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Science Signaling Feb 2021, an important human parasite, has a flagellum that controls cell motility, morphogenesis, proliferation, and cell-cell communication. Inheritance of the newly assembled...
, an important human parasite, has a flagellum that controls cell motility, morphogenesis, proliferation, and cell-cell communication. Inheritance of the newly assembled flagellum during the cell cycle requires the Polo-like kinase homolog TbPLK and the kinetoplastid-specific protein phosphatase KPP1, although whether TbPLK acts on KPP1 or vice versa has been unclear. Here, we showed that dephosphorylation of TbPLK on Thr by KPP1 maintained low TbPLK activity in the flagellum-associated hook complex structure, thereby ensuring proper flagellum positioning and attachment. This dephosphorylation event required the recognition of phosphorylated Thr in the activation loop of TbPLK by the N-terminal Plus3 domain of KPP1 and the dephosphorylation of phosphorylated Thr in TbPLK by the C-terminal catalytic domain of KPP1. Dephosphorylation of TbPLK by KPP1 prevented hyperphosphorylation of the hook complex protein TbCentrin2, thereby allowing timely dephosphorylation of phosphorylated TbCentrin2 for hook complex duplication and flagellum positioning and attachment. Thus, KPP1 attenuates TbPLK activity by dephosphorylating TbPLK to facilitate flagellum inheritance.
Topics: Cell Cycle; Flagella; Phosphoprotein Phosphatases; Protozoan Proteins; Trypanosoma brucei brucei
PubMed: 33563698
DOI: 10.1126/scisignal.abc6435 -
Scientific Reports Aug 2023c-Myc, a transcription factor, induces cell proliferation and is often aberrantly or highly expressed in cancers. However, molecular mechanisms underlying this...
c-Myc, a transcription factor, induces cell proliferation and is often aberrantly or highly expressed in cancers. However, molecular mechanisms underlying this aberrantly high expression remain unclear. Here, we found that intracellular Ca concentration regulates c-Myc oncoprotein stability. We identified that calcineurin, a Ca-dependent protein phosphatase, is a positive regulator of c-Myc expression. Calcineurin depletion suppresses c-Myc targeted gene expression and c-Myc degradation. Calcineurin directly dephosphorylates Thr and Ser in c-Myc, which inhibit binding to the ubiquitin ligase Fbxw7. Mutations within the autoinhibitory domain of calcineurin, most frequently observed in cancer, may increase phosphatase activity, increasing c-Myc transcriptional activity in turn. Notably, calcineurin inhibition with FK506 decreased c-Myc expression with enhanced Thr and Ser phosphorylation in a mouse xenograft model. Thus, calcineurin can stabilize c-Myc, promoting tumor progression. Therefore, we propose that Ca signaling dysfunction affects cancer-cell proliferation via increased c-Myc stability and that calcineurin inhibition could be a new therapeutic target of c-Myc-overexpressing cancers.
Topics: Humans; Mice; Animals; Calcineurin; Transcriptional Activation; Transcription Factors; Gene Expression Regulation; Protein Processing, Post-Translational
PubMed: 37573463
DOI: 10.1038/s41598-023-40412-1 -
Cell Chemical Biology Jun 2022PDE3A-SLFN12 complex formation activates the SLFN12 RNase, but the biochemical details of RNase activation remain mysterious. In this issue of Cell Chemical Biology, Yan...
PDE3A-SLFN12 complex formation activates the SLFN12 RNase, but the biochemical details of RNase activation remain mysterious. In this issue of Cell Chemical Biology, Yan and colleagues report that two phosphoserines on SLFN12 are dephosphorylated in response to PDE3A binding, and this dephosphorylation is required for activation of the SLFN12 RNase.
Topics: Cell Line, Tumor; Phosphorylation; Ribonucleases
PubMed: 35714590
DOI: 10.1016/j.chembiol.2022.05.010 -
EMBO Reports May 2020The tumor suppressor Smad4, a key mediator of the TGF-β/BMP pathways, is essential for development and tissue homeostasis. Phosphorylation of Smad4 in its linker region...
The tumor suppressor Smad4, a key mediator of the TGF-β/BMP pathways, is essential for development and tissue homeostasis. Phosphorylation of Smad4 in its linker region catalyzed by the mitogen-activated protein kinase (MAPK) plays a pivotal role in regulating its transcriptional activity and stability. In contrast, roles of Smad4 dephosphorylation as a control mechanism of TGF-β/BMP signaling and the phosphatases responsible for its dephosphorylation remain so far elusive. Here, we identify Wip1 as a Smad4 phosphatase. Wip1 selectively binds and dephosphorylates Smad4 at Thr277, a key MAPK phosphorylation site, thereby regulating its nuclear accumulation and half-life. In Xenopus embryos, Wip1 limits mesoderm formation and favors neural induction by inhibiting TGF-β/BMP signals. Wip1 restrains TGF-β-induced growth arrest, migration, and invasion in human cells and enhances the tumorigenicity of cancer cells by repressing the antimitogenic activity of Smad4. We propose that Wip1-dependent dephosphorylation of Smad4 is critical for the regulation of TGF-β signaling.
Topics: Animals; Humans; Mitogen-Activated Protein Kinases; Phosphorylation; Protein Phosphatase 2C; Signal Transduction; Smad4 Protein; Transforming Growth Factor beta; Xenopus Proteins; Xenopus laevis
PubMed: 32103600
DOI: 10.15252/embr.201948693 -
Shock (Augusta, Ga.) May 2021We recently demonstrated that fibrinogen stabilizes syndecan-1 on the endothelial cell (EC) surface and contributes to EC barrier protection, though the intracellular...
INTRODUCTION
We recently demonstrated that fibrinogen stabilizes syndecan-1 on the endothelial cell (EC) surface and contributes to EC barrier protection, though the intracellular signaling pathway remains unclear. P21 (Rac1) activated kinase 1 (PAK1) is a protein kinase involved in intracellular signaling leading to actin cytoskeleton rearrangement and plays an important role in maintaining endothelial barrier integrity. We therefore hypothesized that fibrinogen binding to syndecan-1 activated the PAK1 pathway.
METHODS
Primary human lung microvascular endothelial cells were incubated in 10% lactated Ringers (LR) solution or 10% fibrinogen saline solution (5 mg/mL). Protein phosphorylation was determined by Western blot analysis and endothelial permeability measured by fluorescein isothiocyanate (FITC)-dextran. Cells were silenced by siRNA transfection. Protein concentration was measured in the lung lavages of mice.
RESULTS
Fibrinogen treatment resulted in increased syndecan-1, PAK1 activation (phosphorylation), cofilin activation (dephosphorylation), as well as decreased stress fibers and permeability when compared with LR treatment. Cofilin is an actin-binding protein that depolymerizes F-actin to decrease stress fiber formation. Notably, fibrinogen did not influence myosin light chain activation (phosphorylation), a mediator of EC tension. Silencing of PAK1 prevented fibrinogen-induced dephosphorylation of cofilin and barrier integrity. Moreover, to confirm the in vitro findings, mice underwent hemorrhagic shock and were resuscitated with either LR or fibrinogen. Hemorrhage shock decreased lung p-PAK1 levels and caused significant lung vascular leakage. However, fibrinogen administration increased p-PAK1 expression to near sham levels and remarkably prevented the lung leakage.
CONCLUSION
We have identified a novel pathway by which fibrinogen activates PAK1 signaling to stimulate/dephosphorylate cofilin, leading to disassembly of stress fibers and reduction of endothelial permeability.
Topics: Actin Depolymerizing Factors; Animals; Endothelial Cells; Fibrinogen; Male; Mice; Mice, Inbred C57BL; Signal Transduction; p21-Activated Kinases
PubMed: 32433215
DOI: 10.1097/SHK.0000000000001564 -
Autophagy 2018Mitophagy is a main type of selective autophagy, via which damaged mitochondria are selectively degraded via the autophagic pathway. The protein kinase PINK1 and E3...
UNLABELLED
Mitophagy is a main type of selective autophagy, via which damaged mitochondria are selectively degraded via the autophagic pathway. The protein kinase PINK1 and E3 ubiquitin ligase PRKN are the most well studied regulators of mitophagy, via a feedforward mechanism involving ubiquitin phosphorylation (p-Ser65-Ub) and accumulation at the damaged mitochondria. However, it is unknown whether there is a protein phosphatase against PINK1-mediated phosphorylation of ubiquitin. We recently reported that PTEN-L, a newly identified PTEN isoform, is a novel negative regulator of mitophagy through dephosphorylation of p-Ser65-Ub. Our data demonstrate that a significant portion of PTEN-L localizes at the outer mitochondrial membrane and is able to prevent PRKN's mitochondrial translocation, reduce the phosphorylation of PRKN, impair its E3 ligase activity as well as maintain PRKN in a closed/inactive status. Moreover, we found that PTEN-L dephosphorylates p-Ser65-Ub to disrupt the feedforward mechanism of mitophagy. Our findings suggest that PTEN-L acts as a brake in the regulation of mitophagy.
ABBREVIATIONS
ATR: alternatively translated region; CCCP: carbonylcyanide 3-chlorophenylhydrazone; DUBs: deubiquitinating enzymes; MFN2: mitofusion2; MS/MS: tandem mass spectrometry; mtDNA: mitochondrial DNA; MTS: mitochondrial targeting sequences; O/A: oligomycin and antimycin A; PINK1: PTEN induced putative kinase 1; PRKN/parkin: parkin RBR E3 ubiquitin protein ligase; PTEN: phosphatase and tensin homolog; PTEN-L: phosphatase and tensin homolog-long; Ub: ubiquitin; USP: ubiquitin-specific proteases; YFP: yellow fluorescence protein.
Topics: Autophagy; Mitophagy; Phosphoprotein Phosphatases; Protein Kinases; Tandem Mass Spectrometry; Ubiquitin; Ubiquitin-Protein Ligases
PubMed: 30106322
DOI: 10.1080/15548627.2018.1502565 -
PloS One 2020The pivotal role of KCC2 and NKCC1 in development and maintenance of fast inhibitory neurotransmission and their implication in severe human diseases arouse interest in...
The pivotal role of KCC2 and NKCC1 in development and maintenance of fast inhibitory neurotransmission and their implication in severe human diseases arouse interest in posttranscriptional regulatory mechanisms such as (de)phosphorylation. Staurosporine (broad kinase inhibitor) and N-ethylmalemide (NEM) that modulate kinase and phosphatase activities enhance KCC2 and decrease NKCC1 activity. Here, we investigated the regulatory mechanism for this reciprocal regulation by mass spectrometry and immunoblot analyses using phospho-specific antibodies. Our analyses revealed that application of staurosporine or NEM dephosphorylates Thr1007 of KCC2, and Thr203, Thr207 and Thr212 of NKCC1. Dephosphorylation of Thr1007 of KCC2, and Thr207 and Thr212 of NKCC1 were previously demonstrated to activate KCC2 and to inactivate NKCC1. In addition, application of the two agents resulted in dephosphorylation of the T-loop and S-loop phosphorylation sites Thr233 and Ser373 of SPAK, a critical kinase in the WNK-SPAK/OSR1 signaling module mediating phosphorylation of KCC2 and NKCC1. Taken together, these results suggest that reciprocal regulation of KCC2 and NKCC1 via staurosporine and NEM is based on WNK-SPAK/OSR1 signaling. The key regulatory phospho-site Ser940 of KCC2 is not critically involved in the enhanced activation of KCC2 upon staurosporine and NEM treatment, as both agents have opposite effects on its phosphorylation status. Finally, NEM acts in a tissue-specific manner on Ser940, as shown by comparative analysis in HEK293 cells and immature cultured hippocampal neurons. In summary, our analyses identified phospho-sites that are responsive to staurosporine or NEM application. This provides important information towards a better understanding of the cooperative interactions of different phospho-sites.
Topics: Animals; Binding Sites; Cells, Cultured; Ethylmaleimide; HEK293 Cells; Hippocampus; Humans; Neurons; Phosphorylation; Protein Kinases; Protein Serine-Threonine Kinases; Rats; Recombinant Proteins; Signal Transduction; Solute Carrier Family 12, Member 2; Staurosporine; Symporters; Transfection; WNK Lysine-Deficient Protein Kinase 1; K Cl- Cotransporters
PubMed: 32413057
DOI: 10.1371/journal.pone.0232967 -
Life (Basel, Switzerland) Jan 2023Phosphorylation of beta-amyloid peptide (Aβ) at the Ser8 residue affects its neurotoxicity, metal-dependent oligomerisation, amyloidogenicity, and other pathogenic...
Phosphorylation of beta-amyloid peptide (Aβ) at the Ser8 residue affects its neurotoxicity, metal-dependent oligomerisation, amyloidogenicity, and other pathogenic properties. Phosphorylated Aβ (pS8-Aβ) was detected in vivo in AD model mice and in the brains of patients with AD. However, the pS8-Aβ production and the regulation of its levels have not been previously studied in detail. In this paper, immunochemical methods together with radioactive labelling were used to study the Aβ phosphorylation by intracellular and surface protein kinases of HEK293 cells and brain endothelial cells (bEnd.3). It was found that HEK293 robustly phosphorylated Aβ, likely with contribution from casein kinase 2 (CK2), whereas in bEnd.3, the activity of Aβ phosphorylation was relatively low. Further, the study showed that both HEK293 and bEnd.3 could dephosphorylate pS8-Aβ, mainly due to the activity of protein phosphatases PP1 and PP2A. The Aβ dephosphorylation efficiency in bEnd.3 was three times higher than in HEK293, which correlated with the reduced abundance of pS8-Aβ in vascular amyloid deposits of patients with AD compared to senile plaques. These data suggest an important role of CK2, PP1, and PP2A as regulators of Aβ phosphorylation, and point to the involvement of the blood-brain barrier in the control of Aβ modification levels.
PubMed: 36676097
DOI: 10.3390/life13010147 -
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 -
Journal of Virology May 2017The C-terminal domain (CTD) of hepadnavirus core protein is involved in multiple steps of viral replication. In particular, the CTD is initially phosphorylated at...
The C-terminal domain (CTD) of hepadnavirus core protein is involved in multiple steps of viral replication. In particular, the CTD is initially phosphorylated at multiple sites to facilitate viral RNA packaging into immature nucleocapsids (NCs) and the early stage of viral DNA synthesis. For the avian hepadnavirus duck hepatitis B virus (DHBV), CTD is dephosphorylated subsequently to facilitate the late stage of viral DNA synthesis and to stabilize NCs containing mature viral DNA. The role of CTD phosphorylation in virion secretion, if any, has remained unclear. Here, the CTD from the human hepatitis B virus (HBV) was found to be dephosphorylated in association with NC maturation and secretion of DNA-containing virions, as in DHBV. In contrast, the CTD in empty HBV virions (i.e., enveloped capsids with no RNA or DNA) was found to be phosphorylated. The potential role of CTD dephosphorylation in virion secretion was analyzed through mutagenesis. For secretion of empty HBV virions, which is independent of either viral RNA packaging or DNA synthesis, multiple substitutions in the CTD to mimic either phosphorylation or dephosphorylation showed little detrimental effect. Similarly, phospho-mimetic substitutions in the DHBV CTD did not block the secretion of DNA-containing virions. These results indicate that CTD dephosphorylation, though associated with NC maturation in both HBV and DHBV, is not essential for the subsequent NC-envelope interaction to secrete DNA-containing virions, and the CTD state of phosphorylation also does not play an essential role in the interaction between empty capsids and the envelope for secretion of empty virions. The phosphorylation state of the C-terminal domain (CTD) of hepatitis B virus (HBV) core or capsid protein is highly dynamic and plays multiple roles in the viral life cycle. To study the potential role of the state of phosphorylation of CTD in virion secretion, we have analyzed the CTD phosphorylation state in complete (containing the genomic DNA) versus empty (genome-free) HBV virions. Whereas CTD is unphosphorylated in complete virions, it is phosphorylated in empty virions. Mutational analyses indicate that neither phosphorylation nor dephosphorylation of CTD is required for virion secretion. These results demonstrate that while CTD dephosphorylation is associated with HBV DNA synthesis, the CTD state of phosphorylation may not regulate virion secretion.
Topics: Animals; Capsid; Capsid Proteins; Cell Line, Tumor; Chickens; Hep G2 Cells; Hepatitis B Virus, Duck; Hepatitis B virus; Humans; Phosphorylation; Protein Structure, Tertiary; RNA, Viral; Virus Assembly; Virus Replication; Virus Shedding
PubMed: 28228589
DOI: 10.1128/JVI.00092-17