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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 -
The Journal of Biological Chemistry Dec 2019Endothelial cells have key functions in endothelial barrier integrity and in responses to angiogenic signals that promote cell proliferation, cell migration,...
Endothelial cells have key functions in endothelial barrier integrity and in responses to angiogenic signals that promote cell proliferation, cell migration, cytoskeletal reorganization, and formation of new blood vessels. These functions highly depend on protein-protein interactions in cell-cell junction and cell attachment complexes and on interactions with cytoskeletal proteins. Protein phosphatase 2A (PP2A) dephosphorylates several target proteins involved in cytoskeletal dynamics and cell adhesion. Our goal was to find new interacting and substrate proteins of the PP2A-B55α holoenzyme in bovine pulmonary endothelial cells. Using LC-MS/MS analysis, we identified flotillin-1 as a protein that binds recombinant GSH -transferase-tagged PP2A-B55α. Immunoprecipitation experiments, proximity ligation assays, and immunofluorescent staining confirmed the interaction between these two endogenous proteins in endothelial cells. Originally, flotillins were described as regulatory proteins for axon regeneration, but they appear to function in many cellular processes, such as membrane receptor signaling, endocytosis, and cell adhesion. Ser is a known PKC-targeted site in flotillin-1. Utilizing phosphomutants of flotillin-1 and the NanoBiT luciferase assay, we show here that phosphorylation/dephosphorylation of Ser in flotillin-1 significantly affects its interaction with PP2A-B55α and that PP2A-B55α dephosphorylates phospho-Ser Spreading, attachment, migration, and tube formation rates of S315A variant-overexpressing cells were faster than those of nontransfected or S315D-transfected cells. These results indicate that the PP2A-flotillin-1 interaction identified here affects major physiological activities of pulmonary endothelial cells.
Topics: Animals; Carbazoles; Cattle; Cell Movement; Cells, Cultured; Endothelial Cells; Holoenzymes; Membrane Proteins; Mutagenesis, Site-Directed; Neovascularization, Physiologic; Phosphorylation; Protein Interaction Domains and Motifs; Protein Kinase C; Protein Phosphatase 2; Protein Subunits; RNA Interference; RNA, Small Interfering; Up-Regulation
PubMed: 31753918
DOI: 10.1074/jbc.RA119.007980 -
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 -
International Journal of Molecular... Sep 2018The signal transducer and activator of transcription 3 (STAT3) protein is a major transcription factor involved in many cellular processes, such as cell growth and... (Review)
Review
The signal transducer and activator of transcription 3 (STAT3) protein is a major transcription factor involved in many cellular processes, such as cell growth and proliferation, differentiation, migration, and cell death or cell apoptosis. It is activated in response to a variety of extracellular stimuli including cytokines and growth factors. The aberrant activation of STAT3 contributes to several human diseases, particularly cancer. Consequently, STAT3-mediated signaling continues to be extensively studied in order to identify potential targets for the development of new and more effective clinical therapeutics. STAT3 activation can be regulated, either positively or negatively, by different posttranslational mechanisms including serine or tyrosine phosphorylation/dephosphorylation, acetylation, or demethylation. One of the major mechanisms that negatively regulates STAT3 activation is dephosphorylation of the tyrosine residue essential for its activation by protein tyrosine phosphatases (PTPs). There are seven PTPs that have been shown to dephosphorylate STAT3 and, thereby, regulate STAT3 signaling: PTP receptor-type D (PTPRD), PTP receptor-type T (PTPRT), PTP receptor-type K (PTPRK), Src homology region 2 (SH-2) domain-containing phosphatase 1(SHP1), SH-2 domain-containing phosphatase 2 (SHP2), MEG2/PTP non-receptor type 9 (PTPN9), and T-cell PTP (TC-PTP)/PTP non-receptor type 2 (PTPN2). These regulators have great potential as targets for the development of more effective therapies against human disease, including cancer.
Topics: Animals; Humans; Mutation; Neoplasms; Phosphorylation; Protein Processing, Post-Translational; Protein Tyrosine Phosphatases; STAT3 Transcription Factor; Signal Transduction
PubMed: 30208623
DOI: 10.3390/ijms19092708 -
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 -
Open Biology May 2017Ubiquitin-like domain-containing C-terminal domain phosphatase 1 (UBLCP1), an FCP/SCP phosphatase family member, was identified as the first proteasome phosphatase....
Ubiquitin-like domain-containing C-terminal domain phosphatase 1 (UBLCP1), an FCP/SCP phosphatase family member, was identified as the first proteasome phosphatase. UBLCP1 binds to proteasome subunit Rpn1 and dephosphorylates the proteasome However, it is still unclear which proteasome subunit(s) are the substrate(s) of UBLCP1 and the precise mechanism for proteasome regulation remains elusive. Here, we show that UBLCP1 selectively binds to the 19S regulatory particle (RP) through its interaction with Rpn1, but not the 20S core particle (CP) or the 26S proteasome holoenzyme. In the RP, UBLCP1 dephosphorylates the subunit Rpt1, impairs its ATPase activity, and consequently disrupts the 26S proteasome assembly, yet it has no effects on the RP assembly from precursor complexes. The Rpn1-binding and phosphatase activities of UBLCP1 are essential for its function on Rpt1 dephosphorylation and proteasome activity both and Our study establishes the essential role of the UBLCP1/Rpn1/Rpt1 complex in regulating proteasome assembly.
Topics: Adenosine Triphosphatases; HEK293 Cells; HeLa Cells; Humans; Nuclear Proteins; Phosphoprotein Phosphatases; Phosphorylation; Proteasome Endopeptidase Complex; Protein Binding; Protein Subunits
PubMed: 28539385
DOI: 10.1098/rsob.170042 -
PeerJ 2023Golgin subfamily A member 3 (), a member of the golgin subfamily A, is highly expressed in mouse testis. The GOLGA3 protein, which contains eight phosphorylation sites,...
BACKGROUND
Golgin subfamily A member 3 (), a member of the golgin subfamily A, is highly expressed in mouse testis. The GOLGA3 protein, which contains eight phosphorylation sites, is involved in protein transport, cell apoptosis, Golgi localization, and spermatogenesis. Although it has been previously reported that nonsense mutations in cause multiple defects in spermatogenesis, the role of in the testis is yet to be clarified.
METHODS
Immunofluorescence co-localization in cells and protein dephosphorylation experiments were performed. mice were generated using cytosine base editors. Fertility tests as well as computer-assisted sperm analysis (CASA) were then performed to investigate sperm motility within caudal epididymis. Histological and immunofluorescence staining were used to analyze testis and epididymis phenotypes and TUNEL assays were used to measure germ cell apoptosis in spermatogenic tubules.
RESULTS
Immunofluorescence co-localization showed reduced Golgi localization of GOLGA3 with some protein scattered in the cytoplasm of HeLa cells .In addition, protein dephosphorylation experiments indicated a reduced band shift of the dephosphorylated GOLGA3, confirming S461 as the phosphorylation site. is an evolutionarily conserved gene and mice were successfully generated using cytosine base editors. These mice had normal fertility and spermatozoa, and did not differ significantly from wild-type mice in terms of spermatogenesis and apoptotic cells in tubules.
CONCLUSIONS
was found to be highly conserved in the testis, and GOLGA3 was shown to be involved in spermatogenesis, especially in apoptosis and Golgi complex-mediated effects. Infertility was also observed in KO male mice. Although GOLGA3showed reduced localization in the Golgi with some expression in the cytoplasm, this abnormal localization did not adversely affect fertility or spermatogenesis in male C57BL/6 mice. Therefore, mutation of the S461 GOLGA3 phosphorylation site did not affect mouse spermatogenesis.
Topics: Animals; Humans; Male; Mice; Golgi Matrix Proteins; HeLa Cells; Mice, Inbred C57BL; Mutation; Phosphorylation; Proteins; Semen; Sperm Motility; Spermatogenesis
PubMed: 37090114
DOI: 10.7717/peerj.15133 -
The Journal of Biological Chemistry Nov 2021Posttranslational modifications (PTMs) such as phosphorylation of RNA-binding proteins (RBPs) regulate several critical steps in RNA metabolism, including spliceosome...
Posttranslational modifications (PTMs) such as phosphorylation of RNA-binding proteins (RBPs) regulate several critical steps in RNA metabolism, including spliceosome assembly, alternative splicing, and mRNA export. Notably, serine-/arginine- (SR)-rich RBPs are densely phosphorylated compared with the remainder of the proteome. Previously, we showed that dephosphorylation of the splicing factor SRSF2 regulated increased interactions with similar arginine-rich RBPs U1-70K and LUC7L3. However, the large-scale functional and structural impact of these modifications on RBPs remains unclear. In this work, we dephosphorylated nuclear extracts using phosphatase in vitro and analyzed equal amounts of detergent-soluble and -insoluble fractions by mass-spectrometry-based proteomics. Correlation network analysis resolved 27 distinct modules of differentially soluble nucleoplasm proteins. We found classes of arginine-rich RBPs that decrease in solubility following dephosphorylation and enrich the insoluble pelleted fraction, including the SR protein family and the SR-like LUC7L RBP family. Importantly, increased insolubility was not observed across broad classes of RBPs. We determined that phosphorylation regulated SRSF2 structure, as dephosphorylated SRSF2 formed high-molecular-weight oligomeric species in vitro. Reciprocally, phosphorylation of SRSF2 by serine/arginine protein kinase 2 (SRPK2) in vitro decreased high-molecular-weight SRSF2 species formation. Furthermore, upon pharmacological inhibition of SRPKs in mammalian cells, we observed SRSF2 cytoplasmic mislocalization and increased formation of cytoplasmic granules as well as cytoplasmic tubular structures that associated with microtubules by immunocytochemical staining. Collectively, these findings demonstrate that phosphorylation may be a critical modification that prevents arginine-rich RBP insolubility and oligomerization.
Topics: HEK293 Cells; Humans; Nuclear Proteins; Phosphorylation; Protein Multimerization; Protein Serine-Threonine Kinases; Protein Stability; Ribonucleoprotein, U1 Small Nuclear; Serine-Arginine Splicing Factors
PubMed: 34673031
DOI: 10.1016/j.jbc.2021.101306 -
Frontiers in Immunology 2023Macrophages play a critical role in the regulation of inflammation and tissue homeostasis. In addition to their vital functions for cell survival and physiology,...
Macrophages play a critical role in the regulation of inflammation and tissue homeostasis. In addition to their vital functions for cell survival and physiology, mitochondria play a crucial role in innate immunity as a platform for the induction of inflammatory responses by regulating cell signaling and dynamics. Dynamin-related protein 1 (Drp1) plays a role in the induction of inflammatory responses and the subsequent development of various diseases. PGAM5 (phosphoglycerate mutase member 5) is a mitochondrial outer membrane phosphatase that dephosphorylates its substrate, Drp1. Previous studies showed that PGAM5 regulates the phosphorylation of Drp1 for the activation of NKT cells and T cells. However, it is not clear how PGAM5 regulates Drp1 activity for the induction of inflammation in macrophages. Here, we demonstrate that PGAM5 activity regulates the dephosphorylation of Drp1 in macrophages, leading to the induction of proinflammatory responses in macrophages. In TLR signaling, PGAM5 regulates the expression and production of inflammatory cytokines by regulating the activation of downstream signaling pathways, including the NF-κB and MAPK pathways. Upon LPS stimulation, PGAM5 interacts with Drp1 to form a complex, leading to the production of mtROS. Furthermore, PGAM5-Drp1 signaling promotes the polarization of macrophages toward a proinflammatory phenotype. Our study further demonstrates that PGAM5-Drp1 signaling promotes metabolic reprogramming by upregulating glycolysis and mitochondrial metabolism in macrophages. Altogether, PGAM5 signaling is a linker between alterations in Drp1-mediated mitochondrial dynamics and inflammatory responses in macrophages and may be a target for the treatment of inflammatory diseases.
Topics: Humans; Dynamins; Inflammation; Macrophages; Mitochondrial Proteins; Phosphoprotein Phosphatases; Signal Transduction; Animals
PubMed: 37771598
DOI: 10.3389/fimmu.2023.1243548