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Molecular Cell Jan 2023In eukaryotes, cyclin-dependent kinase (CDK) ensures that the genome is duplicated exactly once by inhibiting helicase loading factors before activating origin firing....
In eukaryotes, cyclin-dependent kinase (CDK) ensures that the genome is duplicated exactly once by inhibiting helicase loading factors before activating origin firing. CDK activates origin firing by phosphorylating two substrates, Sld2 and Sld3, forming a transient and limiting intermediate-the pre-initiation complex (pre-IC). Here, we show in the budding yeast Saccharomyces cerevisiae that the CDK phosphorylations of Sld3 and Sld2 are rapidly turned over during S phase by the PP2A and PP4 phosphatases. PP2A targets Sld3 specifically through an Rts1-interaction motif, and this targeted dephosphorylation is important for origin firing genome-wide, for formation of the pre-IC at origins and for ensuring that Sld3 is dephosphorylated in G1 phase. PP2A promotes replication in vitro, and we show that targeted Sld3 dephosphorylation is critical for viability. Together, these studies demonstrate that phosphatases enforce the correct ordering of replication factor phosphorylation and in addition to kinases are also key drivers of replication initiation.
Topics: DNA-Binding Proteins; Saccharomyces cerevisiae Proteins; DNA Replication; Cyclin-Dependent Kinases; Cell Cycle Proteins; Phosphorylation; Saccharomyces cerevisiae; Saccharomycetales; Replication Origin
PubMed: 36543171
DOI: 10.1016/j.molcel.2022.12.001 -
Cell Division 2020Cell division is orchestrated by the phosphorylation and dephosphorylation of thousands of proteins. These post-translational modifications underlie the molecular... (Review)
Review
Cell division is orchestrated by the phosphorylation and dephosphorylation of thousands of proteins. These post-translational modifications underlie the molecular cascades converging to the activation of the universal mitotic kinase, Cdk1, and entry into cell division. They also govern the structural events that sustain the mechanics of cell division. While the role of protein kinases in mitosis has been well documented by decades of investigations, little was known regarding the control of protein phosphatases until the recent years. However, the regulation of phosphatase activities is as essential as kinases in controlling the activation of Cdk1 to enter M-phase. The regulation and the function of phosphatases result from post-translational modifications but also from the combinatorial association between conserved catalytic subunits and regulatory subunits that drive their substrate specificity, their cellular localization and their activity. It now appears that sequential dephosphorylations orchestrated by a network of phosphatase activities trigger Cdk1 activation and then order the structural events necessary for the timely execution of cell division. This review discusses a series of recent works describing the important roles played by protein phosphatases for the proper regulation of meiotic division. Many breakthroughs in the field of cell cycle research came from studies on oocyte meiotic divisions. Indeed, the meiotic division shares most of the molecular regulators with mitosis. The natural arrests of oocytes in G2 and in M-phase, the giant size of these cells, the variety of model species allowing either biochemical or imaging as well as genetics approaches explain why the process of meiosis has served as an historical model to decipher signalling pathways involved in the G2-to-M transition. The review especially highlights how the phosphatase PP2A-B55δ critically orchestrates the timing of meiosis resumption in amphibian oocytes. By opposing the kinase PKA, PP2A-B55δ controls the release of the G2 arrest through the dephosphorylation of their substrate, Arpp19. Few hours later, the inhibition of PP2A-B55δ by Arpp19 releases its opposing kinase, Cdk1, and triggers M-phase. In coordination with a variety of phosphatases and kinases, the PP2A-B55δ/Arpp19 duo therefore emerges as the key effector of the G2-to-M transition.
PubMed: 32508972
DOI: 10.1186/s13008-020-00065-2 -
Cell Chemical Biology Feb 2023Reversible protein phosphorylation, catalyzed by protein kinases and phosphatases, is a fundamental process that controls protein function and intracellular signaling....
Reversible protein phosphorylation, catalyzed by protein kinases and phosphatases, is a fundamental process that controls protein function and intracellular signaling. Failure of phospho-control accounts for many human diseases. While a kinase phosphorylates multiple substrates, a substrate is often phosphorylated by multiple kinases. This renders phospho-control at the substrate level challenging, as it requires inhibition of multiple kinases, which would thus affect other kinase substrates. Here, we describe the development and application of the affinity-directed phosphatase (AdPhosphatase) system for targeted dephosphorylation of specific phospho-substrates. By deploying the Protein Phosphatase 1 or 2A catalytic subunits conjugated to an antigen-stabilized anti-GFP nanobody, we can promote the dephosphorylation of two independent phospho-proteins, FAM83D or ULK1, knocked in with GFP-tags using CRISPR-Cas9, with exquisite specificity. By redirecting protein phosphatases to neo-substrates through nanobody-mediated proximity, AdPhosphatase can alter the phospho-status and function of target proteins and thus, offers a new modality for potential drug discovery approaches.
Topics: Humans; Cell Cycle Proteins; Microtubule-Associated Proteins; Phosphorylation; Protein Kinases; Protein Phosphatase 2; Substrate Specificity; Phosphoric Monoester Hydrolases
PubMed: 36720221
DOI: 10.1016/j.chembiol.2023.01.003 -
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 Biological Rhythms Aug 2019The circadian clock controls 24-h biological rhythms in our body, influencing many time-related activities such as sleep and wake. The simplest circadian clock is found...
The circadian clock controls 24-h biological rhythms in our body, influencing many time-related activities such as sleep and wake. The simplest circadian clock is found in cyanobacteria, with the proteins KaiA, KaiB, and KaiC generating a self-sustained circadian oscillation of KaiC phosphorylation and dephosphorylation. KaiA activates KaiC phosphorylation by binding the A-loop of KaiC, while KaiB attenuates the phosphorylation by sequestering KaiA from the A-loop. Structural analysis revealed that magnesium regulates the phosphorylation and dephosphorylation of KaiC by dissociating from and associating with catalytic Glu residues that activate phosphorylation and dephosphorylation, respectively. High magnesium causes KaiC to dephosphorylate, whereas low magnesium causes KaiC to phosphorylate. KaiC alone behaves as an hourglass timekeeper when the magnesium concentration is alternated between low and high levels in vitro. We suggest that a magnesium-based hourglass timekeeping system may have been used by ancient cyanobacteria before magnesium homeostasis was established.
Topics: Bacterial Proteins; Circadian Rhythm; Cloning, Molecular; Cyanobacteria; Magnesium; Molecular Dynamics Simulation; Phosphorylation
PubMed: 31216910
DOI: 10.1177/0748730419851655 -
Cell & Bioscience 2019Dual-specificity phosphatases (DUSPs) are a subset of protein tyrosine phosphatases (PTPs), many of which dephosphorylate the residues of phosphor-serine/threonine and... (Review)
Review
Dual-specificity phosphatases (DUSPs) are a subset of protein tyrosine phosphatases (PTPs), many of which dephosphorylate the residues of phosphor-serine/threonine and phosphor-tyrosine on mitogen-activated protein kinases (MAPKs), and hence are also referred to as MAPK phosphatases (MKPs). Homologue of Vaccinia virus H1 phosphatase gene clone 5 (HVH-5), also known as DUSP8, is a unique member of the DUSPs family of phosphatases. Accumulating evidence has shown that DUSP8 plays an important role in phosphorylation-mediated signal transduction of MAPK signaling ranging from cell oxidative stress response, cell apoptosis and various human diseases. It is generally believed that DUSP8 exhibits significant dephosphorylation activity against JNK, however, with the deepening of research, plenty of new literature reports that DUSP8 also has effective dephosphorylation activity on p38 MAPK and ERKs, successfully affects the transduction of MAPKs pathway, indicating that DUSP8 presents a unknown diversity of DUSPs family on distinct corresponding dephosphorylated substrates in different biological events. Therefore, the in-depth study of DUSP8 not only throws a new light on the multi-biological function of DUSPs, but also is much valuable for the reveal of complex pathobiology of clinical diseases. In this review, we provide a detail overview of DUSP8 phosphatase structure, biological function and expression regulation, as well as its role in related clinical human diseases, which might be help for the understanding of biological function of DUSP8 and the development of prevention, diagnosis and therapeutics in related human diseases.
PubMed: 31467668
DOI: 10.1186/s13578-019-0329-4 -
Methods in Molecular Biology (Clifton,... 2022SNF1-related protein kinase 2 s (SnRK2s) are major regulators of plant growth, development and responses to environmental stresses. Together with clade A protein...
SNF1-related protein kinase 2 s (SnRK2s) are major regulators of plant growth, development and responses to environmental stresses. Together with clade A protein phosphatases of type 2C (PP2C) and REGULATORY COMPONENTS OF ABA RECEPTOR (RCAR also known as PYRABACTIN RESISTANCE1 (PYR1) or PYR1-LIKE (PYL)) soluble abscisic acid (ABA) receptors they form the core of ABA-signaling. Clade A PP2Cs play a negative role in ABA signaling, primarily by inhibiting SnRK2 activity, through direct interaction and dephosphorylation of SnRK2s. Here, we describe two methods, which can be used for monitoring inhibition of the SnRK2 activity by PP2C phosphatases. One of them is an in vitro dephosphorylation assay using SnRK2 as the substrate followed by a classical in-gel kinase-activity assay and the other is immunocomplex kinase-activity assay, which can be applied for analysis of the SnRK2 activity in plant material.
Topics: Abscisic Acid; Arabidopsis Proteins; Phosphoprotein Phosphatases; Phosphorylation; Signal Transduction
PubMed: 35152377
DOI: 10.1007/978-1-0716-2156-1_2 -
International Journal of Molecular... Dec 2019Protein phosphorylation affects conformational change, interaction, catalytic activity, and subcellular localization of proteins. Because the post-modification of... (Review)
Review
Protein phosphorylation affects conformational change, interaction, catalytic activity, and subcellular localization of proteins. Because the post-modification of proteins regulates diverse cellular signaling pathways, the precise control of phosphorylation states is essential for maintaining cellular homeostasis. Kinases function as phosphorylating enzymes, and phosphatases dephosphorylate their target substrates, typically in a much shorter time. The c-Jun N-terminal kinase (JNK) signaling pathway, a mitogen-activated protein kinase pathway, is regulated by a cascade of kinases and in turn regulates other physiological processes, such as cell differentiation, apoptosis, neuronal functions, and embryonic development. However, the activation of the JNK pathway is also implicated in human pathologies such as cancer, neurodegenerative diseases, and inflammatory diseases. Therefore, the proper balance between activation and inactivation of the JNK pathway needs to be tightly regulated. Dual specificity phosphatases (DUSPs) regulate the magnitude and duration of signal transduction of the JNK pathway by dephosphorylating their substrates. In this review, we will discuss the dynamics of phosphorylation/dephosphorylation, the mechanism of JNK pathway regulation by DUSPs, and the new possibilities of targeting DUSPs in JNK-related diseases elucidated in recent studies.
Topics: Animals; Dual-Specificity Phosphatases; Humans; JNK Mitogen-Activated Protein Kinases; Mitogen-Activated Protein Kinases; Models, Biological; Phosphorylation; Signal Transduction
PubMed: 31817617
DOI: 10.3390/ijms20246157 -
Biochimica Et Biophysica Acta. Gene... Sep 2023The transcription factor E2F1 participates in cell cycle control through transcriptional activation of genes that promote S-phase entry. E2F1 is also linked to the...
The transcription factor E2F1 participates in cell cycle control through transcriptional activation of genes that promote S-phase entry. E2F1 is also linked to the expression of proapoptotic genes, and the loss of E2F1 activity facilitates tumor progression by reducing cellular apoptosis. Phosphorylation controlled by protein kinases and phosphatases is the major posttranslational modification and regulates the cellular levels and transactivator function of E2F1. Here, we characterize the regulatory roles of serine-375 (S375), one of the major phosphorylation sites of E2F1. Cyclin-dependent kinases such as CDK8 phosphorylate at S375 of E2F1, which is dephosphorylated by protein phosphatase 2A (PP2A) containing the B55 regulatory subunit. The PP2A adapter protein IER5 binds to both PP2A/B55 and E2F1 and assists dephosphorylation at S375 by PP2A. S375-dephosphorylated E2F1 exhibits higher DNA-binding affinity than the phosphorylated form. Although the promoter regions of proapoptotic genes are less occupied by E2F1 in cells, an increase in S375-dephosphorylated E2F1 induces preferential binding of E2F1 to the proapoptotic gene promoters and their expression. Our data identify PP2A/B55-IER5 as a critical regulator of E2F1 and suggest that the phosphorylation state of E2F1 is an important determinant for the expression of proapoptotic genes.
Topics: Protein Phosphatase 2; Serine; Protein Processing, Post-Translational; Adaptor Proteins, Signal Transducing; Gene Expression; DNA
PubMed: 37467925
DOI: 10.1016/j.bbagrm.2023.194960 -
Journal of Biochemistry Mar 2021Receptor protein tyrosine phosphatases (RPTPs) are type-I transmembrane proteins and involved in various biological and pathological processes. Their functions are...
Receptor protein tyrosine phosphatases (RPTPs) are type-I transmembrane proteins and involved in various biological and pathological processes. Their functions are supposed to be exerted through tyrosine dephosphorylation of their specific substrates. However, our comprehensive understanding of specific substrates or interacting proteins for RPTPs is poor. PTPRσ belongs to class 2a RPTP family, dephosphorylates cortactin, and leads to autophagy flux disruption and axonal regeneration inhibition in response to its ligand chondroitin sulphate. Here, we applied proximity-dependent biotin identification (BioID) assay, a proximity-labelling assay, to PTPRσ and reproducibly identified the 99 candidates as interactors for PTPRσ including already-known interactors such as Liprin-α and Trio. Of note, cortactin was also listed up in our assay. Our results suggest that the BioID assay is a powerful and reliable tool to identify RPTP-interacting proteins including its specific substrate.
Topics: Autophagy; Biotinylation; Cell Line; Chondroitin Sulfates; HEK293 Cells; Humans; Phosphorylation; Protein Binding; Protein Interaction Domains and Motifs; Proteomics; Receptor-Like Protein Tyrosine Phosphatases, Class 4; Recombinant Fusion Proteins
PubMed: 33313879
DOI: 10.1093/jb/mvaa141