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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 -
American Journal of Physiology. Cell... Feb 2009Mitogen-activated protein (MAP) kinase phosphatase-1 (MKP-1) is a nuclear, dual-specificity phosphatase that has been shown to dephosphorylate MAP kinases. We used a...
Mitogen-activated protein (MAP) kinase phosphatase-1 (MKP-1) is a nuclear, dual-specificity phosphatase that has been shown to dephosphorylate MAP kinases. We used a "substrate-trap" technique involving a mutation in MKP-1 of the catalytically critical cysteine to a serine residue ("CS" mutant) to capture novel MKP-1 substrates. We transfected the MKP-1 (CS) mutant and control (wild-type, WT) constructs into phorbol 12-myristate 13-acetate (PMA)-activated COS-1 cells. MKP-1-substrate complexes were immunoprecipitated, which yielded four bands of 17, 15, 14, and 10 kDa with the CS MKP-1 mutant but not the WT MKP-1. The bands were identified by mass spectrometry as histones H3, H2B, H2A, and H4, respectively. Histone H3 was phosphorylated, and purified MKP-1 dephosphorylated histone H3 (phospho-Ser-10) in vitro; whereas, histone H3 (phospho-Thr-3) was unaffected. We have previously shown that thrombin and vascular endothelial growth factor (VEGF) upregulated MKP-1 in human endothelial cells (EC). We now show that both thrombin and VEGF caused dephosphorylation of histone H3 (phospho-Ser-10) and histone H3 (phospho-Thr-3) in EC with kinetics consistent with MKP-1 induction. Furthermore, MKP-1-specific small interfering RNA (siRNA) prevented VEGF- and thrombin-induced H3 (phospho-Ser-10) dephosphorylation but had no effect on H3 (phospho-Thr-3 or Thr-11) dephosphorylation. In summary, histone H3 is a novel substrate of MKP-1, and VEGF- and thrombin-induced H3 (phospho-Ser-10) dephosphorylation requires MKP-1. We propose that MKP-1-mediated H3 (phospho-Ser-10) dephosphorylation is a key regulatory step in EC activation by VEGF and thrombin.
Topics: Animals; COS Cells; Catalytic Domain; Chlorocebus aethiops; Dual Specificity Phosphatase 1; Endothelial Cells; Epigenesis, Genetic; Histones; Humans; Immunoprecipitation; Molecular Weight; Mutation; Peptide Fragments; Phosphorylation; Protein Binding; Protein Processing, Post-Translational; RNA Interference; Serine; Signal Transduction; Tetradecanoylphorbol Acetate; Thrombin; Time Factors; Transfection; Vascular Endothelial Growth Factor A
PubMed: 19020052
DOI: 10.1152/ajpcell.00492.2008 -
European Journal of Biochemistry May 1983First-order rate constants (50 degrees C; I = 0.1 M, NaClO4) for the dephosphorylation of UTP and TTP (1 mM) in the pH range 2-10 are compared with those of ATP and CTP;... (Comparative Study)
Comparative Study
Metal-ion-promoted dephosphorylation of the 5'-triphosphates of uridine and thymidine, and a comparison with the reactivity in the corresponding cytidine and adenosine nucleotide systems.
First-order rate constants (50 degrees C; I = 0.1 M, NaClO4) for the dephosphorylation of UTP and TTP (1 mM) in the pH range 2-10 are compared with those of ATP and CTP; they all show the same properties indicating that the nucleic base has no influence on the rate. In the presence of Cu2+ or Zn2+ (NTP:M2+ = 1:1) this changes drastically: ATP-M2+ much greater than UTP-M2+ approximately equal to TTP-M2+ approximately equal to CTP-M2+ greater than NTP, the Cu2+ systems being always more reactive than the Zn2+ systems, and these more than the Ni2+ systems. An interaction between the nucleic base and metal ion is important for the Cu2+-ATP and Zn2+-ATP systems, but not for the pyrimidine-nucleotide systems (these behave like methyltriphosphate). Accordingly, prevention of the Cu2+-purine interaction by the addition of one equivalent of 2,2'-bipyridyl, leading to Cu(Bpy) (NTP)2-, strongly reduces the activity and all four ternary Cu2+ systems now show the same dephosphorylation rate. Addition of a second equivalent of Cu2+ to the Cu2+-UTP 1:1 system enhances the dephosphorylation rate significantly and Job's method provides evidence that a 2:1 complex is the most reactive intermediate. The relation between the initial rate, vo = d[PO3-4]/dt, and the concentration of Cu2+-UTP in 1:1 and 2:1 systems was determined. The results suggest that the reactive complex with pyrimidine nucleotides is a monomeric, dinuclear species of the type M2(NTP) (OH)- (its formation is inhibited by ligands like tryptophanate), while with M2+-ATP the reactive complex is a dimer. The connection between the indicated dephosphorylations in vitro, i.e. trans-phosphorylations to H2O, and related reactions in vivo are discussed.
Topics: Adenosine Triphosphate; Chemical Phenomena; Chemistry; Copper; Cytidine Triphosphate; Ligands; Metals; Nickel; Nucleotides; Phosphorylation; Thymine Nucleotides; Uridine Triphosphate; Zinc
PubMed: 6852014
DOI: 10.1111/j.1432-1033.1983.tb07401.x -
Scientific Reports Feb 2018Eyes absent (EYA) proteins are unusual proteins combining in a single polypeptide chain transactivation, threonine phosphatase, and tyrosine phosphatase activities. They...
Eyes absent (EYA) proteins are unusual proteins combining in a single polypeptide chain transactivation, threonine phosphatase, and tyrosine phosphatase activities. They play pivotal roles in organogenesis and are involved in a variety of physiological and pathological processes including innate immunity, DNA damage repair or cancer metastasis. The molecular targets of EYA tyrosine phosphatase activity are still elusive. Therefore, we sought to identify novel EYA substrates and also to obtain further insight into the tyrosine-dephosphorylating role of EYA proteins in various cellular processes. We show here that Src kinase phosphorylates tyrosine residues in two human EYA family members, EYA1 and EYA3. Both can autodephosphorylate these residues and their nuclear and cytoskeletal localization seems to be controlled by Src phosphorylation. Next, using a microarray of phosphotyrosine-containing peptides, we identified a phosphopeptide derived from WD-repeat-containing protein 1 (WDR1) that is dephosphorylated by EYA3. We further demonstrated that several tyrosine residues on WDR1 are phosphorylated by Src kinase, and are efficiently dephosphorylated by EYA3, but not by EYA1. The lack of phosphorylation generates major changes to the cellular actin cytoskeleton. We, therefore, conclude that WDR1 is an EYA3-specific substrate, which implies that EYA3 is a key modulator of the cytoskeletal reorganization.
Topics: Actin Cytoskeleton; Amino Acid Sequence; Biocatalysis; Conserved Sequence; DNA-Binding Proteins; Humans; Microfilament Proteins; Mutation; Phosphorylation; Protein Domains; Protein Tyrosine Phosphatases; src-Family Kinases
PubMed: 29440662
DOI: 10.1038/s41598-018-21155-w -
Retrovirology Jul 2005HIV-1 Tat protein recruits human positive transcription elongation factor P-TEFb, consisting of CDK9 and cyclin T1, to HIV-1 transactivation response (TAR) RNA. CDK9 is...
BACKGROUND
HIV-1 Tat protein recruits human positive transcription elongation factor P-TEFb, consisting of CDK9 and cyclin T1, to HIV-1 transactivation response (TAR) RNA. CDK9 is maintained in dephosphorylated state by TFIIH and undergo phosphorylation upon the dissociation of TFIIH. Thus, dephosphorylation of CDK9 prior to its association with HIV-1 preinitiation complex might be important for HIV-1 transcription. Others and we previously showed that protein phosphatase-2A and protein phosphatase-1 regulates HIV-1 transcription. In the present study we analyze relative contribution of PP2A and PP1 to dephosphorylation of CDK9 and to HIV-1 transcription in vitro and in vivo.
RESULTS
In vitro, PP2A but not PP1 dephosphorylated autophosphorylated CDK9 and reduced complex formation between P-TEFb, Tat and TAR RNA. Inhibition of PP2A by okadaic acid inhibited basal as well as Tat-induced HIV-1 transcription whereas inhibition of PP1 by recombinant nuclear inhibitor of PP1 (NIPP1) inhibited only Tat-induced transcription in vitro. In cultured cells, low concentration of okadaic acid, inhibitory for PP2A, only mildly inhibited Tat-induced HIV-1 transcription. In contrast Tat-mediated HIV-1 transcription was strongly inhibited by expression of NIPP1. Okadaic acid induced phosphorylation of endogenous as well transiently expressed CDK9, but this induction was not seen in the cells expressing NIPP1. Also the okadaic acid did not induce phosphorylation of CDK9 with mutation of Thr 186 or with mutations in Ser-329, Thr-330, Thr-333, Ser-334, Ser-347, Thr-350, Ser-353, and Thr-354 residues involved in autophosphorylation of CDK9.
CONCLUSION
Our results indicate that although PP2A dephosphorylates autophosphorylated CDK9 in vitro, in cultured cells PP1 is likely to dephosphorylate CDK9 and contribute to the regulation of activated HIV-1 transcription.
Topics: Animals; COS Cells; Chlorocebus aethiops; Cyclin-Dependent Kinase 9; Gene Products, tat; HIV Long Terminal Repeat; HIV-1; Humans; Phosphoprotein Phosphatases; Phosphorylation; Protein Phosphatase 1; Protein Phosphatase 2; Transcription, Genetic; tat Gene Products, Human Immunodeficiency Virus
PubMed: 16048649
DOI: 10.1186/1742-4690-2-47 -
Food Research International (Ottawa,... Nov 2023Industrial-scale production of recombinant proteins for food products may become economically feasible but correct post-translational modification of proteins by...
Industrial-scale production of recombinant proteins for food products may become economically feasible but correct post-translational modification of proteins by microbial expression systems remains a challenge. For efficient production of hybrid products from bovine casein and recombinant casein, it is therefore of interest to evaluate the necessity of casein post-translational phosphorylation for the preparation of hybrid casein micelles and study their rennet-induced coagulation. Our results show that dephosphorylated casein was hardly incorporated into artificial casein micelles but was capable of stabilising calcium phosphate nanoclusters with an increased size through adsorption on their surface. Thereby, dephosphorylated casein formed larger colloidal particles with a decreased hydration. Furthermore, the presence of increasing amounts of dephosphorylated casein resulted in increasingly poor rennet coagulation behaviour, where dephosphorylated casein disrupted the formation of a coherent gel network by native casein. These results emphasise that post-translational phosphorylation of casein is crucial for their assembly into micelles and thereby for the production of dairy products for which the casein micelle structure is a prerequisite, such as many cheese varieties and yoghurt. Therefore, phosphorylation of future recombinant casein is essential to allow its use in the production of animal-free dairy products.
Topics: Animals; Cattle; Micelles; Caseins; Phosphorylation; Milk; Cheese
PubMed: 37803629
DOI: 10.1016/j.foodres.2023.113315 -
Plant, Cell & Environment Feb 2021Improving chilling tolerance is a major target of rice breeding. The OsMAPK3-OsbHLH002-OsTPP1 signalling pathway enhances chilling tolerance in rice: the kinase is...
Improving chilling tolerance is a major target of rice breeding. The OsMAPK3-OsbHLH002-OsTPP1 signalling pathway enhances chilling tolerance in rice: the kinase is activated by cold stress, and subsequently the transcription factor is phosphorylated by the activated kinase, triggering the expression of cold response genes. However, it is largely unknown how this pathway is suppressed in time to avoid it being in a continuously activated state. We found that a novel type 2C protein phosphatase, OsPP2C27, functions as a negative regulator of the OsMAPK3-OsbHLH002-OsTPP1 pathway. A dynamic change in OsMAPK3 activity was found during cold treatment. We show that OsPP2C27 interacts physically with and dephosphorylates OsMAPK3 in vitro and in vivo. Interestingly, OsPP2C27 can also directly dephosphorylate OsbHLH002, the target of OsMAPK3. After cold treatment, survival rates were higher in OsPP2C27-RNAi lines and a T-DNA insertion mutant, and lower in OsPP2C27-overexpression lines, compared to wild type. Moreover, expression of the OsTPP1 and OsDREBs were increased in OsPP2C27-RNAi lines and decreased in OsPP2C27-overexpression lines. These results indicate that cold-induced OsPP2C27 negatively regulates the OsMAPK3-OsbHLH002-OsTPP1 signalling pathway by directly dephosphorylating both phospho-OsMAPK3 and phospho-OsbHLH002, preventing the sustained activation of a positive pathway for cold stress and maintaining normal growth under chilling conditions.
Topics: Cold Temperature; Gene Expression Regulation, Plant; Oryza; Phosphoric Monoester Hydrolases; Phosphorylation; Plant Proteins; Plant Roots; Plant Transpiration; Salt Stress; Signal Transduction; Transcription Factors
PubMed: 33150964
DOI: 10.1111/pce.13938 -
Nature Communications Sep 2019Mitotic cells attenuate the DNA damage response (DDR) by phosphorylating 53BP1, a critical DDR mediator, to prevent its localization to damaged chromatin. Timely...
Mitotic cells attenuate the DNA damage response (DDR) by phosphorylating 53BP1, a critical DDR mediator, to prevent its localization to damaged chromatin. Timely dephosphorylation of 53BP1 is critical for genome integrity, as premature recruitment of 53BP1 to DNA lesions impairs mitotic fidelity. Protein phosphatase 4 (PP4) dephosphorylates 53BP1 in late mitosis to allow its recruitment to DNA lesions in G1. How cells appropriately dephosphorylate 53BP1, thereby restoring DDR, is unclear. Here, we elucidate the underlying mechanism of kinetic control of 53BP1 dephosphorylation in mitosis. We demonstrate that CDK5, a kinase primarily functional in post-mitotic neurons, is active in late mitotic phases in non-neuronal cells and directly phosphorylates PP4R3β, the PP4 regulatory subunit that recognizes 53BP1. Specific inhibition of CDK5 in mitosis abrogates PP4R3β phosphorylation and abolishes its recognition and dephosphorylation of 53BP1, ultimately preventing the localization of 53BP1 to damaged chromatin. Our results establish CDK5 as a regulator of 53BP1 recruitment.
Topics: Cell Line, Tumor; Cyclin-Dependent Kinase 5; DNA Damage; DNA Repair; G1 Phase; HEK293 Cells; HeLa Cells; Humans; Mitosis; Phosphoprotein Phosphatases; Phosphorylation; RNA Interference; RNA, Small Interfering; Tumor Suppressor p53-Binding Protein 1
PubMed: 31534152
DOI: 10.1038/s41467-019-12084-x -
Protein phosphatase 2A regulates cytotoxicity and drug resistance by dephosphorylating AHR and MDR1.The Journal of Biological Chemistry May 2022Protein phosphatase 2A (PP2A) is a serine/threonine dephosphorylating enzyme complex that plays numerous roles in biological processes, including cell growth and...
Protein phosphatase 2A (PP2A) is a serine/threonine dephosphorylating enzyme complex that plays numerous roles in biological processes, including cell growth and metabolism. However, its specific actions in many of these critical pathways are unclear. To explore mechanisms underlying metabolic enzyme regulation in the liver, we investigated the key pathways involved in regulation of xenobiotic-metabolizing enzymes in a mouse model with hepatocyte-specific deletion of Ppp2r1a, encoding the Aα subunit of PP2A. We performed transcriptome and phosphoproteome analysis in mouse livers at the age of 3 months and identified 2695 differentially expressed genes and 549 upregulated phosphoproteins in homozygous knockout mouse livers compared with WT littermates. In particular, the expression of metabolic enzymes Cyp2e1, Cyp1a1, Cyp1a2, Mdr1a, and Abcg2 was dramatically altered in homozygous knockout mouse livers. We also demonstrated that activation of PP2A reversed the decline of metabolic enzyme expression in primary mouse hepatocytes. We found that specific PP2A holoenzymes were involved in metabolic enzyme induction through dephosphorylation of transcription factors, nuclear receptors, or the target enzymes themselves, leading to dysregulation of xenobiotic metabolism or drug-induced hepatotoxicity. Notably, we confirmed that a regulatory axis, PP2A B56α-aryl hydrocarbon receptor-Cyp1a1, was involved in benzo(a)pyrene-induced cytotoxicity through dephosphorylation of the metabolic nuclear receptor, aryl hydrocarbon receptor, at serine 36. In addition, we showed that PP2A B56δ complexes directly dephosphorylated the multidrug efflux pump MDR1 (encoded by multi-drug resistance gene 1), contributing to drug resistance against the chemotherapeutic 5-fluorouracil. Taken together, these novel findings demonstrate the involvement of PP2A in the regulation of liver metabolism.
Topics: ATP Binding Cassette Transporter, Subfamily B; Animals; Cytochrome P-450 CYP1A1; Drug Resistance; Mice; Mice, Knockout; Phosphorylation; Protein Phosphatase 2; Receptors, Aryl Hydrocarbon; Xenobiotics
PubMed: 35405096
DOI: 10.1016/j.jbc.2022.101918 -
BMB Reports Jun 2020Since cancer is the leading cause of death worldwide, there is an urgent need to understand the mechanisms underlying cancer progression and the development of cancer...
Since cancer is the leading cause of death worldwide, there is an urgent need to understand the mechanisms underlying cancer progression and the development of cancer inhibitors. Signal transducer and activator of transcription 3 (STAT3) is a major transcription factor that regulates the proliferation and survival of various cancer cells. Here, dual-specificity phosphatase 3 (DUSP3) was identified as a regulator of STAT3 based on an interaction screening performed using the protein tyrosine phosphatase library. DUSP3 interacted with the C-terminal domain of STAT3 and dephosphorylated p-Y705 of STAT3. In vitro dephosphorylation assay revealed that DUSP3 directly dephosphorylated p-STAT3. The suppressive effects of DUSP3 on STAT3 were evaluated by a decreased STAT3-specific promoter activity, which in turn reduced the expression of the downstream target genes of STAT3. In summary, DUSP3 downregulated the transcriptional activity of STAT3 via dephosphorylation at Y705 and also suppressed the migratory activity of cancer cells. This study demonstrated that DUSP3 inhibits interleukin 6 (IL-6)/STAT3 signaling and is expected to regulate cancer development. Novel functions of DUSP3 discovered in IL-6/STAT3 signaling regulation would help expand the understanding of cancer development mechanisms. [BMB Reports 2020; 53(6): 335-340].
Topics: Cells, Cultured; Dual Specificity Phosphatase 3; Humans; Interleukin-6; STAT3 Transcription Factor; Signal Transduction
PubMed: 32475380
DOI: 10.5483/BMBRep.2020.53.6.054