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Cell Death & Disease Sep 2022Mixed lineage leukemia 1 (MLL1) is a histone H3 lysine 4 (H3K4) methyltransferase that interacts with WD repeat domain 5 (WDR5) to regulate cell survival, proliferation,...
Mixed lineage leukemia 1 (MLL1) is a histone H3 lysine 4 (H3K4) methyltransferase that interacts with WD repeat domain 5 (WDR5) to regulate cell survival, proliferation, and senescence. The role of MLL1 in the pathogenesis of acute kidney injury (AKI) is unknown. In this study, we demonstrate that MLL1, WDR5, and trimethylated H3K4 (H3K4me3) were upregulated in renal tubular cells of cisplatin-induced AKI in mice, along with increased phosphorylation of p53 and decreased expression of E-cadherin. Administration of MM102, a selective MLL1/WDR5 complex inhibitor, improved renal function and attenuated tubular injury and apoptosis, while repressing MLL1, WDR5, and H3K4me3, dephosphorylating p53 and preserving E-cadherin. In cultured mouse renal proximal tubular cells (RPTCs) exposed to cisplatin, treatment with MM102 or transfection with siRNAs for either MLL1 or WDR5 also inhibited apoptosis and p53 phosphorylation while preserving E-cadherin expression; p53 inhibition with Pifithrin-α lowered cisplatin-induced apoptosis without affecting expression of MLL1, WDR5, and H3K4me3. Interestingly, silencing of E-cadherin offset MM102's cytoprotective effects, but had no effect on p53 phosphorylation. These findings suggest that MLL1/WDR5 activates p53, which, in turn, represses E-cadherin, leading to apoptosis during cisplatin-induced AKI. Further studies showed that MM102 effectively inhibited cisplatin-triggered DNA damage response (DDR), as indicated by dephosphorylation of ataxia telangiectasia mutated (ATM) and ATM and Rad-3 related (ATR) proteins, dephosphorylation of checkpoint kinase 1 and 2 (Chk1 and Chk2); depression of γ-H2AX; and restrained cell cycle arrest, as evidenced by decreased expression of p21 and phospho-histone H3 at serine 10 in vitro and in vivo. Overall, we identify MLL1 as a novel DDR regulator that drives cisplatin-induced RPTC apoptosis and AKI by modulating the MLL1/WDR5-/ATR/ATM-Chk-p53-E-cadherin axis. Targeting the MLL1/WDR5 complex may have a therapeutic potential for the treatment of AKI.
Topics: Acute Kidney Injury; Animals; Apoptosis; Cadherins; Cisplatin; Histone Methyltransferases; Histones; Kidney; Leukemia; Mice; Myeloid-Lymphoid Leukemia Protein; Tumor Suppressor Protein p53
PubMed: 36068197
DOI: 10.1038/s41419-022-05104-0 -
Plant, Cell & Environment Dec 2022Heat stress (HS) caused by ambient high temperature poses a threat to plants. In the natural and agricultural environment, plants often encounter repeated and changeable...
Heat stress (HS) caused by ambient high temperature poses a threat to plants. In the natural and agricultural environment, plants often encounter repeated and changeable HS. Moderate HS primes plants to establish a molecular 'thermomemory' that enables plants to withstand a later-and possibly more extreme-HS attack. Recent years, brassinosteroids (BRs) have been implicated in HS response, whereas the information is lacking on whether BRs signal transduction modulates thermomemory. Here, we uncover the positive role of BRs signalling in thermomemory of Arabidopsis thaliana. Heat priming induces de novo synthesis and nuclear accumulation of BRI1-Ethyl methyl sulfon-SUPPRESSOR (BES1), which is the key regulator of BRs signalling. BRs promote the accumulation of dephosphorylated BES1 during memory phase, and stoppage of BRs synthesis impairs dephosphorylation. During HS memory, BES1 is required to maintain sustained induction of HS memory genes and directly targets APX2 and HSFA3 for activation. In summary, our results reveal a BES1-required, BRs-enhanced transcriptional control module of thermomemory in Arabidopsis thaliana.
Topics: Arabidopsis; Brassinosteroids; Arabidopsis Proteins; Gene Expression Regulation, Plant; DNA-Binding Proteins; Plants, Genetically Modified
PubMed: 36130868
DOI: 10.1111/pce.14444 -
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 -
Cell Communication and Signaling : CCS Jan 2022G protein-coupled receptors (GPCRs) usually regulate cellular processes via activation of intracellular signaling pathways. However, we have previously shown that in...
BACKGROUND
G protein-coupled receptors (GPCRs) usually regulate cellular processes via activation of intracellular signaling pathways. However, we have previously shown that in several cell lines, GqPCRs induce immediate inactivation of the AKT pathway, which leads to JNK-dependent apoptosis. This apoptosis-inducing AKT inactivation is essential for physiological functions of several GqPCRs, including those for PGF2α and GnRH.
METHODS
Here we used kinase activity assays of PI3K and followed phosphorylation state of proteins using specific antibodies. In addition, we used coimmunoprecipitation and proximity ligation assays to follow protein-protein interactions. Apoptosis was detected by TUNEL assay and PARP1 cleavage.
RESULTS
We identified the mechanism that allows the unique stimulated inactivation of AKT and show that the main regulator of this process is the phosphatase PP2A, operating with the non-canonical regulatory subunit IGBP1. In resting cells, an IGBP1-PP2Ac dimer binds to PI3K, dephosphorylates the inhibitory pSer608-p85 of PI3K and thus maintains its high basal activity. Upon GqPCR activation, the PP2Ac-IGBP1 dimer detaches from PI3K and thus allows the inhibitory dephosphorylation. At this stage, the free PP2Ac together with IGBP1 and PP2Aa binds to AKT, causing its dephosphorylation and inactivation.
CONCLUSION
Our results show a stimulated shift of PP2Ac from PI3K to AKT termed "PP2A switch" that represses the PI3K/AKT pathway, providing a unique mechanism of GPCR-stimulated dephosphorylation. Video Abstract.
Topics: Phosphatidylinositol 3-Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; Receptors, G-Protein-Coupled; Signal Transduction
PubMed: 34998390
DOI: 10.1186/s12964-021-00805-z -
Journal of Cell Science Oct 2022Protein phosphorylation on serine and threonine residues is a widely distributed post-translational modification on proteins that acts to regulate their function.... (Review)
Review
Protein phosphorylation on serine and threonine residues is a widely distributed post-translational modification on proteins that acts to regulate their function. Phosphoprotein phosphatases (PPPs) contribute significantly to a plethora of cellular functions through the accurate dephosphorylation of phosphorylated residues. Most PPPs accomplish their purpose through the formation of complex holoenzymes composed of a catalytic subunit with various regulatory subunits. PPP holoenzymes then bind and dephosphorylate substrates in a highly specific manner. Despite the high prevalence of PPPs and their important role for cellular function, their mechanisms of action in the cell are still not well understood. Nevertheless, substantial experimental advancements in (phospho-)proteomics, structural and computational biology have contributed significantly to a better understanding of PPP biology in recent years. This Review focuses on recent approaches and provides an overview of substantial new insights into the complex mechanism of PPP holoenzyme regulation and substrate selectivity.
Topics: Holoenzymes; Phosphoprotein Phosphatases; Phosphoproteins; Phosphorylation; Protein Phosphatase 2; Serine; Threonine
PubMed: 36205606
DOI: 10.1242/jcs.259618 -
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 -
International Journal of Molecular... Apr 2021More than 70% of eukaryotic proteins are regulated by phosphorylation. However, the mechanism of dephosphorylation that counteracts phosphorylation is less studied.... (Review)
Review
More than 70% of eukaryotic proteins are regulated by phosphorylation. However, the mechanism of dephosphorylation that counteracts phosphorylation is less studied. Phosphatases are classified into 104 distinct groups based on substrate-specific features and the sequence homologies in their catalytic domains. Among them, dual-specificity phosphatases (DUSPs) that dephosphorylate both phosphoserine/threonine and phosphotyrosine are important for cellular homeostasis. Ssu72 is a newly studied phosphatase with dual specificity that can dephosphorylate both phosphoserine/threonine and phosphotyrosine. It is important for cell-growth signaling, metabolism, and immune activation. Ssu72 was initially identified as a phosphatase for the Ser5 and Ser7 residues of the C-terminal domain of RNA polymerase II. It prefers the configuration of the serine-proline motif within its substrate and regulates Pin1, different from other phosphatases. It has recently been reported that Ssu72 can regulate sister chromatid cohesion and the separation of duplicated chromosomes during the cell cycle. Furthermore, Ssu72 appears to be involved in the regulation of T cell receptor signaling, telomere regulation, and even hepatocyte homeostasis in response to a variety of stress and damage signals. In this review, we aim to summarize various functions of the Ssu72 phosphatase, their implications in diseases, and potential therapeutic indications.
Topics: Animals; Chromatids; Chromosomes, Human; Humans; NIMA-Interacting Peptidylprolyl Isomerase; Phosphoprotein Phosphatases; Protein Domains; RNA Polymerase II; Receptors, Antigen, T-Cell; Signal Transduction
PubMed: 33917542
DOI: 10.3390/ijms22073791 -
Cell Apr 2020Protein phosphatase 2A (PP2A) enzymes can suppress tumors, but they are often inactivated in human cancers overexpressing inhibitory proteins. Here, we identify a class...
Protein phosphatase 2A (PP2A) enzymes can suppress tumors, but they are often inactivated in human cancers overexpressing inhibitory proteins. Here, we identify a class of small-molecule iHAPs (improved heterocyclic activators of PP2A) that kill leukemia cells by allosterically assembling a specific heterotrimeric PP2A holoenzyme consisting of PPP2R1A (scaffold), PPP2R5E (B56ε, regulatory), and PPP2CA (catalytic) subunits. One compound, iHAP1, activates this complex but does not inhibit dopamine receptor D2, a mediator of neurologic toxicity induced by perphenazine and related neuroleptics. The PP2A complex activated by iHAP1 dephosphorylates the MYBL2 transcription factor on Ser241, causing irreversible arrest of leukemia and other cancer cells in prometaphase. In contrast, SMAPs, a separate class of compounds, activate PP2A holoenzymes containing a different regulatory subunit, do not dephosphorylate MYBL2, and arrest tumor cells in G1 phase. Our findings demonstrate that small molecules can serve as allosteric switches to activate distinct PP2A complexes with unique substrates.
Topics: Apoptosis; Cell Cycle Proteins; Cell Line, Tumor; Enzyme Activators; G1 Phase; Humans; Multiprotein Complexes; Phenothiazines; Phosphorylation; Protein Phosphatase 2; Protein Subunits; Trans-Activators; Transcription Factors
PubMed: 32315619
DOI: 10.1016/j.cell.2020.03.051 -
Cold Spring Harbor Perspectives in... Mar 2020Biological processes are dynamically regulated by signaling networks composed of protein kinases and phosphatases. Calcineurin, or PP3, is a conserved... (Review)
Review
Biological processes are dynamically regulated by signaling networks composed of protein kinases and phosphatases. Calcineurin, or PP3, is a conserved phosphoserine/phosphothreonine-specific protein phosphatase and member of the PPP family of phosphatases. Calcineurin is unique, however, in its activation by Ca and calmodulin. This ubiquitously expressed phosphatase controls Ca-dependent processes in all human tissues, but is best known for driving the adaptive immune response by dephosphorylating the nuclear factor of the activated T-cells (NFAT) family of transcription factors. Therefore, calcineurin inhibitors, FK506 (tacrolimus), and cyclosporin A serve as immunosuppressants. We describe some of the adverse effects associated with calcineurin inhibitors that result from inhibition of calcineurin in nonimmune tissues, illustrating the many functions of this enzyme that have yet to be elucidated. In fact, calcineurin has essential roles beyond the immune system, from yeast to humans, but since its discovery more than 30 years ago, only a small number of direct calcineurin substrates have been shown (∼75 proteins). This is because of limitations in current methods for identification of phosphatase substrates. Here we discuss recent insights into mechanisms of calcineurin activation and substrate recognition that have been critical in the development of novel approaches for identifying its targets systematically. Rather than comprehensively reviewing known functions of calcineurin, we highlight new approaches to substrate identification for this critical regulator that may reveal molecular mechanisms underlying toxicities caused by calcineurin inhibitor-based immunosuppression.
Topics: Amino Acid Motifs; Animals; Calcineurin; Calcineurin Inhibitors; Calcium; Computer Simulation; Cyclosporine; Gene Expression Regulation; Humans; Hypertension; Immune System; Immunosuppression Therapy; Immunosuppressive Agents; Isoenzymes; NFATC Transcription Factors; Nuclear Proteins; Phosphoric Monoester Hydrolases; Phosphorylation; Protein Conformation; Protein Isoforms; Proteomics; Signal Transduction; Tacrolimus
PubMed: 31308145
DOI: 10.1101/cshperspect.a035436 -
Annals of the New York Academy of... Nov 2019Latrophilin-1 is an adhesion G protein-coupled receptor that mediates the effect of α-latrotoxin, causing massive release of neurotransmitters from nerve terminals and...
Latrophilin-1 is an adhesion G protein-coupled receptor that mediates the effect of α-latrotoxin, causing massive release of neurotransmitters from nerve terminals and endocrine cells. Autoproteolysis cleaves latrophilin-1 into two parts: the extracellular N-terminal fragment (NTF) and the heptahelical C-terminal fragment (CTF). NTF and CTF can exist as independent proteins in the plasma membrane, but α-latrotoxin binding to NTF induces their association and G protein-mediated signaling. We demonstrate here that CTF in synapses is phosphorylated on multiple sites. Phosphorylated CTF has a high affinity for NTF and copurifies with it on affinity columns and sucrose density gradients. Dephosphorylated CTF has a lower affinity for NTF and can behave as a separate protein. α-Latrotoxin (and possibly other ligands of latrophilin-1) binds both to the NTF-CTF complex and receptor-like protein tyrosine phosphatase σ, bringing them together. This leads to CTF dephosphorylation and facilitates CTF release from the complex. We propose that ligand-dependent phosphorylation-dephosphorylation of latrophilin-1 could affect the interaction between its fragments and functions as a G protein-coupled receptor.
Topics: Animals; Binding Sites; Chromatography, Liquid; Humans; Male; Mice; Mice, Inbred C57BL; Phosphorylation; Protein Binding; Rats; Rats, Sprague-Dawley; Receptors, G-Protein-Coupled; Receptors, Peptide
PubMed: 31553068
DOI: 10.1111/nyas.14242