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ELife Sep 2020APC/C-mediated proteolysis of cyclin B and securin promotes anaphase entry, inactivating CDK1 and permitting chromosome segregation, respectively. Reduction of CDK1...
APC/C-mediated proteolysis of cyclin B and securin promotes anaphase entry, inactivating CDK1 and permitting chromosome segregation, respectively. Reduction of CDK1 activity relieves inhibition of the CDK1-counteracting phosphatases PP1 and PP2A-B55, allowing wide-spread dephosphorylation of substrates. Meanwhile, continued APC/C activity promotes proteolysis of other mitotic regulators. Together, these activities orchestrate a complex series of events during mitotic exit. However, the relative importance of regulated proteolysis and dephosphorylation in dictating the order and timing of these events remains unclear. Using high temporal-resolution proteomics, we compare the relative extent of proteolysis and protein dephosphorylation. This reveals highly-selective rapid proteolysis of cyclin B, securin and geminin at the metaphase-anaphase transition, followed by slow proteolysis of other substrates. Dephosphorylation requires APC/C-dependent destruction of cyclin B and was resolved into PP1-dependent categories with unique sequence motifs. We conclude that dephosphorylation initiated by selective proteolysis of cyclin B drives the bulk of changes observed during mitotic exit.
Topics: CDC2 Protein Kinase; Cyclin B; HeLa Cells; Humans; Mitosis; Phosphorylation; Protein Phosphatase 1; Protein Phosphatase 2; Proteolysis
PubMed: 32869743
DOI: 10.7554/eLife.59885 -
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 -
Biochimica Et Biophysica Acta.... Jan 2021Protein phosphatase 2A (PP2A) complex comprises an extended family of intracellular protein serine/threonine phosphatases, that participate in different signaling...
Protein phosphatase 2A (PP2A) complex comprises an extended family of intracellular protein serine/threonine phosphatases, that participate in different signaling transduction pathways. Different functions of PP2As are determined by the variety of regulatory subunits. In this study, CRISPR/Cas9-mediated loss-of-function screen revealed that PPP2R2A downregulation suppressed cell growth in NSCLC cells. AMOTL2 was identified and confirmed as a novel binding partner of PPP2R2A in NSCLC cells by mass spectrometry, CO-IP, GST pull-down and immunofluorescence. Upregulation of AMOTL2 also led to cell proliferation delay in human and mouse lung tumor cells. The proto-oncogene JUN is a key subunit of activator protein-1 (AP-1) transcription factor which plays crucial role in regulating tumorigenesis and its activity is negatively regulated by the phosphorylation at T239. Our results showed that either AMOTL2 upregulation or PPP2R2A downregulation led to great increase in JUN T239 phosphorylation. AMOTL2 bound PPP2R2A in cytoplasm, which reduced nuclear localization of PPP2R2A. In conclusion, AMOTL2 and PPP2R2A act respectively as negative and positive regulator of cell growth in NSCLC cells and function in the AMOTL2-PPP2R2A-JUN axis, in which AMOTL2 inhibits the entry of PPP2R2A into the nucleus to dephosphorylate JUN at T239.
Topics: Angiomotins; Carcinoma, Non-Small-Cell Lung; Carrier Proteins; Cell Line, Tumor; Cell Proliferation; Gene Expression Regulation, Neoplastic; Humans; MAP Kinase Signaling System; Phosphorylation; Protein Phosphatase 2; Proto-Oncogene Mas; Proto-Oncogene Proteins c-jun; Transcription Factor AP-1; Up-Regulation
PubMed: 32950569
DOI: 10.1016/j.bbamcr.2020.118858 -
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 -
Applied and Environmental Microbiology Feb 2016Burkholderia cenocepacia, a member of the B. cepacia complex (Bcc), is an opportunistic pathogen causing serious chronic infections in patients with cystic fibrosis....
Burkholderia cenocepacia, a member of the B. cepacia complex (Bcc), is an opportunistic pathogen causing serious chronic infections in patients with cystic fibrosis. Tyrosine phosphorylation has emerged as an important posttranslational modification modulating the physiology and pathogenicity of Bcc bacteria. Here, we investigated the predicted bacterial tyrosine kinases BCAM1331 and BceF and the low-molecular-weight protein tyrosine phosphatases BCAM0208, BceD, and BCAL2200 of B. cenocepacia K56-2. We show that BCAM1331, BceF, BCAM0208, and BceD contribute to biofilm formation, while BCAL2200 is required for growth under nutrient-limited conditions. Multiple deletions of either tyrosine kinase or low-molecular-weight protein tyrosine phosphatase genes resulted in the attenuation of B. cenocepacia intramacrophage survival and reduced pathogenicity in the Galleria mellonella larval infection model. Experimental evidence indicates that BCAM1331 displays reduced tyrosine autophosphorylation activity compared to that of BceF. With the artificial substrate p-nitrophenyl phosphate, the phosphatase activities of the three low-molecular-weight protein tyrosine phosphatases demonstrated similar kinetic parameters. However, only BCAM0208 and BceD could dephosphorylate BceF. Further, BCAL2200 became tyrosine phosphorylated in vivo and catalyzed its autodephosphorylation. Together, our data suggest that despite having similar biochemical activities, low-molecular-weight protein tyrosine phosphatases and tyrosine kinases have both overlapping and specific roles in the physiology of B. cenocepacia.
Topics: Animals; Bacterial Proteins; Biofilms; Burkholderia cenocepacia; Gene Deletion; Gene Expression Regulation, Bacterial; Humans; Larva; Macrophages; Mice; Moths; Phosphorylation; Phosphotyrosine; Protein Tyrosine Phosphatases; Protein-Tyrosine Kinases; RAW 264.7 Cells; Virulence
PubMed: 26590274
DOI: 10.1128/AEM.03513-15 -
Molecular Cell Oct 2022Neuronal activity induces topoisomerase IIβ (Top2B) to generate DNA double-strand breaks (DSBs) within the promoters of neuronal early response genes (ERGs) and...
Neuronal activity induces topoisomerase IIβ (Top2B) to generate DNA double-strand breaks (DSBs) within the promoters of neuronal early response genes (ERGs) and facilitate their transcription, and yet, the mechanisms that control Top2B-mediated DSB formation are unknown. Here, we report that stimulus-dependent calcium influx through NMDA receptors activates the phosphatase calcineurin to dephosphorylate Top2B at residues S1509 and S1511, which stimulates its DNA cleavage activity and induces it to form DSBs. Exposing mice to a fear conditioning paradigm also triggers Top2B dephosphorylation at S1509 and S1511 in the hippocampus, indicating that calcineurin also regulates Top2B-mediated DSB formation following physiological neuronal activity. Furthermore, calcineurin-Top2B interactions following neuronal activity and sites that incur activity-induced DSBs are preferentially localized at the nuclear periphery in neurons. Together, these results reveal how radial gene positioning and the compartmentalization of activity-dependent signaling govern the position and timing of activity-induced DSBs and regulate gene expression patterns in neurons.
Topics: Animals; Mice; Calcineurin; Calcium; DNA; DNA Breaks, Double-Stranded; DNA Topoisomerases, Type II; DNA-Binding Proteins; Neurons; Receptors, N-Methyl-D-Aspartate
PubMed: 36206766
DOI: 10.1016/j.molcel.2022.09.012 -
The Journal of Cell Biology May 2022Autophagy is a conserved eukaryotic lysosomal degradation pathway that responds to environmental and cellular cues. Autophagy is essential for the meiotic exit and...
Autophagy is a conserved eukaryotic lysosomal degradation pathway that responds to environmental and cellular cues. Autophagy is essential for the meiotic exit and sporulation in budding yeast, but the underlying molecular mechanisms remain unknown. Here, we show that autophagy is maintained during meiosis and stimulated in anaphase I and II. Cells with higher levels of autophagy complete meiosis faster, and genetically enhanced autophagy increases meiotic kinetics and sporulation efficiency. Strikingly, our data reveal that the conserved phosphatase Cdc14 regulates meiosis-specific autophagy. Cdc14 is activated in anaphase I and II, accompanying its subcellular relocation from the nucleolus to the cytoplasm, where it dephosphorylates Atg13 to stimulate Atg1 kinase activity and thus autophagy. Together, our findings reveal a meiosis-tailored mechanism that spatiotemporally controls meiotic autophagy activity to ensure meiosis progression, exit, and sporulation.
Topics: Adaptor Proteins, Signal Transducing; Anaphase; Autophagy; Autophagy-Related Proteins; Cell Cycle Proteins; Meiosis; Protein Tyrosine Phosphatases; Saccharomyces cerevisiae Proteins
PubMed: 35238874
DOI: 10.1083/jcb.202107151 -
Frontiers in Cellular and Infection... 2023During infection, the virulence factor PtpA belonging to the protein tyrosine phosphatase family is delivered into the cytosol of the macrophage. PtpA interacts with...
During infection, the virulence factor PtpA belonging to the protein tyrosine phosphatase family is delivered into the cytosol of the macrophage. PtpA interacts with numerous eukaryotic proteins modulating phagosome maturation, innate immune response, apoptosis, and potentially host-lipid metabolism, as previously reported by our group. , the human trifunctional protein enzyme (TFP) is a PtpA substrate, a key enzyme of mitochondrial β-oxidation of long-chain fatty acids, containing two alpha and two beta subunits arranged in a tetramer structure. Interestingly, it has been described that the alpha subunit of TFP (ECHA, TFPα) is no longer detected in mitochondria during macrophage infection with the virulent H37Rv. To better understand if PtpA could be the bacterial factor responsible for this effect, in the present work, we studied in-depth the PtpA activity and interaction with TFP. With this aim, we performed docking and dephosphorylation assays defining the P-Tyr-271 as the potential target of mycobacterial PtpA, a residue located in the helix-10 of TFP, previously described as relevant for its mitochondrial membrane localization and activity. Phylogenetic analysis showed that Tyr-271 is absent in TFP of bacteria and is present in more complex eukaryotic organisms. These results suggest that this residue is a specific PtpA target, and its phosphorylation state is a way of regulating its subcellular localization. We also showed that phosphorylation of Tyr-271 can be catalyzed by Jak kinase. In addition, we found by molecular dynamics that PtpA and TFP form a stable protein complex through the PtpA active site, and we determined the dissociation equilibrium constant. Finally, a detailed study of PtpA interaction with ubiquitin, a reported PtpA activator, showed that additional factors are required to explain a ubiquitin-mediated activation of PtpA. Altogether, our results provide further evidence supporting that PtpA could be the bacterial factor that dephosphorylates TFP during infection, potentially affecting its mitochondrial localization or β-oxidation activity.
Topics: Humans; Lipid Metabolism; Mycobacterium tuberculosis; Phylogeny; Ubiquitins; Mitochondrial Trifunctional Protein; Bacterial Proteins
PubMed: 37424790
DOI: 10.3389/fcimb.2023.1095060 -
International Journal of Molecular... Mar 2023We previously showed that the phosphatases PP1/PP2A and PP2B dephosphorylate the water channel, AQP2, suggesting their role in water reabsorption. In this study, we...
We previously showed that the phosphatases PP1/PP2A and PP2B dephosphorylate the water channel, AQP2, suggesting their role in water reabsorption. In this study, we investigated whether protein phosphatase 2A (PP2A) and protein phosphatase 2B (PP2B or calcineurin), which are present in the inner medullary collecting duct (IMCD), are regulators of urea and water permeability. Inhibition of calcineurin by tacrolimus increased both basal and vasopressin-stimulated osmotic water permeability in perfused rat IMCDs. However, tacrolimus did not affect osmotic water permeability in the presence of aldosterone. Inhibition of PP2A by calyculin increased both basal and vasopressin-stimulated osmotic water permeability, and aldosterone reversed the increase by calyculin. Previous studies showed that adrenomedullin (ADM) activates PP2A and decreases osmotic water permeability. Inhibition of PP2A by calyculin prevented the ADM-induced decrease in water reabsorption. ADM reduced the phosphorylation of AQP2 at serine 269 (pSer269 AQP2). Urea is linked to water reabsorption by building up hyperosmolality in the inner medullary interstitium. Calyculin increased urea permeability and phosphorylated UT-A1. Our results indicate that phosphatases regulate water reabsorption. Aldosterone and adrenomedullin decrease urea or osmotic water permeability by acting through calcineurin and PP2A, respectively. PP2A may regulate water reabsorption by dephosphorylating pSer269, AQP2, and UT-A1.
Topics: Rats; Animals; Rats, Sprague-Dawley; Membrane Transport Proteins; Phosphoric Monoester Hydrolases; Tacrolimus; Water; Adrenomedullin; Aquaporin 2; Calcineurin; Urea; Aldosterone; Vasopressins; Permeability; Kidney Tubules, Collecting
PubMed: 37047509
DOI: 10.3390/ijms24076537 -
Journal of Cancer Research and Clinical... Jan 2022Cyclase-associated protein 1 (CAP1) is a ubiquitous protein which regulates actin dynamics. Previous studies have shown that S308 and S310 are the two major...
PURPOSE
Cyclase-associated protein 1 (CAP1) is a ubiquitous protein which regulates actin dynamics. Previous studies have shown that S308 and S310 are the two major phosphorylated sites in human CAP1. In the present study, we aimed to investigate the role of CAP1 phosphorylation in lung cancer.
METHODS
Massive bioinformatics analysis was applied to determine CAP1's role in different cancers and especially in lung cancer. Lung cancer patients' serum and tissue were collected and analyzed in consideration of clinical background. CAP1 shRNA-lentivirus and siRNA were applied to CAP1 gene knockdown, and plasmids were constructed for CAP1 phosphorylation and de-phosphorylation. Microarray analysis was used for CAP1-associated difference analysis. Both in vitro and in vivo experiments were performed to investigate the roles of CAP1 phosphorylation and de-phosphorylation in lung cancer A549 cells.
RESULTS
CAP1 is a kind of cancer-related protein. Its mRNA was overexpressed in most types of cancer tissues when compared with normal tissues. CAP1 high expression correlated with poor prognosis. Our results showed that serum CAP1 protein concentrations were significantly upregulated in non-small cell lung cancer (NSCLC) patients when compared with the healthy control group, higher serum CAP1 protein concentration correlated with shorter overall survival (OS) in NSCLC patients, and higher pCAP1 and CAP1 protein level were observed in lung cancer patients' tumor tissue compared with adjacent normal tissue. Knockdown CAP1 in A549 cells can inhibit proliferation and migration, and the effect is validated in H1975 cells. It can also lead to an increase ratio of F-actin/G-actin. In addition, phosphorylated S308 and S310 in CAP1 promoted lung cancer cell proliferation, migration, and metastasis both in vitro and in vivo. When de-phosphorylated, these two sites in CAP1 showed the opposite effect. Phosphorylation of CAP1 can promote epithelial-mesenchymal transition (EMT).
CONCLUSION
These findings indicated that CAP1 phosphorylation can promote lung cancer proliferation, migration, and invasion. Phosphorylation sites of CAP1 might be a novel target for lung cancer treatment.
Topics: A549 Cells; Aged; Animals; Carcinoma, Non-Small-Cell Lung; Cell Cycle Proteins; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cytoskeletal Proteins; Epithelial-Mesenchymal Transition; Female; Humans; Lung Neoplasms; Male; Mice; Mice, Nude; Neoplasm Invasiveness; Neoplasm Transplantation; Phosphorylation; RNA Interference; RNA, Small Interfering; Transplantation, Heterologous
PubMed: 34636991
DOI: 10.1007/s00432-021-03819-9