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Molecular Plant Pathology Jun 2018Phosphorylation and O-GlcNAcylation are two widespread post-translational modifications (PTMs), often affecting the same eukaryotic target protein. Plum pox virus (PPV)...
Phosphorylation and O-GlcNAcylation are two widespread post-translational modifications (PTMs), often affecting the same eukaryotic target protein. Plum pox virus (PPV) is a member of the genus Potyvirus which infects a wide range of plant species. O-GlcNAcylation of the capsid protein (CP) of PPV has been studied extensively, and some evidence of CP phosphorylation has also been reported. Here, we use proteomics analyses to demonstrate that PPV CP is phosphorylated in vivo at the N-terminus and the beginning of the core region. In contrast with the 'yin-yang' mechanism that applies to some mammalian proteins, PPV CP phosphorylation affects residues different from those that are O-GlcNAcylated (serines Ser-25, Ser-81, Ser-101 and Ser-118). Our findings show that PPV CP can be concurrently phosphorylated and O-GlcNAcylated at nearby residues. However, an analysis using a differential proteomics strategy based on iTRAQ (isobaric tags for relative and absolute quantitation) showed a significant enhancement of phosphorylation at Ser-25 in virions recovered from O-GlcNAcylation-deficient plants, suggesting that crosstalk between O-GlcNAcylation and phosphorylation in PPV CP takes place. Although the preclusion of phosphorylation at the four identified phosphotarget sites only had a limited impact on viral infection, the mimicking of phosphorylation prevents PPV infection in Prunus persica and weakens infection in Nicotiana benthamiana and other herbaceous hosts, prompting the emergence of potentially compensatory second mutations. We postulate that the joint action of phosphorylation and O-GlcNAcylation in the N-proximal segment of CP allows a fine-tuning of protein stability, providing the amount of CP required in each step of viral infection.
Topics: Phosphorylation; Plant Diseases; Plant Viruses; Viral Proteins; Virus Diseases
PubMed: 29024291
DOI: 10.1111/mpp.12626 -
Calcified Tissue International Oct 2021It is widely accepted that cellular processes are controlled by protein phosphorylation and has become increasingly clear that protein degradation, localization and...
It is widely accepted that cellular processes are controlled by protein phosphorylation and has become increasingly clear that protein degradation, localization and conformation as well as protein-protein interaction are the examples of subsequent cellular events modulated by protein phosphorylation. Enamel matrix proteins belong to members of the secretory calcium binding phosphoprotein (SCPP) family clustered on chromosome 4q21, and most of the SCPP phosphoproteins have at least one S-X-E motifs (S; serine, X; any amino acid, E; glutamic acid). It has been reported that mutations in C4orf26 gene, located on chromosome 4q21, are associated with autosomal recessive type of Amelogenesis Imperfecta (AI), a hereditary condition that affects enamel formation/mineralization. The enamel phenotype observed in patients with C4orf26 mutations is hypomineralized and partially hypoplastic, indicating that C4orf26 protein may function at both secretory and maturation stages of amelogenesis. The previous in vitro study showed that the synthetic phosphorylated peptide based on C4orf26 protein sequence accelerates hydroxyapatite nucleation. Here we show the molecular cloning of Gm1045, mouse homologue of C4orf26, which has 2 splicing isoforms. Immunohistochemical analysis demonstrated that the immunolocalization of Gm1045 is mainly observed in enamel matrix in vivo. Our report is the first to show that FAM20C, the Golgi casein kinase, phosphorylates C4orf26 and Gm1045 in cell cultures. The extracellular localization of C4orf26/Gm1045 was regulated by FAM20C kinase activity. Thus, our data point out the biological importance of enamel matrix-kinase control of SCPP phosphoproteins and may have a broad impact on the regulation of amelogenesis and AI.
Topics: Amelogenesis; Amelogenesis Imperfecta; Amino Acid Sequence; Animals; Calcium-Binding Proteins; Casein Kinase I; Cloning, Molecular; Extracellular Matrix Proteins; Humans; Mice; Phosphorylation
PubMed: 33884476
DOI: 10.1007/s00223-021-00847-y -
Life Science Alliance Oct 2022Yeast use the G-protein-coupled receptor signaling pathway to detect and track the mating pheromone. The G-protein-coupled receptor pathway is inhibited by the regulator...
Yeast use the G-protein-coupled receptor signaling pathway to detect and track the mating pheromone. The G-protein-coupled receptor pathway is inhibited by the regulator of G-protein signaling (RGS) Sst2 which induces Gα GTPase activity and inactivation of downstream signaling. G-protein signaling activates the MAPK Fus3, which phosphorylates the RGS; however, the role of this modification is unknown. We found that pheromone-induced RGS phosphorylation peaks early; the phospho-state of RGS controls its localization and influences MAPK spatial distribution. Surprisingly, phosphorylation of the RGS promotes completion of cytokinesis before pheromone-induced growth. Completion of cytokinesis in the presence of pheromone is promoted by the kelch-repeat protein, Kel1 and antagonized by the formin Bni1. We found that RGS complexes with Kel1 and prefers the unphosphorylatable RGS mutant. We also found overexpression of unphosphorylatable RGS exacerbates cytokinetic defects, whereas they are rescued by overexpression of Kel1. These data lead us to a model where Kel1 promotes completion of cytokinesis before pheromone-induced polarity but is inhibited by unphosphorylated RGS binding.
Topics: Cytokinesis; GTP-Binding Proteins; GTPase-Activating Proteins; Microfilament Proteins; Mitogen-Activated Protein Kinases; Pheromones; Phosphorylation; RGS Proteins; Receptors, G-Protein-Coupled; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 35985794
DOI: 10.26508/lsa.202101245 -
Molecular Biology of the Cell Jul 2016Multisite phosphorylation cycles are ubiquitous in cell regulation systems and are studied at multiple levels of complexity, from molecules to organisms, with the...
Multisite phosphorylation cycles are ubiquitous in cell regulation systems and are studied at multiple levels of complexity, from molecules to organisms, with the ultimate goal of establishing predictive understanding of the effects of genetic and pharmacological perturbations of protein phosphorylation in vivo. Achieving this goal is essentially impossible without mathematical models, which provide a systematic framework for exploring dynamic interactions of multiple network components. Most of the models studied to date do not discriminate between the distinct partially phosphorylated forms and focus on two limiting reaction regimes, distributive and processive, which differ in the number of enzyme-substrate binding events needed for complete phosphorylation or dephosphorylation. Here we use a minimal model of extracellular signal-related kinase regulation to explore the dynamics of a reaction network that includes all essential phosphorylation forms and arbitrary levels of reaction processivity. In addition to bistability, which has been studied extensively in distributive mechanisms, this network can generate periodic oscillations. Both bistability and oscillations can be realized at high levels of reaction processivity. Our work provides a general framework for systematic analysis of dynamics in multisite phosphorylation systems.
Topics: Models, Biological; Molecular Dynamics Simulation; Phosphorylation; Protein Binding; Proteins
PubMed: 27226482
DOI: 10.1091/mbc.E16-03-0137 -
Biophysical Journal Nov 2020Proteins carry out a wide range of functions that are tightly regulated in space and time. Protein phosphorylation is the most common post-translation modification of...
Proteins carry out a wide range of functions that are tightly regulated in space and time. Protein phosphorylation is the most common post-translation modification of proteins and plays a key role in the regulation of many biological processes. The finding that many phosphorylated residues are not solvent exposed in the unphosphorylated state opens several questions for understanding the mechanism that underlies phosphorylation and how phosphorylation may affect protein structures. First, because kinases need access to the phosphorylated residue, how do such buried residues become modified? Second, once phosphorylated, what are the structural effects of phosphorylation of buried residues, and do they lead to changed conformational dynamics? We have used the ternary complex between p27 (p27), Cdk2, and cyclin A to study these questions using enhanced sampling molecular dynamics simulations. In line with previous NMR and single-molecule fluorescence experiments, we observe transient exposure of Tyr88 in p27, even in its unphosphorylated state. Once Tyr88 is phosphorylated, we observe a coupling to a second site, thus making Tyr74 more easily exposed and thereby the target for a second phosphorylation step. Our observations provide atomic details on how protein dynamics plays a role in modulating multisite phosphorylation in p27, thus supplementing previous experimental observations. More generally, we discuss how the observed phenomenon of transient exposure of buried residues may play a more general role in regulating protein function.
Topics: Cell Cycle Proteins; Cyclin A; Cyclin-Dependent Kinase 2; Cyclins; Phosphorylation; Proliferating Cell Nuclear Antigen; Protein Processing, Post-Translational
PubMed: 33147476
DOI: 10.1016/j.bpj.2020.06.040 -
International Journal of Molecular... Nov 2019Autophagy is a catabolic cellular recycling pathway that is essential for maintaining intracellular homeostasis. Autophagosome formation is achieved via the coordination...
BACKGROUND
Autophagy is a catabolic cellular recycling pathway that is essential for maintaining intracellular homeostasis. Autophagosome formation is achieved via the coordination of the Beclin-1 protein complex. Rubicon is a Beclin-1 associated protein that suppresses autophagy by impairing the activity of the class III PI3K, Vps34. However, very little is known about the molecular mechanisms that regulate Rubicon function.
METHODS
In this study, co-immunoprecipitation and kinase assays were used to investigate the ability of Hormonally Upregulated Neu-associated Kinase (HUNK) to bind to and phosphorylate Rubicon. LC3B was monitored by immunofluorescence and immunoblotting to determine whether phosphorylation of Rubicon by HUNK controls the autophagy suppressive function of Rubicon.
RESULTS
Findings from this study identify Rubicon as a novel substrate of HUNK and show that phosphorylation of Rubicon inhibits its function, promoting autophagy.
Topics: Apoptosis Regulatory Proteins; Autophagy; Autophagy-Related Proteins; Cells, Cultured; HEK293 Cells; Humans; Phagosomes; Phosphorylation; Protein Serine-Threonine Kinases
PubMed: 31752345
DOI: 10.3390/ijms20225813 -
Frontiers in Bioscience (Landmark... Jan 2022Psychoactive substances are a class of chemical substances which could cause public health threats. Cognitive disorders are a category of mental health disorders that... (Review)
Review
Psychoactive substances are a class of chemical substances which could cause public health threats. Cognitive disorders are a category of mental health disorders that primarily affect cognitive abilities. Tau protein could maintain neuronal cytoskeleton stabilization. Post-translational modification of tau, especially phosphorylation, is an important way to regulate the structure and function of tau and phosphorylated tau is closely related to cognitive function. Lots of studies have reported the phenomenon that psychoactive substances can cause cognitive function impairment. We reviewed recent related studies and discussed them by drug classification. We mainly focused on cognitive disorders caused by acute or chronic exposure of each drugs, animal experiments and the mechanisms associated with tau phosphorylation, then compared the similarities and differences among them, trying to find out the common rules. The results suggested that tau phosphorylation is involved in psychoactive substance-induced cognitive disorder and different psychoactive substances may act by affecting amount or activity of different kinases and phosphatases in the metabolic pathway of tau. We demonstrated that tau protein is a potential target for psychoactive substances induced cognitive disorder treatments.
Topics: Animals; Cognition; Cognitive Dysfunction; Phosphorylation; Substance-Related Disorders; tau Proteins
PubMed: 35090311
DOI: 10.31083/j.fbl2701006 -
Bioconjugate Chemistry Jun 2023Phosphorylation of proteins by kinase enzymes is a post-translational modification involved in a myriad of biological events, including cell signaling and disease...
Phosphorylation of proteins by kinase enzymes is a post-translational modification involved in a myriad of biological events, including cell signaling and disease development. Identifying the interactions between a kinase and its phosphorylated substrate(s) is necessary to characterize phosphorylation-mediated cellular events and encourage development of kinase-targeting drugs. One method for substrate-kinase identification utilizes photocrosslinking γ-phosphate-modified ATP analogues to covalently link kinases to their substrates for subsequent monitoring. Because photocrosslinking ATP analogues require UV light, which could influence cell biology, we report here two ATP analogues, ATP-aryl fluorosulfate (ATP-AFS) and ATP-hexanoyl bromide (ATP-HexBr), that crosslink kinase-substrate pairs via proximity-mediated reactions without the need for UV irradiation. Both ATP-AFS and ATP-HexBr acted as cosubstrates with a variety of kinases for affinity-based crosslinking, with ATP-AFS showing more robust complexes. Importantly, ATP-AFS promoted crosslinking in lysates, which demonstrates compatibility with complex cellular mixtures for future application to kinase-substrate identification.
Topics: Phosphorylation; Protein Processing, Post-Translational; Proteins; Catalysis; Adenosine Triphosphate
PubMed: 37279085
DOI: 10.1021/acs.bioconjchem.3c00131 -
The Biochemical Journal Mar 2022How cellular functions are regulated through protein phosphorylation events that promote or inhibit protein-protein interactions (PPIs) is key to understanding...
How cellular functions are regulated through protein phosphorylation events that promote or inhibit protein-protein interactions (PPIs) is key to understanding regulatory molecular mechanisms. Whilst phosphorylation can orthosterically or allosterically influence protein recognition, phospho-driven changes in the conformation of recognition motifs are less well explored. We recently discovered that clathrin heavy chain recognizes phosphorylated TACC3 through a helical motif that, in the unphosphorylated protein, is disordered. However, it was unclear whether and how phosphorylation could stabilize a helix in a broader context. In the current manuscript, we address this challenge using poly-Ala-based model peptides and a suite of circular dichroism and nuclear magnetic resonance spectroscopies. We show that phosphorylation of a Ser residue stabilizes the α-helix in the context of an Arg(i-3)pSeri Lys(i+4) triad through charge-reinforced side chain interactions with positive co-operativity, whilst phosphorylation of Thr induces an opposing response. This is significant as it may represent a general method for control of PPIs by phosphorylation; basic kinase-substrate motifs are common with 55 human protein kinases recognizing an Arg at a position -3 from the phosphorylated Ser, whilst the Arg(i-3)Seri Lys(i+4) is a motif found in over 2000 human proteins.
Topics: Cell Cycle Proteins; Circular Dichroism; Humans; Microtubule-Associated Proteins; Phosphorylation; Phosphoserine; Protein Conformation, alpha-Helical
PubMed: 35212726
DOI: 10.1042/BCJ20210812 -
The Journal of Biological Chemistry Mar 2019Twist1 is a basic helix-loop-helix transcription factor that plays a key role in embryonic development, and its expression is down-regulated in adult cells. However,...
Twist1 is a basic helix-loop-helix transcription factor that plays a key role in embryonic development, and its expression is down-regulated in adult cells. However, Twist1 is highly expressed during cancer development, conferring a proliferative, migratory, and invasive phenotype to malignant cells. Twist1 expression can be regulated post-translationally by phosphorylation or ubiquitination events. We report in this study a previously unknown and relevant Twist1 phosphorylation site that controls its stability. To identify candidate phosphorylation sites in Twist1, we first conducted an analysis of the Twist1 protein, which yielded several potential sites. Because most of these sites were predicted to be phosphorylated by protein kinase C (PKC), we overexpressed PKCα in several cell lines and found that it phosphorylates Twist1 on Ser-144. Using a combination of immunoblotting, immunoprecipitation, protein overexpression, and CRISPR/Cas9-mediated PKCα knockout experiments, we observed that PKCα-mediated Twist1 phosphorylation at Ser-144 inhibits Twist1 ubiquitination and consequently stabilizes it. These results provide evidence for a direct association between PKCα and Twist1 and yield critical insights into the PKCα/Twist1 signaling axis that governs cancer aggressiveness.
Topics: Epithelial-Mesenchymal Transition; HEK293 Cells; Humans; Models, Molecular; Nuclear Proteins; Phosphorylation; Protein Interaction Domains and Motifs; Protein Kinase C-alpha; Protein Stability; Twist-Related Protein 1; Ubiquitination
PubMed: 30733340
DOI: 10.1074/jbc.RA118.005921