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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 -
Biophysics and Physicobiology 2024KaiC is a multifunctional enzyme functioning as the core of the circadian clock system in cyanobacteria: its N-terminal domain has adenosine triphosphatase (ATPase)...
KaiC is a multifunctional enzyme functioning as the core of the circadian clock system in cyanobacteria: its N-terminal domain has adenosine triphosphatase (ATPase) activity, and its C-terminal domain has autokinase and autophosphatase activities targeting own S431 and T432. The coordination of these multiple biochemical activities is the molecular basis for robust circadian rhythmicity. Therefore, much effort has been devoted to elucidating the cooperative relationship between the two domains. However, structural and functional relationships between the two domains remain unclear especially with respect to the dawn phase, at which KaiC relieves its nocturnal history through autodephosphorylation. In this study, we attempted to design a double mutation of S431 and T432 that can capture KaiC as a fully dephosphorylated form with minimal impacts on its structure and function, and investigated the cooperative relationship between the two domains in the night to morning phases from many perspectives. The results revealed that both domains cooperate at the dawn phase through salt bridges formed between the domains, thereby non-locally co-activating two events, ATPase de-inhibition and S431 dephosphorylation. Our further analysis using existing crystal structures of KaiC suggests that the states of both domains are not always in one-to-one correspondence at every phase of the circadian cycle, and their coupling is affected by the interactions with KaiA or adjacent subunits within a KaiC hexamer.
PubMed: 38803331
DOI: 10.2142/biophysico.bppb-v21.0001 -
Frontiers in Immunology 2023Macrophages play a critical role in the regulation of inflammation and tissue homeostasis. In addition to their vital functions for cell survival and physiology,...
Macrophages play a critical role in the regulation of inflammation and tissue homeostasis. In addition to their vital functions for cell survival and physiology, mitochondria play a crucial role in innate immunity as a platform for the induction of inflammatory responses by regulating cell signaling and dynamics. Dynamin-related protein 1 (Drp1) plays a role in the induction of inflammatory responses and the subsequent development of various diseases. PGAM5 (phosphoglycerate mutase member 5) is a mitochondrial outer membrane phosphatase that dephosphorylates its substrate, Drp1. Previous studies showed that PGAM5 regulates the phosphorylation of Drp1 for the activation of NKT cells and T cells. However, it is not clear how PGAM5 regulates Drp1 activity for the induction of inflammation in macrophages. Here, we demonstrate that PGAM5 activity regulates the dephosphorylation of Drp1 in macrophages, leading to the induction of proinflammatory responses in macrophages. In TLR signaling, PGAM5 regulates the expression and production of inflammatory cytokines by regulating the activation of downstream signaling pathways, including the NF-κB and MAPK pathways. Upon LPS stimulation, PGAM5 interacts with Drp1 to form a complex, leading to the production of mtROS. Furthermore, PGAM5-Drp1 signaling promotes the polarization of macrophages toward a proinflammatory phenotype. Our study further demonstrates that PGAM5-Drp1 signaling promotes metabolic reprogramming by upregulating glycolysis and mitochondrial metabolism in macrophages. Altogether, PGAM5 signaling is a linker between alterations in Drp1-mediated mitochondrial dynamics and inflammatory responses in macrophages and may be a target for the treatment of inflammatory diseases.
Topics: Humans; Dynamins; Inflammation; Macrophages; Mitochondrial Proteins; Phosphoprotein Phosphatases; Signal Transduction; Animals
PubMed: 37771598
DOI: 10.3389/fimmu.2023.1243548 -
International Journal of Molecular... Jan 2022Calcineurin, a calcium-dependent serine/threonine phosphatase, integrates the alterations in intracellular calcium levels into downstream signaling pathways by... (Review)
Review
Calcineurin, a calcium-dependent serine/threonine phosphatase, integrates the alterations in intracellular calcium levels into downstream signaling pathways by regulating the phosphorylation states of several targets. Intracellular Ca is essential for normal cellular physiology and cell cycle progression at certain critical stages of the cell cycle. Recently, it was reported that calcineurin is activated in a variety of cancers. Given that abnormalities in calcineurin signaling can lead to malignant growth and cancer, the calcineurin signaling pathway could be a potential target for cancer treatment. For example, NFAT, a typical substrate of calcineurin, activates the genes that promote cell proliferation. Furthermore, cyclin D1 and estrogen receptors are dephosphorylated and stabilized by calcineurin, leading to cell proliferation. In this review, we focus on the cell proliferative functions and regulatory mechanisms of calcineurin and summarize the various substrates of calcineurin. We also describe recent advances regarding dysregulation of the calcineurin activity in cancer cells. We hope that this review will provide new insights into the potential role of calcineurin in cancer development.
Topics: Calcineurin; Calcium; Cell Cycle; Cell Proliferation; Gene Expression Regulation, Neoplastic; Humans; NFATC Transcription Factors; Neoplasms; Phosphorylation; Signal Transduction
PubMed: 35163061
DOI: 10.3390/ijms23031122 -
Current Opinion in Structural Biology Oct 2016Glucan phosphatases are functionally conserved at the enzymatic level, dephosphorylating glycogen in animals and starch in plants. The human glucan phosphatase laforin... (Review)
Review
Glucan phosphatases are functionally conserved at the enzymatic level, dephosphorylating glycogen in animals and starch in plants. The human glucan phosphatase laforin is the founding member of the family and it is comprised of a carbohydrate binding module (CBM) domain followed by a dual specificity phosphatase (DSP) domain. Plants encode two glucan phosphatases: Starch EXcess4 (SEX4) and Like Sex Four2 (LSF2). SEX4 contains a DSP domain followed by a CBM domain, while LSF2 contains a DSP domain and lacks a CBM. This review demonstrates how glucan phosphatase function is conserved and highlights how each family member employs a unique mechanism to bind and dephosphorylate glucan substrates.
Topics: Animals; Glucans; Humans; Phosphoric Monoester Hydrolases; Phosphorylation; Plants; Substrate Specificity
PubMed: 27498086
DOI: 10.1016/j.sbi.2016.07.015 -
Frontiers in Oncology 2022Pyruvate dehydrogenase (PDH) complex converts pyruvate into acetyl-CoA by pyruvate decarboxylation, which drives energy metabolism during cell growth, including prostate...
BACKGROUND
Pyruvate dehydrogenase (PDH) complex converts pyruvate into acetyl-CoA by pyruvate decarboxylation, which drives energy metabolism during cell growth, including prostate cancer (PCa) cell growth. The major catalytic subunit of PDH, PDHA1, is regulated by phosphorylation/dephosphorylation by pyruvate dehydrogenase kinases (PDKs) and pyruvate dehydrogenase phosphatases (PDPs). There are four kinases, PDK1, PDK2, PDK3 and PDK4, which can phosphorylate and inactivate PDH; and two phosphatases, PDP1 and PDP2, that dephosphorylate and activate PDH.
METHODS
We have analyzed by immunohistochemistry the expression and clinicopathological correlations of PDHA1, PDP1, PDP2, PDK1, PDK2, PDK3, and PDK4, as well as of androgen receptor (AR), in a retrospective PCa cohort of patients. A total of 120 PCa samples of representative tumor areas from all patients were included in tissue microarray (TMA) blocks for analysis. In addition, we studied the subcellular localization of PDK2 and PDK3, and the effects of the PDK inhibitor dichloroacetate (DCA) in the growth, proliferation, and mitochondrial respiration of PCa cells.
RESULTS
We found heterogeneous expression of the PDH complex components in PCa tumors. PDHA1, PDP1, PDK1, PDK2, and PDK4 expression correlated positively with AR expression. A significant correlation of PDK2 immunostaining with biochemical recurrence and disease-free survival was revealed. In PCa tissue specimens, PDK2 displayed cytoplasmic and nuclear immunostaining, whereas PDK1, PDK3 and PDK4 showed mostly cytoplasmic staining. In cells, ectopically expressed PDK2 and PDK3 were mainly localized in mitochondria compartments. An increase in maximal mitochondrial respiration was observed in PCa cells upon PDK inhibition by DCA, in parallel with less proliferative capacity.
CONCLUSION
Our findings support the notion that expression of specific PDH complex components is related with AR signaling in PCa tumors. Furthermore, PDK2 expression associated with poor PCa prognosis. This highlights a potential for PDH complex components as targets for intervention in PCa.
PubMed: 35692804
DOI: 10.3389/fonc.2022.873516 -
International Journal of Molecular... Sep 2018The signal transducer and activator of transcription 3 (STAT3) protein is a major transcription factor involved in many cellular processes, such as cell growth and... (Review)
Review
The signal transducer and activator of transcription 3 (STAT3) protein is a major transcription factor involved in many cellular processes, such as cell growth and proliferation, differentiation, migration, and cell death or cell apoptosis. It is activated in response to a variety of extracellular stimuli including cytokines and growth factors. The aberrant activation of STAT3 contributes to several human diseases, particularly cancer. Consequently, STAT3-mediated signaling continues to be extensively studied in order to identify potential targets for the development of new and more effective clinical therapeutics. STAT3 activation can be regulated, either positively or negatively, by different posttranslational mechanisms including serine or tyrosine phosphorylation/dephosphorylation, acetylation, or demethylation. One of the major mechanisms that negatively regulates STAT3 activation is dephosphorylation of the tyrosine residue essential for its activation by protein tyrosine phosphatases (PTPs). There are seven PTPs that have been shown to dephosphorylate STAT3 and, thereby, regulate STAT3 signaling: PTP receptor-type D (PTPRD), PTP receptor-type T (PTPRT), PTP receptor-type K (PTPRK), Src homology region 2 (SH-2) domain-containing phosphatase 1(SHP1), SH-2 domain-containing phosphatase 2 (SHP2), MEG2/PTP non-receptor type 9 (PTPN9), and T-cell PTP (TC-PTP)/PTP non-receptor type 2 (PTPN2). These regulators have great potential as targets for the development of more effective therapies against human disease, including cancer.
Topics: Animals; Humans; Mutation; Neoplasms; Phosphorylation; Protein Processing, Post-Translational; Protein Tyrosine Phosphatases; STAT3 Transcription Factor; Signal Transduction
PubMed: 30208623
DOI: 10.3390/ijms19092708 -
The FEBS Journal Apr 2016Tyr phosphatases are, by definition, enzymes that dephosphorylate phospho-Tyr (pTyr) from proteins. This activity is found in several structurally diverse protein... (Review)
Review
Tyr phosphatases are, by definition, enzymes that dephosphorylate phospho-Tyr (pTyr) from proteins. This activity is found in several structurally diverse protein families, including the protein Tyr phosphatase (PTP), arsenate reductase, rhodanese, haloacid dehalogenase (HAD) and His phosphatase (HP) families. Most of these families include members with substrate specificity for non-pTyr substrates, such as phospho-Ser/phospho-Thr, phosphoinositides, phosphorylated carbohydrates, mRNAs, or inorganic moieties. A Cys is essential for catalysis in PTPs, rhodanese and arsenate reductase enzymes, whereas this work is performed by an Asp in HAD phosphatases and by a His in HPs, via a catalytic mechanism shared by all of the different families. The category that contains most Tyr phosphatases is the PTP family, which, although it received its name from this activity, includes Ser, Thr, inositide, carbohydrate and RNA phosphatases, as well as some inactive pseudophosphatase proteins. Here, we propose an extended collection of human Tyr phosphatases, which we call the extended human PTPome. The addition of new members (SACs, paladin, INPP4s, TMEM55s, SSU72, and acid phosphatases) to the currently categorized PTP group of enzymes means that the extended human PTPome contains up to 125 proteins, of which ~ 40 are selective for pTyr. We set criteria to ascribe proteins to the extended PTPome, and summarize the more important features of the new PTPome members in the context of their phosphatase activity and their relationship with human disease.
Topics: Animals; Catalysis; Humans; Phosphoprotein Phosphatases; Phosphorylation; Phosphotyrosine; Protein Tyrosine Phosphatases; Substrate Specificity
PubMed: 26573778
DOI: 10.1111/febs.13600 -
Frontiers in Cellular and Infection... 2021Protein phosphorylation and dephosphorylation are increasingly recognized as important processes for regulating multiple physiological mechanisms. Phosphorylation is...
Protein phosphorylation and dephosphorylation are increasingly recognized as important processes for regulating multiple physiological mechanisms. Phosphorylation is carried out by protein kinases and dephosphorylation by protein phosphatases. Phosphoprotein phosphatases (PPPs), one of three families of protein serine/threonine phosphatases, have great structural diversity and are involved in regulating many cell functions. PP2C, a type of PPP, is found in , a dimorphic protozoan parasite and the causal agent of leishmaniasis. The aim of this study was to clone, purify, biochemically characterize and quantify the expression of PP2C in (PP2C). Recombinant PP2C dephosphorylated a specific threonine (with optimal activity at pH 8) in the presence of the manganese divalent cation (Mn). PP2C activity was inhibited by sanguinarine (a specific inhibitor) but was unaffected by protein tyrosine phosphatase inhibitors. Western blot analysis indicated that anti-PP2C antibodies recognized a molecule of 45.2 kDa. Transmission electron microscopy with immunodetection localized PP2C in the flagellar pocket and flagellum of promastigotes but showed poor staining in amastigotes. Interestingly, PP2C belongs to the ortholog group OG6_142542, which contains only protozoa of the family Trypanosomatidae. This suggests a specific function of the enzyme in the flagellar pocket of these microorganisms.
Topics: Humans; Leishmania; Leishmania mexicana; Leishmaniasis; Phosphoprotein Phosphatases; Phosphorylation; Serine
PubMed: 33937094
DOI: 10.3389/fcimb.2021.641356 -
Molecular Cell Feb 2024Regulated protein phosphorylation controls most cellular processes. The protein phosphatase PP1 is the catalytic subunit of many holoenzymes that dephosphorylate...
Regulated protein phosphorylation controls most cellular processes. The protein phosphatase PP1 is the catalytic subunit of many holoenzymes that dephosphorylate serine/threonine residues. How these enzymes recruit their substrates is largely unknown. Here, we integrated diverse approaches to elucidate how the PP1 non-catalytic subunit PPP1R15B (R15B) captures its full trimeric eIF2 substrate. We found that the substrate-recruitment module of R15B is largely disordered with three short helical elements, H1, H2, and H3. H1 and H2 form a clamp that grasps the substrate in a region remote from the phosphorylated residue. A homozygous N423D variant, adjacent to H1, reducing substrate binding and dephosphorylation was discovered in a rare syndrome with microcephaly, developmental delay, and intellectual disability. These findings explain how R15B captures its 125 kDa substrate by binding the far end of the complex relative to the phosphosite to present it for dephosphorylation by PP1, a paradigm of broad relevance.
Topics: Humans; Catalytic Domain; Eukaryotic Initiation Factor-2; Phosphorylation; Protein Phosphatase 1
PubMed: 38159565
DOI: 10.1016/j.molcel.2023.12.011