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ELife Apr 2017The small phosphoprotein pCPI-17 inhibits myosin light-chain phosphatase (MLCP). Current models postulate that during muscle relaxation, phosphatases other than MLCP...
The small phosphoprotein pCPI-17 inhibits myosin light-chain phosphatase (MLCP). Current models postulate that during muscle relaxation, phosphatases other than MLCP dephosphorylate and inactivate pCPI-17 to restore MLCP activity. We show here that such hypotheses are insufficient to account for the observed rapidity of pCPI-17 inactivation in mammalian smooth muscles. Instead, MLCP itself is the critical enzyme for pCPI-17 dephosphorylation. We call the mutual sequestration mechanism through which pCPI-17 and MLCP interact MLCP protects pCPI-17 from other phosphatases, while pCPI-17 blocks other substrates from MLCP's active site. MLCP dephosphorylates pCPI-17 at a slow rate that is, nonetheless, both sufficient and necessary to explain the speed of pCPI-17 dephosphorylation and the consequent MLCP activation during muscle relaxation.
Topics: HEK293 Cells; Humans; Intracellular Signaling Peptides and Proteins; Muscle Proteins; Myosin-Light-Chain Phosphatase; Phosphoprotein Phosphatases; Phosphorylation; Protein Processing, Post-Translational
PubMed: 28387646
DOI: 10.7554/eLife.24665 -
The Plant Cell Mar 2020Phosphate (Pi) uptake in plants depends on plasma membrane (PM)-localized phosphate transporters (PTs). OsCK2 phosphorylates PTs and inhibits their trafficking from the...
Phosphate (Pi) uptake in plants depends on plasma membrane (PM)-localized phosphate transporters (PTs). OsCK2 phosphorylates PTs and inhibits their trafficking from the endoplasmic reticulum (ER) to the PM in rice (), but how PTs are dephosphorylated is unknown. We demonstrate that the protein phosphatase type 2C (PP2C) protein phosphatase OsPP95 interacts with OsPT2 and OsPT8 and dephosphorylates OsPT8 at Ser-517. Rice plants overexpressing reduced OsPT8 phosphorylation and promoted OsPT2 and OsPT8 trafficking from the ER to the PM, resulting in Pi accumulation. Under Pi-sufficient conditions, Pi levels were lower in young leaves and higher in old leaves in mutants than in those of the wild type, even though the overall shoot Pi levels were the same in the mutant and the wild type. In the wild type, OsPP95 accumulated under Pi starvation but was rapidly degraded under Pi-sufficient conditions. We show that OsPHO2 interacts with and induces the degradation of OsPP95. We conclude that OsPP95, a protein phosphatase negatively regulated by OsPHO2, positively regulates Pi homeostasis and remobilization by dephosphorylating PTs and affecting their trafficking to the PM, a reversible process required for adaptation to variable Pi conditions.
Topics: Endoplasmic Reticulum; Epistasis, Genetic; Gene Expression Regulation, Plant; Homeostasis; Membrane Transport Proteins; Models, Biological; Mutation; Organ Specificity; Oryza; Phosphates; Phosphorylation; Plant Proteins; Plant Roots; Plant Shoots; Protein Binding; Subcellular Fractions
PubMed: 31919298
DOI: 10.1105/tpc.19.00685 -
Journal of Cellular and Molecular... Jul 2021Protein kinases and phosphatases regulate cellular processes by reversible phosphorylation and dephosphorylation events. CPPED1 is a recently identified serine/threonine...
Protein kinases and phosphatases regulate cellular processes by reversible phosphorylation and dephosphorylation events. CPPED1 is a recently identified serine/threonine protein phosphatase that dephosphorylates AKT1 of the PI3K-AKT signalling pathway. We previously showed that CPPED1 levels are down-regulated in the human placenta during spontaneous term birth. In this study, based on sequence comparisons, we propose that CPPED1 is a member of the class III phosphodiesterase (PDE) subfamily within the calcineurin-like metallophosphoesterase (MPE) superfamily rather than a member of the phosphoprotein phosphatase (PPP) or metal-dependent protein phosphatase (PPM) protein families. We used a human proteome microarray to identify 36 proteins that putatively interact with CPPED1. Of these, GRB2, PAK4 and PIK3R2 are known to regulate the PI3K-AKT pathway. We further confirmed CPPED1 interactions with PAK4 and PIK3R2 by coimmunoprecipitation analyses. We characterized the effect of CPPED1 on phosphorylation of PAK4 and PIK3R2 in vitro by mass spectrometry. CPPED1 dephosphorylated specific serine residues in PAK4, while phosphorylation levels in PIK3R2 remained unchanged. Our findings indicate that CPPED1 may regulate PI3K-AKT pathway activity at multiple levels. Higher CPPED1 levels may inhibit PI3K-AKT pathway maintaining pregnancy. Consequences of decreased CPPED1 expression during labour remain to be elucidated.
PubMed: 34009729
DOI: 10.1111/jcmm.16607 -
Frontiers in Pharmacology 2015Decades of cardiovascular research have shown that variable and flexible levels of protein phosphorylation are necessary to maintain cardiac function. A delicate balance... (Review)
Review
Decades of cardiovascular research have shown that variable and flexible levels of protein phosphorylation are necessary to maintain cardiac function. A delicate balance between phosphorylated and dephosphorylated states of proteins is guaranteed by a complex interplay of protein kinases (PKs) and phosphatases. Serine/threonine phosphatases, in particular members of the protein phosphatase (PP) family govern dephosphorylation of the majority of these cardiac proteins. Recent findings have however shown that PPs do not only dephosphorylate previously phosphorylated proteins as a passive control mechanism but are capable to actively control PK activity via different direct and indirect signaling pathways. These control mechanisms can take place on (epi-)genetic, (post-)transcriptional, and (post-)translational levels. In addition PPs themselves are targets of a plethora of proteinaceous interaction partner regulating their endogenous activity, thus adding another level of complexity and feedback control toward this system. Finally, novel approaches are underway to achieve spatiotemporal pharmacologic control of PPs which in turn can be used to fine-tune misleaded PK activity in heart disease. Taken together, this review comprehensively summarizes the major aspects of PP-mediated PK regulation and discusses the subsequent consequences of deregulated PP activity for cardiovascular diseases in depth.
PubMed: 26617522
DOI: 10.3389/fphar.2015.00270 -
PloS One 2015Excessive cytokine inflammatory response due to chronic or superphysiological level of microbial infection during pregnancy leads to pregnancy complications such as...
Excessive cytokine inflammatory response due to chronic or superphysiological level of microbial infection during pregnancy leads to pregnancy complications such as early pregnancy defects/loss and preterm birth. Bacterial toxin lipopolysaccharide (LPS), long recognized as a potent proinflammatory mediator, has been identified as a risk factor for pregnancy complications. Alkaline phosphatase (AP) isozymes have been shown to detoxify LPS by dephosphorylation. In this study, we examined the role of alkaline phosphatase (AP) in mitigating LPS-induced early pregnancy complications in mice. We found that 1) the uterus prior to implantation and implantation sites following embryo implantation produce LPS recognition and dephosphorylation molecules TLR4 and tissue non-specific AP (TNAP) isozyme, respectively; 2) uterine TNAP isozyme dephosphorylates LPS at its sites of production; 3) while LPS administration following embryo implantation elicits proinflammatory cytokine mRNA levels at the embryo implantation sites (EISs) and causes early pregnancy loss, dephosphorylated LPS neither triggers proinflammatory cytokine mRNA levels at the EISs nor induces pregnancy complications; 4) AP isozyme supplementation to accelerate LPS detoxification attenuates LPS-induced pregnancy complications following embryo implantation. These findings suggest that a LPS dephosphorylation strategy using AP isozyme may have a unique therapeutic potential to mitigate LPS- or Gram-negative bacteria-induced pregnancy complications in at-risk women.
Topics: Alkaline Phosphatase; Animals; Disease Models, Animal; Embryo Implantation; Enzyme Activation; Female; Gene Expression; In Situ Hybridization; Inflammation; Isoenzymes; Lipopolysaccharide Receptors; Lipopolysaccharides; Mice; Myeloid Differentiation Factor 88; Phosphorylation; Pregnancy; Pregnancy Complications; RNA, Messenger; Real-Time Polymerase Chain Reaction; Toll-Like Receptor 4; Uterus
PubMed: 25910276
DOI: 10.1371/journal.pone.0123243 -
The EMBO Journal Aug 2012The anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase is tightly regulated to ensure programmed proteolysis in cells. The activity of the APC/C is positively...
The anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase is tightly regulated to ensure programmed proteolysis in cells. The activity of the APC/C is positively controlled by cyclin-dependent kinase (CDK), but a second level of control must also exist because phosphorylation inactivates Cdc20, a mitotic APC/C co-activator. How Cdc20 is dephosphorylated specifically, when CDK is high, has remained unexplained. Here, we show that phosphatases are crucial to activate the APC/C. Cdc20 is phosphorylated at six conserved residues (S50/T64/T68/T79/S114/S165) by CDK in Xenopus egg extracts. When all the threonine residues are phosphorylated, Cdc20 binding to and activation of the APC/C are inhibited. Their dephosphorylation is regulated depending on the sites and protein phosphatase 2A, active in mitosis, is essential to dephosphorylate the threonine residues and activate the APC/C. Consistently, most of the Cdc20 bound to the APC/C in anaphase evades phosphorylation at T79. Furthermore, we show that the 'activation domain' of Cdc20 associates with the Apc6 and Apc8 core subunits. Our data suggest that dephosphorylation of Cdc20 is required for its loading and activation of the APC/C ubiquitin ligase.
Topics: Anaphase-Promoting Complex-Cyclosome; Animals; Cdc20 Proteins; Cell Cycle Proteins; Cells, Cultured; Enzyme Activation; Female; Mice; Mice, Inbred BALB C; Mitosis; Models, Biological; Phosphoprotein Phosphatases; Phosphorylation; Protein Binding; Protein Processing, Post-Translational; Protein Structure, Tertiary; Spodoptera; Ubiquitin-Protein Ligase Complexes; Xenopus Proteins; Xenopus laevis
PubMed: 22713866
DOI: 10.1038/emboj.2012.168 -
Journal of Hematology & Oncology Feb 2011The BCR-ABL1 translocation occurs in chronic myeloid leukemia (CML) and in 25% of cases with acute lymphoblastic leukemia (ALL). The advent of tyrosine kinase inhibitors...
BACKGROUND
The BCR-ABL1 translocation occurs in chronic myeloid leukemia (CML) and in 25% of cases with acute lymphoblastic leukemia (ALL). The advent of tyrosine kinase inhibitors (TKI) has fundamentally changed the treatment of CML. However, TKI are not equally effective for treating ALL. Furthermore, de novo or secondary TKI-resistance is a significant problem in CML. We screened a panel of BCR-ABL1 positive ALL and CML cell lines to find models for imatinib-resistance.
RESULTS
Five of 19 BCR-ABL1 positive cell lines were resistant to imatinib-induced apoptosis (KCL-22, MHH-TALL1, NALM-1, SD-1, SUP-B15). None of the resistant cell lines carried mutations in the kinase domain of BCR-ABL1 and all showed resistance to second generation TKI, nilotinib or dasatinib. STAT5, ERK1/2 and the ribosomal S6 protein (RPS6) are BCR-ABL1 downstream effectors, and all three proteins are dephosphorylated by imatinib in sensitive cell lines. TKI-resistant phosphorylation of RPS6, but responsiveness as regards JAK/STAT5 and ERK1/2 signalling were characteristic for resistant cell lines. PI3K pathway inhibitors effected dephosphorylation of RPS6 in imatinib-resistant cell lines suggesting that an oncogene other than BCR-ABL1 might be responsible for activation of the PI3K/AKT1/mTOR pathway, which would explain the TKI resistance of these cells. We show that the TKI-resistant cell line KCL-22 carries a PI3Kα E545G mutation, a site critical for the constitutive activation of the PI3K/AKT1 pathway. Apoptosis in TKI-resistant cells could be induced by inhibition of AKT1, but not of mTOR.
CONCLUSION
We introduce five Philadelphia-chromosome positive cell lines as TKI-resistance models. None of these cell lines carries mutations in the kinase domain of BCR-ABL1 or other molecular aberrations previously indicted in the context of imatinib-resistance. These cell lines are unique as they dephosphorylate ERK1/2 and STAT5 after treatment with imatinib, while PI3K/AKT1/mTOR activity remains unaffected. Inhibition of AKT1 leads to apoptosis in the imatinib-resistant cell lines. In conclusion, Ph+ cell lines show a form of imatinib-resistance attributable to constitutive activation of the PI3K/AKT1 pathway. Mutations in PIK3CA, as observed in cell line KCL-22, or PI3K activating oncogenes may undelie TKI-resistance in these cell lines.
Topics: Apoptosis; Benzamides; Drug Resistance, Neoplasm; Enzyme Activation; Fusion Proteins, bcr-abl; Humans; Imatinib Mesylate; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Phosphatidylinositol 3-Kinases; Phosphorylation; Piperazines; Protein Kinase Inhibitors; Pyrimidines; Signal Transduction
PubMed: 21299849
DOI: 10.1186/1756-8722-4-6 -
Calcified Tissue International Jul 2017Phosphorylated osteopontin (OPN) inhibits hydroxyapatite crystal formation and growth, and bone alkaline phosphatase (BALP) promotes extracellular mineralization via the...
Phosphorylated osteopontin (OPN) inhibits hydroxyapatite crystal formation and growth, and bone alkaline phosphatase (BALP) promotes extracellular mineralization via the release of inorganic phosphate from the mineralization inhibitor inorganic pyrophosphate (PPi). Tartrate-resistant acid phosphatase (TRAP), produced by osteoclasts, osteoblasts, and osteocytes, exhibits potent phosphatase activity towards OPN; however, its potential capacity as a regulator of mineralization has not previously been addressed. We compared the efficiency of BALP and TRAP towards the endogenous substrates for BALP, i.e., PPi and pyridoxal 5'-phosphate (PLP), and their impact on mineralization in vitro via dephosphorylation of bovine milk OPN. TRAP showed higher phosphatase activity towards phosphorylated OPN and PPi compared to BALP, whereas the activity of TRAP and BALP towards PLP was comparable. Bovine milk OPN could be completely dephosphorylated by TRAP, liberating all its 28 phosphates, whereas BALP dephosphorylated at most 10 phosphates. OPN, dephosphorylated by either BALP or TRAP, showed a partially or completely attenuated phosphorylation-dependent inhibitory capacity, respectively, compared to native OPN on the formation of mineralized nodules. Thus, there are phosphorylations in OPN important for inhibition of mineralization that are removed by TRAP but not by BALP. In conclusion, our data indicate that both BALP and TRAP can alleviate the inhibitory effect of OPN on mineralization, suggesting a potential role for TRAP in skeletal mineralization. Further studies are warranted to explore the possible physiological relevance of TRAP in bone mineralization.
Topics: Alkaline Phosphatase; Animals; Calcification, Physiologic; Cattle; Cell Line; Diphosphates; Humans; Osteoblasts; Osteopontin; Tartrate-Resistant Acid Phosphatase
PubMed: 28303318
DOI: 10.1007/s00223-017-0259-2 -
FEBS Letters Dec 2017PP2A is composed of a scaffolding subunit (A), a catalytic subunit (C) and a regulatory subunit (B) that is classified into four families including B, B', B'' and...
PP2A is composed of a scaffolding subunit (A), a catalytic subunit (C) and a regulatory subunit (B) that is classified into four families including B, B', B'' and B'''/striatin. Here, we found that a distinct PP2A complex regulates NF-κB signalling by dephosphorylation of IKKβ, IκBα and RelA/p65. The PP2A core enzyme AC dimer and the holoenzyme AB'''C trimer dephosphorylate IKKβ, IκBα and RelA, whereas the ABC trimer dephosphorylates IκBα but not IKKβ and RelA in cells. In contrast, AB'C and AB''C trimers have little effect on dephosphorylation of these signalling proteins. These results suggest that different forms of PP2A regulate NF-κB pathway signalling through multiple steps each in a different manner, thereby finely tuning NF-κB- and IKKβ-mediated cellular responses.
Topics: Cells, Cultured; Humans; I-kappa B Kinase; NF-KappaB Inhibitor alpha; NF-kappa B; Phosphorylation; Protein Phosphatase 2; Protein Subunits; Signal Transduction; Transcription Factor RelA
PubMed: 29139553
DOI: 10.1002/1873-3468.12912 -
The Biochemical Journal Feb 1990Insulin stimulates autophosphorylation of the insulin receptor on multiple tyrosines in three domains: tyrosines 1316 and 1322 in the C-terminal tail, 1146, 1150 and...
Insulin stimulates autophosphorylation of the insulin receptor on multiple tyrosines in three domains: tyrosines 1316 and 1322 in the C-terminal tail, 1146, 1150 and 1151 in the tyrosine-1150 domain, and possibly 953, 960 or 972 in the juxtamembrane domain. In the present work the sequence of dephosphorylation of the various autophosphorylation sites by particulate and cytosolic preparations of phosphotyrosyl-protein phosphatase from rat liver was studied with autophosphorylated human placental insulin receptor as substrate. Both phosphatase preparations elicited a broadly similar pattern of dephosphorylation. The tyrosine-1150 domain in triphosphorylated form was found to be exquisitely sensitive to dephosphorylation, and was dephosphorylated 3-10-fold faster than the di- and monophosphorylated forms of the tyrosine-1150 domain or phosphorylation sites in other domains. The major route for dephosphorylation of the triphosphorylated tyrosine-1150 domain involved dephosphorylation of one of the phosphotyrosyl pair, 1150/1151, followed by phosphotyrosyl 1146 to generate a species monophosphorylated mainly (greater than 80%) at tyrosine 1150 or 1151. Insulin receptors monophosphorylated in the tyrosine-1150 domain disappeared slowly, and overall the other domains were completely dephosphorylated faster than the tyrosine-1150 domain. Dephosphorylation of the diphosphorylated C-terminal domain yielded insulin receptor in which the domain was singly phosphorylated at tyrosine 1322. Triphosphorylation of the insulin receptor in the tyrosine-1150 domain appears important in activating the receptor tyrosine kinase to phosphorylate other proteins. The extreme sensitivity of the triphosphorylated form of the tyrosine-1150 domain to dephosphorylation may thus be important in terminating or regulating insulin-receptor tyrosine kinase action and insulin signalling.
Topics: Animals; Binding Sites; Edetic Acid; Female; Humans; Kinetics; Liver; Peptide Mapping; Phosphoprotein Phosphatases; Phosphorylation; Phosphotyrosine; Placenta; Pregnancy; Protein Tyrosine Phosphatases; Rats; Receptor, Insulin; Serine Endopeptidases; Trypsin; Tyrosine
PubMed: 1689998
DOI: 10.1042/bj2660251