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Trends in Molecular Medicine Jun 2018A key site of translation control is the phosphorylation of the eukaryotic translation initiation factor 2α (eIF2α), which reduces the rate of GDP to GTP exchange by... (Review)
Review
A key site of translation control is the phosphorylation of the eukaryotic translation initiation factor 2α (eIF2α), which reduces the rate of GDP to GTP exchange by eIF2B, leading to altered translation. The extent of eIF2α phosphorylation within neurons can alter synaptic plasticity. Phosphorylation of eIF2α is triggered by four stress-responsive kinases, and as such eIF2α is often phosphorylated during neurological perturbations or disease. Moreover, in some cases decreasing eIF2α phosphorylation mitigates neurodegeneration, suggesting that this could be a therapeutic target. Mutations in the γ subunit of eIF2, the guanine exchange factor eIF2B, an eIF2α phosphatase, or in two eIF2α kinases can cause disease in humans, demonstrating the importance of proper regulation of eIF2α phosphorylation for health.
Topics: Animals; Biomarkers; Eukaryotic Initiation Factor-2; Gene Expression Regulation; Humans; Mutation; Nervous System Diseases; Neuronal Plasticity; Neurons; Phosphorylation
PubMed: 29716790
DOI: 10.1016/j.molmed.2018.04.001 -
Endocrine-related Cancer Aug 2014The androgen receptor (AR) is a ligand-regulated transcription factor that belongs to the family of nuclear receptors. In addition to regulation by steroid, the AR is... (Review)
Review
The androgen receptor (AR) is a ligand-regulated transcription factor that belongs to the family of nuclear receptors. In addition to regulation by steroid, the AR is also regulated by post-translational modifications generated by signal transduction pathways. Thus, the AR functions not only as a transcription factor but also as a node that integrates multiple extracellular signals. The AR plays an important role in many diseases, including complete androgen insensitivity syndrome, spinal bulbar muscular atrophy, prostate and breast cancer, etc. In the case of prostate cancer, dependence on AR signaling has been exploited for therapeutic intervention for decades. However, the effectiveness of these therapies is limited in advanced disease due to restoration of AR signaling. Greater understanding of the molecular mechanisms involved in AR action will enable the development of improved therapeutics to treat the wide range of AR-dependent diseases. The AR is subject to regulation by a number of kinases through post-translational modifications on serine, threonine, and tyrosine residues. In this paper, we review the AR phosphorylation sites, the kinases responsible for these phosphorylations, as well as the biological context and the functional consequences of these phosphorylations. Finally, what is known about the state of AR phosphorylation in clinical samples is discussed.
Topics: Animals; Humans; Phosphorylation; Protein Kinases; Protein Structure, Tertiary; Receptors, Androgen
PubMed: 24424504
DOI: 10.1530/ERC-13-0472 -
Journal of Biomolecular Structure &... Aug 2022Glyoxalase I (GLO1) is a dimeric esterase of the glyoxalase system. Phosphorylation of the residue T106 has been found to inhibit GLO1 activity, and contribute to the...
Glyoxalase I (GLO1) is a dimeric esterase of the glyoxalase system. Phosphorylation of the residue T106 has been found to inhibit GLO1 activity, and contribute to the onset of oxidative stress and cellular damage. This research uses multiple molecular dynamics simulations and automated docking of both GLO1 and dimerically phosphorylated GLO1 (p2-GLO1) to predict the initial structural differences induced by phosphorylation, and their interaction with the intermediate substrate Hemimercaptal. This research indicates that immediately following phosphorylation, GLO1 exhibits reduced sphericity, partly caused by outward splaying of the loop region surrounding T106. Phosphorylation induces enhanced concerted motions in the loop composed of residues immediately surrounding T106, which are correlated with motions at the active site pocket at the distant, opposite end of the dimer. These T106 region loop motions result in the distortion of the shape of the active site, and potentially alter its accessibility. Phosphorylation alters the manner in which GLO1 interacts with Hemimercaptal. For GLO1, Hemimercaptal is predicted to bind to T106, which we propose constitutes a novel, highly accessible 'capture site' responsible for initial contact with the substrate. In contrast, for p2-GLO1, Hemimercaptal is unable to bind favourably to (phosphorylated) position T106, suggesting that this proposed transient 'capture site' is abolished upon phosphorylation of GLO1. Hence, a novel physiological role is here proposed for the known essential GLO1 residue T106. These results may further contribute to understanding the inhibition mechanism of GLO1 upon phosphorylation.Communicated by Ramaswamy H. Sarma.
Topics: Catalytic Domain; Lactoylglutathione Lyase; Oxidative Stress; Phosphorylation
PubMed: 33459186
DOI: 10.1080/07391102.2021.1873186 -
Biochimica Et Biophysica Acta Apr 2015Yeast cells have developed a variety of mechanisms to regulate the activity of metabolic enzymes in order to adjust their metabolism in response to genetic and... (Review)
Review
BACKGROUND
Yeast cells have developed a variety of mechanisms to regulate the activity of metabolic enzymes in order to adjust their metabolism in response to genetic and environmental perturbations. This can be achieved by a massive reprogramming of gene expression. However, the transcriptional response cannot explain the complexity of metabolic regulation, and mRNA stability regulation, non-covalent binding of allosteric effectors and post-translational modifications of enzymes (such as phosphorylation, acetylation and ubiquitination) are also involved, especially as short term responses, all converging in modulating enzyme activity.
SCOPE OF REVIEW
The functional significance of post-translational modifications (PTMs) to the regulation of the central carbon metabolism is the subject of this review.
MAJOR CONCLUSIONS
A genome wide analysis of PTMs indicates that several metabolic enzymes are subjected to multiple PTMs, suggesting that yeast cells can use different modifications and/or combinations of them to specifically respond to environmental changes. Glycolysis and fermentation are the pathways where phosphorylation, acetylation and ubiquitination are most frequent, while enzymes of storage carbohydrate metabolism are especially phosphorylated. Interestingly, some enzymes, such as the 6-phosphofructo-2-kinase Pfk26, the phosphofructokinases Pfk1 and Pfk2 and the pyruvate kinase Cdc19, are hubs of PTMs, thus representing central key regulation nodes. For the functionally better characterized enzymes, the role of phosphorylations and lysine modifications is discussed.
GENERAL SIGNIFICANCE
This review focuses on the regulatory mechanisms of yeast carbon metabolism, highlighting the requirement of quantitative, systematical studies to better understand PTM contribution to metabolic regulation.
Topics: Allosteric Regulation; Carbon; Fermentation; Gluconeogenesis; Glycolysis; Phosphorylation; Protein Processing, Post-Translational; RNA Stability; Saccharomyces cerevisiae; Transcription, Genetic
PubMed: 25512067
DOI: 10.1016/j.bbagen.2014.12.010 -
Journal of the American Chemical Society Oct 2014Protein phosphorylation controls major processes in cells. Although phosphorylation of serine, threonine, and tyrosine and also recently histidine and arginine are...
Protein phosphorylation controls major processes in cells. Although phosphorylation of serine, threonine, and tyrosine and also recently histidine and arginine are well-established, the extent and biological significance of lysine phosphorylation has remained elusive. Research in this area has been particularly limited by the inaccessibility of peptides and proteins that are phosphorylated at specific lysine residues, which are incompatible with solid-phase peptide synthesis (SPPS) due to the intrinsic acid lability of the P(═O)-N phosphoramidate bond. To address this issue, we have developed a new synthetic route for the synthesis of site-specifically phospholysine (pLys)-containing peptides by employing the chemoselectivity of the Staudinger-phosphite reaction. Our synthetic approach relies on the SPPS of unprotected ε-azido lysine-containing peptides and their subsequent reaction to phosphoramidates with phosphite esters before they are converted into the natural modification via UV irradiation or basic deprotection. With these peptides in hand, we demonstrate that electron-transfer dissociation tandem mass spectrometry can be used for unambiguous assignment of phosphorylated-lysine residues within histone peptides and that these peptides can be detected in cell lysates using a bottom-up proteomic approach. This new tagging method is expected to be an essential tool for evaluating the biological relevance of lysine phosphorylation.
Topics: Lysine; Molecular Structure; Peptides; Phosphorylation
PubMed: 25196693
DOI: 10.1021/ja507886s -
Topics in Current Chemistry (Cham) Jun 2017The complexity of phosphorylation pathways and their downstream effects is vast. Synthetic chemistry has been working side by side with biology to develop phosphate... (Review)
Review
The complexity of phosphorylation pathways and their downstream effects is vast. Synthetic chemistry has been working side by side with biology to develop phosphate labels for biological processes involving phosphorylated compounds. This chapter discusses recently employed methods for the preparation of several phosphate labels. Synthesis of biomolecules and their analogs and other useful or potentially useful phosphate derivatives is discussed.
Topics: Molecular Structure; Phosphates; Phosphorylation
PubMed: 28444630
DOI: 10.1007/s41061-017-0135-6 -
Journal of Visualized Experiments : JoVE Jun 2020Cardiac-specific myosin regulatory light chain kinase (cMLCK) regulates cardiac sarcomere structure and contractility by phosphorylating the ventricular isoform of the...
Cardiac-specific myosin regulatory light chain kinase (cMLCK) regulates cardiac sarcomere structure and contractility by phosphorylating the ventricular isoform of the myosin regulatory light chain (MLC2v). MLC2v phosphorylation levels are significantly reduced in failing hearts, indicating the clinical importance of assessing the activity of cMLCK and the phosphorylation level of MLC2v to elucidate the pathogenesis of heart failure. This paper describes nonradioactive methods to assess both the activity of cMLCK and MLC2v phosphorylation levels. In vitro kinase reactions are performed using recombinant cMLCK with recombinant calmodulin and MLC2v in the presence of ATP and calcium at 25 °C, which are followed by either a bioluminescent ADP detection assay or a phosphate-affinity SDS-PAGE. In the representative study, the bioluminescent ADP detection assay showed a strict linear increase of the signal at cMLCK concentrations between 1.25 nM to 25 nM. Phosphate-affinity SDS-PAGE also showed a linear increase of phosphorylated MLC2v in the same cMLCK concentration range. Next, the time-dependency of the reactions was examined at the concentration of 5 nM cMLCK. A bioluminescent ADP detection assay showed a linear increase in the signal during 90 min of the reaction. Similarly, phosphate-affinity SDS-PAGE showed a time-dependent increase of phosphorylated MLC2v. The biochemical parameters of cMLCK for MLC2v were determined by a Michaelis-Menten plot using the bioluminescent ADP detection assay. The Vmax was 1.65 ± 0.10 mol/min/mol kinase and the average Km was around 0.5 USA µM at 25 °C. Next, the activity of wild type and the dilated cardiomyopathy-associated p.Pro639Valfs*15 mutant cMLCK were measured. The bioluminescent ADP detection assay and phosphate-affinity SDS-PAGE correctly detected defects in cMLCK activity and MLC2v phosphorylation, respectively. In conclusion, a combination of the bioluminescent ADP detection assay and the phosphate-affinity SDS-PAGE is a simple, accurate, safe, low-cost, and flexible method to measure cMLCK activity and the phosphorylation level of MLC2v.
Topics: Animals; Calcium; Enzyme Assays; Humans; Myocytes, Cardiac; Myosin-Light-Chain Kinase; Phosphorylation
PubMed: 32658182
DOI: 10.3791/61168 -
Scientific Reports Apr 2022The role of TIRAP (toll/interleukin-1 receptor (TIR) domain-containing adapter protein) in macrophage inflammatory signalling has been significantly evolved since its...
The role of TIRAP (toll/interleukin-1 receptor (TIR) domain-containing adapter protein) in macrophage inflammatory signalling has been significantly evolved since its discovery in 2001 due to its dynamic nature and subcellular localization to regulate multiple signaling through several protein-protein interactions (PPIs). Structural analysis of these interactions can reveal a better understanding of their conformational dynamics and the nature of their binding. Tyrosine phosphorylation in the TIR domain of TIRAP is very critical for its function. In toll-like receptor (TLR) 4/2 signalling, Bruton's tyrosine kinase (BTK) and Protein kinase C delta (PKCδ) are known to phosphorylate the Y86, Y106, Y159, and Y187 of TIRAP which is crucial for the downstream function of MAPKs (mitogen-activated protein kinases) activation. The objective of this study is to understand the interaction of TIRAP with p38 MAPK through molecular docking and identify the importance of TIRAP tyrosine phosphorylation in p38 MAPK interaction. In this structural study, we performed an in-silico molecular docking using HADDOCK 2.4, pyDockWEB, ClusPro 2.0, and ZDOCK 3.0.2 tools to unravel the interaction between TIRAP and p38 MAPK. Further, manual in-silico phosphorylations of TIRAP tyrosines; Y86, Y106, Y159, and Y187 was created in the Discovery Studio tool to study the conformational changes in protein docking and their binding affinities with p38 MAPK in comparison to non-phosphorylated state. Our molecular docking and 500 ns of molecular dynamic (MD) simulation study demonstrates that the Y86 phosphorylation (pY86) in TIRAP is crucial in promoting the higher binding affinity (∆G) with p38 MAPK. The conformational changes due to the tyrosine phosphorylation mainly at the Y86 site pull the TIRAP closer to the active site in the kinase domain of p38 MAPK and plays a significant role at the interface site which is reversed in its dephosphorylated state. The heatmap of interactions between the TIRAP and p38 MAPK after the MD simulation shows that the TIRAP pY86 structure makes the highest number of stable hydrogen bonds with p38 MAPK residues. Our findings may further be validated in an in-vitro system and would be crucial for targeting the TIRAP and p38 MAPK interaction for therapeutic purposes against the chronic inflammatory response and associated diseases.
Topics: Membrane Glycoproteins; Mitogen-Activated Protein Kinases; Molecular Docking Simulation; Phosphorylation; Receptors, Interleukin-1; Signal Transduction; p38 Mitogen-Activated Protein Kinases
PubMed: 35379857
DOI: 10.1038/s41598-022-09528-8 -
Advances in Protein Chemistry and... 2023Protein phosphorylation is a vital reversible post-translational modification. This process is established by two classes of enzymes: protein kinases and protein... (Review)
Review
Protein phosphorylation is a vital reversible post-translational modification. This process is established by two classes of enzymes: protein kinases and protein phosphatases. Protein kinases phosphorylate proteins while protein phosphatases dephosphorylate phosphorylated proteins, thus, functioning as 'critical regulators' in signaling pathways. The eukaryotic protein phosphatases are classified as phosphoprotein phosphatases (PPP), metallo-dependent protein phosphatases (PPM), protein tyrosine (Tyr) phosphatases (PTP), and aspartate (Asp)-dependent phosphatases. The PPP and PPM families are serine (Ser)/threonine (Thr) specific phosphatases (STPs) that dephosphorylate Ser and Thr residues. The PTP family dephosphorylates Tyr residues while dual-specificity phosphatases (DsPTPs/DSPs) dephosphorylate Ser, Thr, and Tyr residues. The composition of these enzymes as well as their substrate specificity are important determinants of their functional significance in a number of cellular processes and stress responses. Their role in animal systems is well-understood and characterized. The functional characterization of protein phosphatases has been extensively covered in plants, although the comprehension of their mechanistic basis is an ongoing pursuit. The nature of their interactions with other key players in the signaling process is vital to our understanding. The substrates or targets determine their potential as well as magnitude of the impact they have on signaling pathways. In this article, we exclusively overview the various substrates of protein phosphatases in plant signaling pathways, which are a critical determinant of the outcome of various developmental and stress stimuli.
Topics: Animals; Phosphoprotein Phosphatases; Phosphorylation; Protein Kinases; Protein Processing, Post-Translational; Signal Transduction
PubMed: 36858740
DOI: 10.1016/bs.apcsb.2022.11.003 -
International Journal of Molecular... Feb 2023Our previous research suggests an important regulatory role of CK2-mediated phosphorylation of enzymes involved in the thymidylate biosynthesis cycle, i.e., thymidylate...
Our previous research suggests an important regulatory role of CK2-mediated phosphorylation of enzymes involved in the thymidylate biosynthesis cycle, i.e., thymidylate synthase (TS), dihydrofolate reductase (DHFR), and serine hydroxymethyltransferase (SHMT). The aim of this study was to show whether silencing of the CK2α gene affects TS and DHFR expression in A-549 cells. Additionally, we attempted to identify the endogenous kinases that phosphorylate TS and DHFR in CCRF-CEM and A-549 cells. We used immunodetection, immunofluorescence/confocal analyses, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), in-gel kinase assay, and mass spectrometry analysis. Our results demonstrate that silencing of the CK2α gene in lung adenocarcinoma cells significantly increases both TS and DHFR expression and affects their cellular distribution. Additionally, we show for the first time that both TS and DHFR are very likely phosphorylated by endogenous CK2 in two types of cancer cells, i.e., acute lymphoblastic leukaemia and lung adenocarcinoma. Moreover, our studies indicate that DHFR is phosphorylated intracellularly by CK2 to a greater extent in leukaemia cells than in lung adenocarcinoma cells. Interestingly, in-gel kinase assay results indicate that the CK2α' isoform was more active than the CK2α subunit. Our results confirm the previous studies concerning the physiological relevance of CK2-mediated phosphorylation of TS and DHFR.
Topics: Humans; Adenocarcinoma of Lung; Phosphorylation; Tetrahydrofolate Dehydrogenase; Thymidylate Synthase
PubMed: 36769342
DOI: 10.3390/ijms24033023