminal tail domains of larger molecular mass subunits (NF-M and NF-H) of neurofilaments (NFs), which are the highly phosphorylated moieties, were observed as thin flexible filaments projecting from NF core filaments by rotary shadowing (Hisanaga and Hirokawa, 1988). Dephosphorylation of NFs has been suspected to affect the structures and the functions of the carboxy-terminal tail projections. We report here the effects of the dephosphorylation on the structure of NFs studied by electron microscopy. (1) The structures of carboxy-terminal tail projections after dephosphorylation were compared with those of the control NFs by low-angle rotary shadowing. This was examined with 2 samples; the isolated neurofilaments and the short filaments assembled from NF-H. Both the dephosphorylated NFs and the short filaments showed many projections laterally extending from core filaments similar to those observed in the control samples. (2) With respect to the structure of NF in physiological solution, the density of NFs in the precipitates was examined by thin-section electron microscopy. No difference in the density was noted between control and dephosphorylated NFs. (3) The ability to form cross-bridges in vitro was examined by quick-freeze, deep-etch electron microscopy. The structure and frequency of cross-bridges appeared to be similar in both control and dephosphorylated NFs. (4) Phosphate determination revealed that about 90% of the phosphate groups of NF-H subunit were removed by treatment with E. coli alkaline phosphatase. These results indicated that the dephosphorylation of NF did not affect the structure and the ability to form cross-bridges of the carboxy-terminal tail projections in vitro.
Topics: Animals; Cattle; Cytoskeleton; Intermediate Filament Proteins; Intermediate Filaments; Microscopy, Electron; Neural Pathways; Neurofilament Proteins; Phosphates; Phosphorylation; Synaptic Transmission
PubMed: 2538587
DOI: 10.1523/JNEUROSCI.09-03-00959.1989 -
Molecular and Cellular Biology Apr 1991Simian virus 40 (SV40) large-T antigen and the cellular protein p53 were phosphorylated in vivo by growing cells in the presence of 32Pi. The large-T/p53 complex was...
Simian virus 40 (SV40) large-T antigen and the cellular protein p53 were phosphorylated in vivo by growing cells in the presence of 32Pi. The large-T/p53 complex was isolated by immunoprecipitation and used as a substrate for protein phosphatase 2A (PP2A) consisting of the catalytic subunit (C) and the two regulatory subunits, A and B. Three different purified forms of PP2A, including free C, the AC form, and the ABC form, could readily dephosphorylate both proteins. With both large-T and p53, the C subunit was most active, followed by the AC form, which was more active than the ABC form. The activity of all three forms of PP2A toward these proteins was strongly stimulated by manganese ions and to a lesser extent by magnesium ions. The presence of complexed p53 did not affect the dephosphorylation of large-T antigen by PP2A. The dephosphorylation of individual phosphorylation sites of large-T and p53 were determined by two-dimensional peptide mapping. Individual sites within large-T and p53 were dephosphorylated at different rates by all three forms of PP2A. The phosphates at Ser-120 and Ser-123 of large-T, which affect binding to the origin of SV40 DNA, were removed most rapidly. Three of the six major phosphopeptides of p53 were readily dephosphorylated, while the remaining three were relatively resistant to PP2A. Dephosphorylation of most of the sites in large-T and p53 by the AC form was inhibited by SV40 small-t antigen. The inhibition was most apparent for those sites which were preferentially dephosphorylated. Inhibition was specific for the AC form; no effect was observed on the dephosphorylation of either protein by the free C subunit or the ABC form. The inhibitory effect of small-t on dephosphorylation by PP2A could explain its role in transformation.
Topics: Antigens, Polyomavirus Transforming; Peptide Mapping; Phosphoprotein Phosphatases; Phosphorylation; Precipitin Tests; Protein Phosphatase 2; Simian virus 40; Substrate Specificity; Tumor Suppressor Protein p53
PubMed: 1848668
DOI: 10.1128/mcb.11.4.1996-2003.1991 -
The Journal of Biological Chemistry Mar 2019Na-H exchanger regulatory factor-1 (NHERF1) is a PDZ protein that scaffolds membrane proteins, including sodium-phosphate co-transport protein 2A (NPT2A) at the plasma...
Na-H exchanger regulatory factor-1 (NHERF1) is a PDZ protein that scaffolds membrane proteins, including sodium-phosphate co-transport protein 2A (NPT2A) at the plasma membrane. NHERF1 is a phosphoprotein with 40 Ser and Thr residues. Here, using tandem MS analysis, we characterized the sites of parathyroid hormone (PTH)-induced NHERF1 phosphorylation and identified 10 high-confidence phosphorylation sites. Ala replacement at Ser, Ser, Ser, Ser, Ser, Ser, Thr, Ser, and Ser did not affect phosphate uptake, but S290A substitution abolished PTH-dependent phosphate transport. Unexpectedly, Ser was rapidly dephosphorylated and rephosphorylated after PTH stimulation, and we found that protein phosphatase 1α (PP1α), which binds NHERF1 through a conserved VxF/W PP1 motif, dephosphorylates Ser Mutating VPF eliminated PP1 binding and blunted dephosphorylation. Tautomycetin blocked PP1 activity and abrogated PTH-sensitive phosphate transport. Using fluorescence lifetime imaging (FLIM), we observed that PTH paradoxically and transiently elevates intracellular phosphate. Added phosphate blocked PP1α-mediated Ser dephosphorylation of recombinant NHERF1. Hydrogen-deuterium exchange MS revealed that β-sheets in NHERF1's PDZ2 domain display lower deuterium uptake than those in the structurally similar PDZ1, implying that PDZ1 is more cloistered. Dephosphorylated NHERF1 exhibited faster exchange at C-terminal residues suggesting that NHERF1 dephosphorylation precedes Ser rephosphorylation. Our results show that PP1α and NHERF1 form a holoenzyme and that a multiprotein kinase cascade involving G protein-coupled receptor kinase 6A controls the Ser phosphorylation status of NHERF1 and regulates PTH-sensitive, NPT2A-mediated phosphate uptake. These findings reveal how reversible phosphorylation modifies protein conformation and function and the biochemical mechanisms underlying PTH control of phosphate transport.
Topics: Amino Acid Sequence; Crystallography, X-Ray; Furans; HEK293 Cells; Humans; Ion Transport; Lipids; Parathyroid Hormone; Phosphates; Phosphoproteins; Phosphorylation; Protein Conformation; Receptors, Neuropeptide Y; Serine; Sodium-Hydrogen Exchangers; Sodium-Phosphate Cotransporter Proteins, Type IIa
PubMed: 30696771
DOI: 10.1074/jbc.RA119.007421 -
Biology May 2023mTOR is constitutively activated in acute myeloid leukemia (AML) cells, as indicated by the phosphorylation of its substrates, 4EBP1 and P70S6K. Here, we found that...
mTOR is constitutively activated in acute myeloid leukemia (AML) cells, as indicated by the phosphorylation of its substrates, 4EBP1 and P70S6K. Here, we found that quercetin (Q) and rapamycin (Rap) inhibited P70S6K phosphorylation, partially dephosphorylated 4EBP1, and activated ERK1/2 in U937 and THP1, two leukemia cell lines. ERK1/2 inhibition by U0126 induced a stronger dephosphorylation of mTORC1 substrates and activated AKT. The concomitant inhibition of ERK1/2 and AKT further dephosphorylated 4EBP1 and further increased Q- or Rap-mediated cytotoxicity, compared to the single ERK1/2 or AKT inhibition in cells undergoing Q- or Rap-treatments. Moreover, quercetin or rapamycin reduced autophagy, particularly when used in combination with the ERK1/2 inhibitor, U0126. This effect was not dependent on TFEB localization in nuclei or cytoplasm or on the transcription of different autophagy genes, but did correlate with the reduction in protein translation due to a strong eIF2α-Ser51 phosphorylation. Thus, ERK1/2, by limiting 4EBP1 de-phosphorylation and eIF2α phosphorylation, behaves as a paladin of protein synthesis. Based on these findings, the combined inhibition of mTORC1, ERK1/2, and AKT should be considered in treatment of AML.
PubMed: 37237490
DOI: 10.3390/biology12050676