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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 -
Biochimica Et Biophysica Acta Mar 2013Yeast Pah1p phosphatidate phosphatase (PAP) catalyzes the penultimate step in the synthesis of triacylglycerol. PAP plays a crucial role in lipid homeostasis by... (Review)
Review
Yeast Pah1p phosphatidate phosphatase (PAP) catalyzes the penultimate step in the synthesis of triacylglycerol. PAP plays a crucial role in lipid homeostasis by controlling the relative proportions of its substrate phosphatidate and its product diacylglycerol. The cellular amounts of these lipid intermediates influence the synthesis of triacylglycerol and the pathways by which membrane phospholipids are synthesized. Physiological functions affected by PAP activity include phospholipid synthesis gene expression, nuclear/endoplasmic reticulum membrane growth, lipid droplet formation, and vacuole homeostasis and fusion. Yeast lacking Pah1p PAP activity are acutely sensitive to fatty acid-induced toxicity and exhibit respiratory deficiency. PAP is distinguished in its cellular location, catalytic mechanism, and physiological functions from Dpp1p and Lpp1p lipid phosphate phosphatases that utilize a variety of substrates that include phosphatidate. Phosphorylation/dephosphorylation is a major mechanism by which Pah1p PAP activity is regulated. Pah1p is phosphorylated by cytosolic-associated Pho85p-Pho80p, Cdc28p-cyclin B, and protein kinase A and is dephosphorylated by the endoplasmic reticulum-associated Nem1p-Spo7p phosphatase. The dephosphorylation of Pah1p stimulates PAP activity and facilitates the association with the membrane/phosphatidate allowing for its reaction and triacylglycerol synthesis. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism.
Topics: Animals; Cyclic AMP-Dependent Protein Kinases; Cyclin-Dependent Kinases; Diglycerides; Gene Expression Regulation, Fungal; Lipid Metabolism; Membrane Proteins; Nuclear Proteins; Phosphatidate Phosphatase; Phosphatidic Acids; Phosphorylation; Pyrophosphatases; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Triglycerides
PubMed: 22910056
DOI: 10.1016/j.bbalip.2012.08.006 -
Nature Communications Apr 2023The Hsp90 molecular chaperone collaborates with the phosphorylated Cdc37 cochaperone for the folding and activation of its many client kinases. As with many kinases, the...
The Hsp90 molecular chaperone collaborates with the phosphorylated Cdc37 cochaperone for the folding and activation of its many client kinases. As with many kinases, the Hsp90 client kinase CRaf is activated by phosphorylation at specific regulatory sites. The cochaperone phosphatase PP5 dephosphorylates CRaf and Cdc37 in an Hsp90-dependent manner. Although dephosphorylating Cdc37 has been proposed as a mechanism for releasing Hsp90-bound kinases, here we show that Hsp90 bound kinases sterically inhibit Cdc37 dephosphorylation indicating kinase release must occur before Cdc37 dephosphorylation. Our cryo-EM structure of PP5 in complex with Hsp90:Cdc37:CRaf reveals how Hsp90 both activates PP5 and scaffolds its association with the bound CRaf to dephosphorylate phosphorylation sites neighboring the kinase domain. Thus, we directly show how Hsp90's role in maintaining protein homeostasis goes beyond folding and activation to include post translationally modifying its client kinases.
Topics: Humans; Cell Cycle Proteins; Protein Binding; HSP90 Heat-Shock Proteins; Molecular Chaperones
PubMed: 37069154
DOI: 10.1038/s41467-023-37659-7 -
Molecular & Cellular Proteomics : MCP Aug 2023Protein phosphorylation is an essential regulatory mechanism that controls most cellular processes, including cell cycle progression, cell division, and response to...
Protein phosphorylation is an essential regulatory mechanism that controls most cellular processes, including cell cycle progression, cell division, and response to extracellular stimuli, among many others, and is deregulated in many diseases. Protein phosphorylation is coordinated by the opposing activities of protein kinases and protein phosphatases. In eukaryotic cells, most serine/threonine phosphorylation sites are dephosphorylated by members of the Phosphoprotein Phosphatase (PPP) family. However, we only know for a few phosphorylation sites which specific PPP dephosphorylates them. Although natural compounds such as calyculin A and okadaic acid inhibit PPPs at low nanomolar concentrations, no selective chemical PPP inhibitors exist. Here, we demonstrate the utility of endogenous tagging of genomic loci with an auxin-inducible degron (AID) as a strategy to investigate specific PPP signaling. Using Protein Phosphatase 6 (PP6) as an example, we demonstrate how rapidly inducible protein degradation can be employed to identify dephosphorylation sites and elucidate PP6 biology. Using genome editing, we introduce AID-tags into each allele of the PP6 catalytic subunit (PP6c) in DLD-1 cells expressing the auxin receptor Tir1. Upon rapid auxin-induced degradation of PP6c, we perform quantitative mass spectrometry-based proteomics and phosphoproteomics to identify PP6 substrates in mitosis. PP6 is an essential enzyme with conserved roles in mitosis and growth signaling. Consistently, we identify candidate PP6c-dependent dephosphorylation sites on proteins implicated in coordinating the mitotic cell cycle, cytoskeleton, gene expression, and mitogen-activated protein kinase (MAPK) and Hippo signaling. Finally, we demonstrate that PP6c opposes the activation of large tumor suppressor 1 (LATS1) by dephosphorylating Threonine 35 (T35) on Mps One Binder (MOB1), thereby blocking the interaction of MOB1 and LATS1. Our analyses highlight the utility of combining genome engineering, inducible degradation, and multiplexed phosphoproteomics to investigate signaling by individual PPPs on a global level, which is currently limited by the lack of tools for specific interrogation.
Topics: Humans; Proteolysis; Protein Serine-Threonine Kinases; Phosphoprotein Phosphatases; Phosphorylation; Threonine; Colorectal Neoplasms; Protein Phosphatase 2
PubMed: 37392812
DOI: 10.1016/j.mcpro.2023.100614 -
The Biochemical Journal Dec 1988Calmodulin-dependent protein phosphatase has been proposed to be an important phosphotyrosyl-protein phosphatase. The ability of the enzyme to attack autophosphorylated...
Calmodulin-dependent protein phosphatase has been proposed to be an important phosphotyrosyl-protein phosphatase. The ability of the enzyme to attack autophosphorylated insulin receptor was examined and compared with the known ability of the enzyme to act on autophosphorylated epidermal-growth-factor (EGF) receptor. Purified calmodulin-dependent protein phosphatase was shown to catalyse the complete dephosphorylation of phosphotyrosyl-(insulin receptor). When compared at similar concentrations, 32P-labelled EGF receptor was dephosphorylated at greater than 3 times the rate of 32P-labelled insulin receptor; both dephosphorylations exhibited similar dependence on metal ions and calmodulin. Native phosphotyrosyl-protein phosphatases in cell extracts were also characterized. With rat liver, heart or brain, most (75%) of the native phosphatase activity against both 32P-labelled insulin and EGF receptors was recovered in the particulate fraction of the cell, with only 25% in the soluble fraction. This subcellular distribution contrasts with results of previous studies using artificial substrates, which found most of the phosphotyrosyl-protein phosphatase activity in the soluble fraction of the cell. Properties of particulate and soluble phosphatase activity against 32P-labelled insulin and EGF receptors are reported. The contribution of calmodulin-dependent protein phosphatase activity to phosphotyrosyl-protein phosphatase activity in cell fractions was determined by utilizing the unique metal-ion dependence of calmodulin-dependent protein phosphatase. Whereas Ni2+ (1 mM) markedly activated the calmodulin-dependent protein phosphatase, it was found to inhibit potently both particulate and soluble phosphotyrosyl-protein phosphatase activity. In fractions from rat liver, brain and heart, total phosphotyrosyl-protein phosphatase activity against both 32P-labelled receptors was inhibited by 99.5 +/- 6% (mean +/- S.E.M., 30 observations) by Ni2+. Results of Ni2+ inhibition studies were confirmed by other methods. It is concluded that in cell extracts phosphotyrosyl-protein phosphatases other than calmodulin-dependent protein phosphatase are the major phosphotyrosyl-(insulin receptor) and -(EGF receptor) phosphatases.
Topics: Animals; Calcineurin; Calmodulin-Binding Proteins; ErbB Receptors; Liver; Phosphoprotein Phosphatases; Phosphorylation; Protein Tyrosine Phosphatases; Rats; Receptor, Insulin
PubMed: 2852008
DOI: 10.1042/bj2560893 -
Molecular Cell Mar 2009The control of biological events requires strict regulation using complex protein phosphorylation and dephosphorylation strategies. The bulk of serine-threonine... (Review)
Review
The control of biological events requires strict regulation using complex protein phosphorylation and dephosphorylation strategies. The bulk of serine-threonine dephosphorylations are catalyzed by a handful of phosphatase catalytic subunits, giving rise to the misconception that these phosphatases are promiscuous and unregulated enzymes in vivo. The reality is much more nuanced: PP1 and PP2A, the most abundant serine-threonine phosphatases, are, in fact, families of hundreds of protein serine/threonine phosphatases, assembled from a few catalytic subunits in combination with a highly diverse array of regulators. As recent publications illustrate, these regulatory subunits confer specificity, selectivity, localization, and regulation on these important enzymes.
Topics: Animals; Catalytic Domain; Humans; Intracellular Signaling Peptides and Proteins; Isoenzymes; Models, Molecular; Phosphoprotein Phosphatases; Phosphorylation; Protein Conformation; Protein Phosphatase 1; Protein Phosphatase 2; Protein Processing, Post-Translational; Signal Transduction; Substrate Specificity; Transforming Growth Factor beta; tau Proteins
PubMed: 19285938
DOI: 10.1016/j.molcel.2009.02.015 -
Frontiers in Plant Science 2022Serine/threonine protein phosphatase 2C (PP2C) dephosphorylates proteins and plays crucial roles in plant growth, development, and stress response. In this study, we...
Serine/threonine protein phosphatase 2C (PP2C) dephosphorylates proteins and plays crucial roles in plant growth, development, and stress response. In this study, we characterized a clade B member of maize PP2C family, i.e., ZmPP2C26, that negatively regulated drought tolerance by dephosphorylating ZmMAPK3 and ZmMAPK7 in maize. The gene generated and isoforms through untypical alternative splicing. ZmPP2C26S lost 71 amino acids including an MAPK interaction motif and showed higher phosphatase activity than ZmPP2C26L. ZmPP2C26L directly interacted with, dephosphorylated ZmMAPK3 and ZmMAPK7, and localized in chloroplast and nucleus, but ZmPP2C26S only dephosphorylated ZmMAPK3 and localized in cytosol and nucleus. The expression of and was significantly inhibited by drought stress. Meanwhile, the maize mutant exhibited enhancement of drought tolerance with higher root length, root weight, chlorophyll content, and photosynthetic rate compared with wild type. However, overexpression of and significantly decreased drought tolerance in and rice with lower root length, chlorophyll content, and photosynthetic rate. Phosphoproteomic analysis revealed that the ZmPP2C26 protein also altered phosphorylation level of proteins involved in photosynthesis. This study provides insights into understanding the mechanism of PP2C in response to abiotic stress.
PubMed: 35463404
DOI: 10.3389/fpls.2022.851531 -
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 -
The Journal of Biological Chemistry 2021Histidine phosphorylation is a posttranslational modification that alters protein function and also serves as an intermediate of phosphoryl transfer. Although...
Histidine phosphorylation is a posttranslational modification that alters protein function and also serves as an intermediate of phosphoryl transfer. Although phosphohistidine is relatively unstable, enzymatic dephosphorylation of this residue is apparently needed in some contexts, since both prokaryotic and eukaryotic phosphohistidine phosphatases have been reported. Here we identify the mechanism by which a bacterial phosphohistidine phosphatase dephosphorylates the nitrogen-related phosphotransferase system, a broadly conserved bacterial pathway that controls diverse metabolic processes. We show that the phosphatase SixA dephosphorylates the phosphocarrier protein NPr and that the reaction proceeds through phosphoryl transfer from a histidine on NPr to a histidine on SixA. In addition, we show that Escherichia coli lacking SixA are outcompeted by wild-type E. coli in the context of commensal colonization of the mouse intestine. Notably, this colonization defect requires NPr and is distinct from a previously identified in vitro growth defect associated with dysregulation of the nitrogen-related phosphotransferase system. The widespread conservation of SixA, and its coincidence with the phosphotransferase system studied here, suggests that this dephosphorylation mechanism may be conserved in other bacteria.
Topics: Bacterial Proteins; Escherichia coli; Histidine; Phosphoric Monoester Hydrolases; Phosphorylation; Signal Transduction
PubMed: 33199374
DOI: 10.1074/jbc.RA120.015121 -
The Biochemical Journal Jul 2015Activating mutations in the leucine rich repeat protein kinase 2 (LRRK2) gene are the most common cause of inherited Parkinson's disease (PD). LRRK2 is phosphorylated on...
Activating mutations in the leucine rich repeat protein kinase 2 (LRRK2) gene are the most common cause of inherited Parkinson's disease (PD). LRRK2 is phosphorylated on a cluster of phosphosites including Ser(910), Ser(935), Ser(955) and Ser(973), which are dephosphorylated in several PD-related LRRK2 mutants (N1437H, R1441C/G, Y1699C and I2020T) linking the regulation of these sites to PD. These serine residues are also dephosphorylated after kinase inhibition and lose 14-3-3 binding, which serves as a pharmacodynamic marker for inhibited LRRK2. Loss of 14-3-3 binding is well established, but the consequences of dephosphorylation are only now being uncovered. In the present study, we found that potent and selective inhibition of LRRK2 kinase activity leads to dephosphorylation of Ser(935) then ubiquitination and degradation of a significant fraction of LRRK2. GNE1023 treatment decreased the phosphorylation and stability of LRRK2 in expression systems and endogenous LRRK2 in A549 cells and in mouse dosing studies. We next established that LRRK2 is ubiquitinated through at least Lys(48) and Lys(63) ubiquitin linkages in response to inhibition. To investigate the link between dephosphorylation induced by inhibitor treatment and LRRK2 ubiquitination, we studied LRRK2 in conditions where it is dephosphorylated such as expression of PD mutants [R1441G, Y1699C and I2020T] or by blocking 14-3-3 binding to LRRK2 via difopein expression, and found LRRK2 is hyper-ubiquitinated. Calyculin A treatment prevents inhibitor and PD mutant induced dephosphorylation and reverts LRRK2 to a lesser ubiquitinated species, thus directly implicating phosphatase activity in LRRK2 ubiquitination. This dynamic dephosphorylation-ubiquitination cycle could explain detrimental loss-of-function phenotypes found in peripheral tissues of LRRK2 kinase inactive mutants, LRRK2 KO (knockout) animals and following LRRK2 inhibitor administration.
Topics: 14-3-3 Proteins; Amino Acid Substitution; Animals; Enzyme Inhibitors; HEK293 Cells; Humans; Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; Marine Toxins; Mice; Mutation, Missense; Oxazoles; Phosphorylation; Protein Serine-Threonine Kinases; Ubiquitination
PubMed: 25939886
DOI: 10.1042/BJ20141305