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In contrast to the well-studied canonical regulatory mechanisms, the way by which the recently discovered Src N-terminal regulatory element (SNRE) modulates Src activity is not yet well understood. Phosphorylation of serine and threonine residues modulates the charge distribution along the disordered region of the SNRE and may affect a fuzzy complex with the SH3 domain that is believed to act as an information transduction element. The pre-existing positively charged sites can interact with the newly introduced phosphate groups by modulating their acidity, introducing local conformational restrictions, or by coupling various phosphosites into a functional unit. In this paper, we use pH-dependent NMR measurements combined with single point mutations to identify the interactions of basic residues with physiologically important phosphorylated residues and to characterize the effect of these interactions in neighbor residues, thus providing insight into the electrostatic network in the isolated disordered regions and in the entire SNRE. From a methodological point of view, the linear relationships observed between the mutation-induced pKa changes of the phosphate groups of phosphoserine and phosphothreonine and the pH-induced chemical shifts of the NH groups of these residues provide a very convenient alternative to identify interacting phosphate groups without the need to introduce point mutations on specific basic residues. [ABSTRACT FROM AUTHOR]

The forkhead-associated (FHA) domain, a well-characterized small protein module that mediates protein–protein interactions by targeting motifs containing phosphothreonine, is present in many regulatory molecules like protein kinase, phosphatases, transcription factors, and other functional proteins. FHA-domain containing proteins in yeast and human are involved in a large variety of cellular processes such as DNA repair, cell cycle arrest, or pre-mRNA processing. Since the first FHA-domain protein, kinase-associated protein phosphatase (KAPP) was found in plants, the interest in plant FHA-containing proteins has increased dramatically, mainly due to the important role of FHA domain-containing proteins in plant growth and development. In this review, we provide a comprehensive overview of the fundamental properties of FHA domain-containing proteins in plants, and systematically summarized and analyzed the research progress of proteins containing the FHA domain in plants. We also emphasized that AT5G47790 and its homologs may play an important role as the regulatory subunit of protein phosphatase 1 (PP1) in plants. [ABSTRACT FROM AUTHOR]

We have successfully designed a uranyl ion (U(VI)-specific peptide and used it in the fabrication of an electrochemical sensor. The 12-amino acid peptide sequence, (n) DKDGDGYIpTAAE (c), originates from calmodulin, a Ca(II)-binding protein, and contains a phosphothreonine that enhances the sequence's affinity for U(VI) over Ca(II). The sensing mechanism of this U(VI) sensor is similar to other electrochemical peptide-based sensors, which relies on the change in the flexibility of the peptide probe upon interacting with the target. The sensor was systematically characterized using alternating current voltammetry (ACV) and cyclic voltammetry. Its limit of detection was 50 nM, which is lower than the United States Environmental Protection Agency maximum contaminant level for uranium. The signal saturation time was ~40 min. In addition, it showed minimal cross-reactivity when tested against nine different metal ions, including Ca(II), Mg(II), Pb(II), Hg(II), Cu(II), Fe(II), Zn(II), Cd(II), and Cr(VI). Its reusability and ability to function in diluted aquifer and drinking water samples were further confirmed and validated. The response of the sensor fabricated with the same peptide sequence but with a nonphosphorylated threonine was also analyzed, substantiating the positive effects of threonine phosphorylation on U(VI) binding. This study places emphasis on strategic utilization of non-standard amino acids in the design of metal ion-chelating peptides, which will further diversify the types of peptide recognition elements available for metal ion sensing applications. [ABSTRACT FROM AUTHOR]

In contrast to the well-studied canonical regulatory mechanisms, the way by which the recently discovered Src N-terminal regulatory element (SNRE) modulates Src activity is not yet well understood. Phosphorylation of serine and threonine residues modulates the charge distribution along the disordered region of the SNRE and may affect a fuzzy complex with the SH3 domain that is believed to act as an information transduction element. The pre-existing positively charged sites can interact with the newly introduced phosphate groups by modulating their acidity, introducing local conformational restrictions, or by coupling various phosphosites into a functional unit. In this paper, we use pH-dependent NMR measurements combined with single point mutations to identify the interactions of basic residues with physiologically important phosphorylated residues and to characterize the effect of these interactions in neighbor residues, thus providing insight into the electrostatic network in the isolated disordered regions and in the entire SNRE. From a methodological point of view, the linear relationships observed between the mutation-induced pKa changes of the phosphate groups of phosphoserine and phosphothreonine and the pH-induced chemical shifts of the NH groups of these residues provide a very convenient alternative to identify interacting phosphate groups without the need to introduce point mutations on specific basic residues.

Actin cables, composed of actin filament bundles nucleated by formins, mediate intracellular transport for cell polarity establishment and maintenance. We previously observed that metaphase cells preferentially promote actin cable assembly through cyclin-dependent kinase 1 (Cdk1) activity. However, the relevant metaphase Cdk1 targets were not known. Here we show that the highly conserved actin filament crosslinking protein fimbrin is a critical Cdk1 target for actin cable assembly regulation in budding yeast. Fimbrin is specifically phosphorylated on threonine 103 by the metaphase cyclin-Cdk1 complex, in vivo and in vitro. On the basis of conformational simulations, we suggest that this phosphorylation stabilizes fimbrin's N-terminal domain, and modulates actin filament binding to regulate actin cable assembly and stability in cells. Overall, this work identifies fimbrin as a key target for cell cycle regulation of actin cable assembly in budding yeast, and suggests an underlying mechanism.

Autism spectrum disorders (ASDs) comprise a highly heritable, multifarious group of neurodevelopmental disorders, which are characterized by repetitive behaviors and impairments in social interactions. Point mutations have been identified in X-linked Neuroligin (NLGN) 3 and 4X genes in patients with ASDs and all of these reside in their extracellular domains except for a single point mutation in the cytoplasmic domain of NLGN4X in which an arginine is mutated to a cysteine (R704C). Here we show that endogenous NLGN4X is robustly phosphorylated by protein kinase C (PKC) at T707, and R704C completely eliminates T707 phosphorylation. Endogenous NLGN4X is intensely phosphorylated on T707 upon PKC stimulation in human neurons. Furthermore, a phospho-mimetic mutation at T707 has a profound effect on NLGN4X-mediated excitatory potentiation. Our results now establish an important interplay between a genetic mutation, a key posttranslational modification, and robust synaptic changes, which can provide insights into the synaptic dysfunction of ASDs.

We have successfully designed a uranyl ion (U(VI)-specific peptide and used it in the fabrication of an electrochemical sensor. The 12-amino acid peptide sequence, (n) DKDGDGYIpTAAE (c), originates from calmodulin, a Ca(II)-binding protein, and contains a phosphothreonine that enhances the sequence’s affinity for U(VI) over Ca(II). The sensing mechanism of this U(VI) sensor is similar to other electrochemical peptide-based sensors, which relies on the change in the flexibility of the peptide probe upon interacting with the target. The sensor was systematically characterized using alternating current voltammetry (ACV) and cyclic voltammetry. Its limit of detection was 50 nM, which is lower than the United States Environmental Protection Agency maximum contaminant level for uranium. The signal saturation time was ~40 min. In addition, it showed minimal cross-reactivity when tested against nine different metal ions, including Ca(II), Mg(II), Pb(II), Hg(II), Cu(II), Fe(II), Zn(II), Cd(II), and Cr(VI). Its reusability and ability to function in diluted aquifer and drinking water samples were further confirmed and validated. The response of the sensor fabricated with the same peptide sequence but with a nonphosphorylated threonine was also analyzed, substantiating the positive effects of threonine phosphorylation on U(VI) binding. This study places emphasis on strategic utilization of non-standard amino acids in the design of metal ion-chelating peptides, which will further diversify the types of peptide recognition elements available for metal ion sensing applications.

β-Catenin phosphorylation plays important roles in modulating its functions, but the effects of different phosphorylated forms of β-catenin in response to heterocellular interaction are unclear. Here we investigated whether distinct modes of phosphorylation on β-catenin could be triggered through heterocellular interactions between endothelial cells (ECs) and smooth muscle cells (SMCs), and the consequent modulation of EC functions. ECs were cocultured with SMCs to initiate direct contact and paracrine interaction. EC-SMC coculture induced EC β-catenin phosphorylations simultaneously at tyrosine 142 (Tyr142) and serine 45/threonine 41 (Ser45/Thr41) at the cytoplasm/nuclei and the membrane, respectively. Treating ECs with SMC-conditional medium induced β-catenin phosphorylation only at Ser45/Thr41. These findings indicate that different phosphorylation effects of EC-SMC coculture were induced through heterocellular direct contact and paracrine effects, respectively. Using specific blocking peptides, antagonists, and siRNAs, we found that the β-catenin Tyr142-phosphorylation was mediated by connexin 43/Fer and that the β-catenin Ser45/Thr41-phosphorylation was mediated by SMC-released bone morphogenetic proteins through VE-cadherin and bone morphogenetic protein receptor-II/Smad5. Transfecting ECs with β-catenin-Tyr142 or -Ser45 mutants showed that these two phosphorylated forms of β-catenin modulate differential EC function: The Tyr142-phosphorylated β-catenin stimulates vascular cell-adhesion molecule-1 expression to increase EC-monocytic adhesion, but the Ser45/Thr41-phosphorylated β-catenin attenuates VE-cadherin-dependent junction structures to increase EC permeability. Our findings provide new insights into the understanding of regulatory complexities of distinct modes of β-catenin phosphorylations under EC-SMC interactions and suggest that different phosphorylated forms of β-catenin play important roles in modulating vascular pathophysiology through different heterocellular interactions.

The order and timing of cell-cycle events is controlled by changing substrate specificity and different activity thresholds of cyclin-dependent kinases (CDKs). However, it is not understood how a single protein kinase can trigger hundreds of switches in a sufficiently time-resolved fashion. We show that cyclin-Cdk1-Cks1-dependent phosphorylation of multisite targets in Saccharomyces cerevisiae is controlled by key substrate parameters including distances between phosphorylation sites, distribution of serines and threonines as phosphoacceptors and positioning of cyclin-docking motifs. The component mediating the key interactions in this process is Cks1, the phosphoadaptor subunit of the cyclin-Cdk1-Cks1 complex. We propose that variation of these parameters within networks of phosphorylation sites in different targets provides a wide range of possibilities for differential amplification of Cdk1 signals, thus providing a mechanism to generate a wide range of thresholds in the cell cycle.

In dementia, Alzheimer's disease (AD) is the most common type, characterized by the deposits of neurofibrillary tangles and senile plaques with concomitant deterioration in spatial memory and other cognitive functions. Till date, although no cure is available for AD, a few treatment options offer help in reducing the symptoms. In the present study, the sequence 111-137 in the distal N-terminal charge transition region of tau, harbouring the pathologically relevant phospho-serine (pSer 113) and phosphothreonine (pThr 123) (111TPpSLEDEAAGHVpTQARMVSKSKD GTGS137) was selected as a potential immunotherapeutic peptide. Polyclonal anti-peptide antibodies raised in rabbits effectively dissociated the oligomers/aggregates of recombinant human tau in vitro. Administration of affinity purified anti-peptide antibodies to the okadaic acid induced tauopathy model rats resulted in a significant progress in spatial memory functions in Barnes maze task with concomitant reduction in p-tau levels in the hippocampal homogenates. Thus, targeting the phospho-residue sequence 111-137 in tau may be therapeutically relevant for AD and other related tauopathies. These antibodies may also have a clinical value in terms of immunosassay development for quantitation of pathology associated pSer113 and pThr 123 in AD samples. [ABSTRACT FROM AUTHOR]

Antibodies play a crucial role in monitoring post-translational modifications, like phosphorylation, which regulates protein activity and location; however, commercial polyclonal and monoclonal antibodies have limitations in renewability and engineering compared to recombinant affinity reagents. A scaffold based on the Forkhead-associated domain (FHA) has potential as a selective affinity reagent for this post-translational modification. Engineered FHA domains, termed phosphothreonine-binding domains (pTBDs), with limited cross-reactivity were isolated from an M13 bacteriophage display library by affinity selection with phosphopeptides corresponding to human mTOR, Chk2, 53BP1, and Akt1 proteins. To determine the specificity of the representative pTBDs, we focused on binders to the pT543 phosphopeptide (536-IDEDGENpTQIEDTEP-551) of the DNA repair protein 53BP1. ELISA and western blot experiments have demonstrated the pTBDs are specific to phosphothreonine, demonstrating the potential utility of pTBDs for monitoring the phosphorylation of specific threonine residues in clinically relevant human proteins., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Importance: This work has global significance in the catfish industry, which provides food for increasing global populations. E. ictaluri is a leading cause of disease loss, and EseN is an important player in E. ictaluri virulence. The E. ictaluri T3SS effector EseN plays an essential role in establishing infection, but the specific role EseN plays is not well characterized. EseN belongs to a family of phosphothreonine lyase effectors that specifically target host mitogen activated protein kinase (MAPK) pathways important in regulating host responses to infection. No phosphothreonine lyase equivalents are known in eukaryotes, making this family of effectors an attractive target for indirect narrow-spectrum antibiotics. Targeting of major vault protein and PDK1 kinase by EseN has not been reported in EseN homologs in other pathogens and may indicate unique functions of E. ictaluri EseN. EseN targeting of PDK1 is particularly interesting in that it is linked to an extraordinarily diverse group of cellular functions., Competing Interests: The authors declare no conflict of interest.

The aim of this study was to determine if the achievement of the "in vitro" capacitation (IVC) status and subsequent progesterone‐induced "in vitro" acrosome exocytosis (IVAE) was accompanied with overall changes in threonine phosphorylation (pThre) of boar spermatozoa. For this purpose, mono‐ and bi‐dimensional Western blot analyses as well as immunocytochemistry studies against pThre were performed in boar sperm subjected to IVC and subsequent IVAE. Mono‐dimensional Western blot in non‐capacitated samples showed that launching of IVC did induce an overall increase in signal intensity in all observed bands that was followed by a subsequent decrease afterwards. Bi‐dimensional Western blot analysis showed the presence of four main signal protein clusters. The attainment of IVC induced an overall decrease in the number and intensity of spots of Clusters A, B and C and a concomitant increase in the intensity of spots of Cluster D. The IVAE launching caused a rapid increase in the intensity of spots of Clusters B, C and D, which was followed by a subsequent decrease of the intensity together with a concomitant pI displacement of Cluster C. Finally, immunocytochemistry showed that the pThre signal of non‐capacitated cells was located at the whole sperm. The IVC did not induce prominent changes in this location. In contrast, the induction of IVAE caused the appearance of an additional an intense acrosome and tail pThre signal that subsequently decreased. In conclusion, our results indicate that IVC and further IVAE induced specific changes in the intensity and appearance of pThre protein phosphorylation which were linked to changes of specific protein characteristics as pI. These results support, thus, the existence of a specific role of pThre in IVC/IVAE of boar sperm. [ABSTRACT FROM AUTHOR]

Phosphorylation and dephosphorylation of proteins by kinases and phosphatases are central to cellular responses and function. The structural effects of serine and threonine phosphorylation were examined in peptides and in proteins, by circular dichroism, NMR spectroscopy, bioinformatics analysis of the PDB, small-molecule X-ray crystallography, and computational investigations. Phosphorylation of both serine and threonine residues induces substantial conformational restriction in their physiologically more important dianionic forms. Threonine exhibits a particularly strong disorder-to-order transition upon phosphorylation, with dianionic phosphothreonine preferentially adopting a cyclic conformation with restricted ϕ (ϕ ∼ -60°) stabilized by three noncovalent interactions: a strong intraresidue phosphate-amide hydrogen bond, an n → π* interaction between consecutive carbonyls, and an n → σ* interaction between the phosphate Oγ lone pair and the antibonding orbital of C-Hβ that restricts the χ 2 side-chain conformation. Proline is unique among the canonical amino acids for its covalent cyclization on the backbone. Phosphothreonine can mimic proline's backbone cyclization via noncovalent interactions. The preferred torsions of dianionic phosphothreonine are ϕ,ψ = polyproline II helix > α-helix (ϕ ∼ -60°); χ 1 = g - ; χ 2 ∼ +115° (eclipsed C-H/O-P bonds). This structural signature is observed in diverse proteins, including in the activation loops of protein kinases and in protein-protein interactions. In total, these results suggest a structural basis for the differential use and evolution of threonine versus serine phosphorylation sites in proteins, with serine phosphorylation typically inducing smaller, rheostat-like changes, versus threonine phosphorylation promoting larger, step function-like switches, in proteins.

Transcription factor c-Myc is an oncoprotein that is regulated at the post-translational level through phosphorylation of two conserved residues, Serine 62 (Ser62) and Threonine 58 (Thr58). A highly specific tool capable of recognizing Myc via pThr58 is needed to monitor activation and localization. Through phage display, we have isolated 10 engineered Forkhead-associated (FHA) domains that selectively bind to a phosphothreonine (pThr)-containing peptide (53-FELLP pT PPLSPS-64) segment of human c-Myc. One domain variant was observed to bind to the Myc-pThr58 peptide with a K D value of 800 nM and had >1000-fold discrimination between the phosphorylated and non-phosphorylated peptide. The crystal structure of the engineered FHA Myc-pThr-binding domain (Myc-pTBD) was solved in complex with its cognate ligand. The Myc-pTBD was observed to be structurally similar to the yeast Rad9 FHA1 domain, except that its β4-β5 and β10-β11 loops form a hydrophobic pocket to facilitate the interaction between the domain and the peptide ligand. The Myc-pTBD’s specificity for its cognate ligand was demonstrated to be on a par with 3 commercial polyclonal antibodies, suggesting that this recombinant reagent is a viable alternative to antibodies for monitoring Myc regulation. [ABSTRACT FROM AUTHOR]

Activating Transcription Factor 2 (ATF2) plays an important role in mammalian cell proliferation, apoptosis and DNA repair. Its activation is dependent on the sequential phosphorylation of residue threonine 71 (T71) followed by threonine 69 (T69) in its transactivation domain. While these modifications can be directed by a variety of kinases, the time to reach full phosphorylation is dependent on which signaling pathway has been activated, which is thought to be important for proper temporal regulation. To explore this phenomenon further, there have been ongoing efforts to generate affinity reagents for monitoring phosphorylation events in cellular assays. While phospho-specific antibodies have been valuable tools for monitoring cell signaling events, those raised against a peptide containing two or more adjacent phosphosites tend to cross-react with that peptide’s various phospho-states, rendering such reagents unusable for studying sequential phosphorylation. As an alternative, we have employed the N-terminal Forkhead-associated 1 (FHA1) domain of yeast Rad53p as a scaffold to generate recombinant affinity reagents via phage display and were successful in generating a set of reagents that can distinguish between the dual-phosphorylated epitope, 63-IVADQ pT P pT PTRFLK-77, and the mono-phosphorylated epitope, 63-IVADQ pT PTPTRFLK-77, in the human ATF2 transactivation domain. [ABSTRACT FROM AUTHOR]

Dietary nutrients have emerged as potential therapeutic adjuncts for inflammatory bowel disease (IBD) given their impact on intestinal homeostasis through the modulation of immune response, gut microbiota composition and epithelial barrier stability. Several nutrients have already been associated with a protective phenotype. Yet, there is a lack of knowledge toward the most promising ones as well as the most adequate phase of action. To unveil the most prominent therapy candidates we characterized the colon metabolic profile during colitis development. We have observed a twofold decrease in threonine levels in mice subjected to DSS-induced colitis. We then assessed the effect of threonine supplementation in the beginning of the inflammatory process (DSS + Thr) or when inflammation is already established (DSS + Thr D8). Colitis progression was similar between the treated groups and control colitic mice, yet threonine had a surprisingly detrimental effect when administered in the beginning of the disease, with mice displaying a delayed recovery when compared to control mice and mice supplemented with threonine after day 8. Although no major changes were found in their metabolic profile, DSS + Thr mice displayed altered expression in mucin-encoding genes, as well as in goblet cell counts, unveiling an impaired ability to produce mucus. Moreover, IL-22 secretion was decreased in DSS + Thr mice when compared to DSS + Thr D8 mice. Overall, these results suggest that supplementation with threonine during colitis induction impact goblet cell number and delays the recovery period. This reinforces the importance of a deeper understanding regarding threonine supplementation in IBD. [ABSTRACT FROM AUTHOR]

Blastocladiella emersonii is an interesting model for studding the evolution of cell differentiation in eukaryotic cell because of its taxonomic position towards the base of the fungal phylogenetic tree and because it undergoes radical morphological and biochemical changes throughout its life cycle. In this work, we biochemically characterized a high alkaline phosphotyrosine phosphatase activity present on the cell surface (ectophosphatase) of B. emersonii . The ectophosphatase activity was strongly inhibited at acidic pH values as well as by specific phosphatase inhibitors, such as sodium orthovanadate and bpv-PHEN. In addition, the enzyme activity was modulated by the extracellular concentration of inorganic phosphate (P i ) present in both reaction mixture and culture medium. Phosphotyrosine was hydrolysed at the same extent of its analog, p- NPP, while the hydrolysis of phosphothreonine was 2-fold lower, suggesting that a phosphotyrosine ectophosphatase activity is present on the cell surface of B. emersonii . The ectophosphatase activity was also strongly inhibited by EGTA, indicating the participation of Ca 2+ ions on catalysis. The hydrolysis of p- NPP was differentially regulated throughout the B. emersonii life cycle, suggesting that the ectophosphatase activity could be involved in cell differentiation processes. In support of this, the addition of bpv-PHEN or vanadate at the beginning of germination inhibited the differentiation of zoospores to germ cells, compared to control or tartrate-treated cells. On the other hand, if the inhibitors are added 15 or 30 min after initiation of germination the inhibitory effect on zoospore germination decreases significantly, suggesting that the phosphotyrosine ectophosphatase activity is important at the first minutes of germination. The addition of vanadate, molybdate and bpv-PHEN during vegetative growth inhibited the enlargement of the cells compared to control or tartrate-treated cells. Finally, vanadate or bpv-PHEN added during sporulation strongly inhibited zoospore biogenesis, indicating an important role of such ectophosphatases in this differentiation process. Taken together, these data show the existence of a high alkaline ectophosphotyrosine phosphatase activity in B. emersonii that is probably tied to cell differentiation processes of the fungus. [ABSTRACT FROM AUTHOR]

A novel and facile synthetic strategy for α,α-difluorinated phosphonate mimetics of phosphoserine/phosphothreonine utilizing rhodium-catalyzed asymmetric hydrogenation was developed. The dehydrogenated substrate β-difluorophosphonomethyl α-(acylamino)acrylates were first prepared from protected serine/threonine followed by asymmetric hydrogenation using the rhodium–DuPhos catalytic system to generate the chiral center(s). These important phosphonate building blocks were successfully incorporated into phosphatase-resistant peptides, which displayed similar inhibition to the 14-3-3 ζ protein as the parent pSer/pThr peptides. [ABSTRACT FROM AUTHOR]

Among all the post-translational modifications (PTMs) of proteins, Phosphorylation is known to be the most important and highly occurring PTM in eukaryotes and prokaryotes. It has an important regulatory mechanism which is required in most of the pathological and physiological processes including neural activity and cell signalling transduction. The process of threonine phosphorylation modifies the threonine by the addition of a phosphoryl group to the polar side chain, and generates phosphothreonine sites. The investigation and prediction of phosphorylation sites is important and various methods have been developed based on high throughput mass-spectrometry but such experimentations are time consuming and laborious therefore, an efficient and accurate novel method is proposed in this study for the prediction of phosphothreonine sites. The proposed method uses context-based data to calculate statistical moments. Position relative statistical moments are combined together to train neural networks. Using 10-fold cross validation, 94.97% accurate result has been obtained whereas for Jackknife testing, 96% accurate results have been obtained. The overall accuracy of the system is 94.4% to sensitivity value 94% and specificity 94.6%. These results suggest that the proposed method may play an essential role to the other existing methods for phosphothreonine sites prediction. [ABSTRACT FROM AUTHOR]

The carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) orchestrates dynamic recruitment of specific cellular machines during different stages of transcription. Signature phosphorylation patterns of Y1S2P3T4S5P6S7 heptapeptide repeats of the CTD engage specific "readers." Whereas phospho-Ser5 and phospho-Ser2 marks are ubiquitous, phospho-Thr4 is reported to only impact specific genes. Here, we identify a role for phospho-Thr4 in transcription termination at noncoding small nucleolar RNA (snoRNA) genes. Quantitative proteomics reveals an interactome of known readers as well as protein complexes that were not known to rely on Thr4 for association with Pol II. The data indicate a key role for Thr4 in engaging the machinery used for transcription elongation and termination. We focus on Rtt103, a protein that binds phospho-Ser2 and phospho-Thr4 marks and facilitates transcription termination at protein-coding genes. To elucidate how Rtt103 engages two distinct CTD modifications that are differentially enriched at noncoding genes, we relied on NMR analysis of Rtt103 in complex with phospho-Thr4- or phospho-Ser2-bearing CTD peptides. The structural data reveal that Rtt103 interacts with phospho-Thr4 in a manner analogous to its interaction with phospho-Ser2-modified CTD. The same set of hydrogen bonds involving either the oxygen on phospho-Thr4 and the hydroxyl on Ser2, or the phosphate on Ser2 and the Thr4 hydroxyl, can be formed by rotation of an arginine side chain, leaving the intermolecular interface otherwise unperturbed. This economy of design enables Rtt103 to engage Pol II at distinct sets of genes with differentially enriched CTD marks. [ABSTRACT FROM AUTHOR]

Old long-lived proteins contain dehydroalanine (Dha) and dehydrobutyrine (Dhb), two amino acids engendered by dehydration of serines and threonines, respectively. Although these residues have a suspected role in protein cross-linking and aggregation, their direct implication has yet to be determined. Here, we have taken advantage of the ability of the enteropathogen Shigella to convert the phosphothreonine residue of the pTX- pY consensus sequence ofERKand p38 intoDhband followed the impact of dehydration on the fate of the two MAPKs. To that end, we have generated the first antibodies recognizing Dhbmodified proteins and allowing tracing them as they form. We showed that Dhb modifications accumulate in a long-lasting manner in Shigella-infected cells, causing subsequent formation of covalent cross-links of MAPKs. Moreover, the Dhb signal correlates precisely with the activation of the Shigella type III secretion apparatus, thus evidencing injectisome activity. This observation is the first to document a causal link between Dhb formation and protein cross-linking in live cells. Detection of eliminylation is a new avenue to phosphoproteome regulation in eukaryotes that will be instrumental for the development of type III secretion inhibitors. [ABSTRACT FROM AUTHOR]

EYA1 is known to be overexpressed in human breast cancer, in which the Myc protein is also accumulated in association with decreased phospho-T58 (pT58) levels. We have recently reported that EYA1 functions as a unique protein phosphatase to dephosphorylate Myc at pT58 to regulate Myc levels. However, it remains unclear whether EYA1-mediated Myc dephosphorylation on T58 is a critical function in regulating Myc protein stability in breast cancer. Furthermore, EYA1's substrate specificity has remained elusive. In this study, we have investigated these questions, and here, we report that depletion of EYA1 using short hairpin RNA (shRNA) in breast cancer cells destabilizes the Myc protein and increases pT58 levels, leading to an increase in the doubling time and impairment of cell cycle progression. In correlation with EYA1-mediated stabilization of cMyc and reduced levels of pT58, EYA1 greatly reduced cMyc-FBW7 binding and cMyc ubiquitination, thus providing novel insight into how EYA1 acts to regulate the FBW7-mediated Myc degradation machinery. We found that the conserved C-terminal haloacid dehalogenase domain of EYA1, which has been reported to have only tyrosine phosphatase activity, has dual phosphatase activities, and both the N- and C-terminal domains interact with substrates to increase the catalytic activity of EYA1. Enzymatic assay and nuclear magnetic resonance (NMR) analysis demonstrated that EYA1 has a striking conformation preference for phospho-T58 of Myc. Together, our results not only provide novel structural evidence about the conformation specificity of EYA1 in dephosphorylating phosphothreonine in Myc but also reveal an important mechanism contributing to Myc deregulation in human breast cancer. [ABSTRACT FROM AUTHOR]

The transmembrane protein Ci-VSP from the ascidian Ciona intestinalis was described as first member of a fascinating family of enzymes, the voltage sensitive phosphatases (VSPs). Ci-VSP and its voltage-activated homologs from other species are stimulated by positive membrane potentials and dephosphorylate the head groups of negatively charged phosphoinositide phosphates (PIPs). In doing so, VSPs act as control centers at the cytosolic membrane surface, because they intervene in signaling cascades that are mediated by PIP lipids. The characteristic motif CX5RT/S in the active site classifies VSPs as members of the huge family of cysteine-based protein tyrosine phosphatases (PTPs). Although PTPs have already been well characterized regarding both, structure and function, their relationship to VSPs has drawn only limited attention so far. Therefore, the intention of this review is to give a short overview about the extensive knowledge about PTPs in relation to the facts known about VSPs. Here, we concentrate on the structural features of the catalytic domain which are similar between both classes of phosphatases and their consequences for the enzymatic function. By discussing results obtained from crystal structures, molecular dynamics simulations, and mutagenesis studies, a possible mechanism for the catalytic cycle of VSPs is presented based on that one proposed for PTPs. In this way, we want to link the knowledge about the catalytic activity of VSPs and PTPs.

Phosphorylation is an important post-translational event that has a wide array of functional consequences. With advances in the ability of various technologies in revealing and mapping new phosphosites in proteins, it is equally important to develop affinity reagents that can monitor such post-translational modifications in eukaryotic cells. While monoclonal and polyclonal antibodies have been shown to be useful in assessing the phosphoproteome, we have expanded our efforts to exploit the Forkhead-associated 1 (FHA1) domain as scaffold for generating recombinant affinity reagents that recognize phosphothreonine-containing peptides. A phage display library of FHA1 variants was screened by affinity selection with 15 phosphothreonine-containing peptides corresponding to various human transcription factors and kinases, including human Myc, calmodulin-dependent protein kinase II (CaMKII), and extracellular-signal regulated kinases 1 and 2 (ERK1/2). The library yielded binding variants against 10 targets (66% success rate); success was largely determined by what residue occurred at the +3 position (C-terminal) to the pThr moiety (i.e., pT+3). The FHA domains binding Myc, CaMKII, and ERK1/2 were characterized and compared against commercially available antibodies. All FHA domains were shown to be phosphorylation-dependent and phosphothreonine-specific in their binding, unlike several commercial monoclonal and polyclonal antibodies. Both the pThr and the residue at the pT+3 position were major factors in defining the specificity of the FHA domains. [ABSTRACT FROM AUTHOR]

The Saccharomyces cerevisiae Fkh1 protein has roles in cell-cycle regulated transcription as well as a transcription-independent role in recombination donor preference during mating-type switching. The conserved FHA domain of Fkh1 regulates donor preference by juxtaposing two distant regions on chromosome III to promote their recombination. A model posits that this Fkh1-mediated long-range chromosomal juxtaposition requires an interaction between the FHA domain and a partner protein(s), but to date no relevant partner has been described. In this study, we used structural modeling, 2-hybrid assays, and mutational analyses to show that the predicted phosphothreonine-binding FHA domain of Fkh1 interacted with multiple partner proteins. The Fkh1 FHA domain was important for its role in cell-cycle regulation, but no single interaction partner could account for this role. In contrast, Fkh1’s interaction with the Mph1 DNA repair helicase regulated donor preference during mating-type switching. Using 2-hybrid assays, co-immunoprecipitation, and fluorescence anisotropy, we mapped a discrete peptide within the regulatory Mph1 C-terminus required for this interaction and identified two threonines that were particularly important. In vitro binding experiments indicated that at least one of these threonines had to be phosphorylated for efficient Fkh1 binding. Substitution of these two threonines with alanines (mph1-2TA) specifically abolished the Fkh1-Mph1 interaction in vivo and altered donor preference during mating-type switching to the same degree as mph1Δ. Notably, the mph1-2TA allele maintained other functions of Mph1 in genome stability. Deletion of a second Fkh1-interacting protein encoded by YMR144W also resulted in a change in Fkh1-FHA-dependent donor preference. We have named this gene FDO1 for orkhead one interacting protein involved in nor preference. We conclude that a phosphothreonine-mediated protein-protein interface between Fkh1-FHA and Mph1 contributes to a specific long-range chromosomal interaction required for mating-type switching, but that Fkh1-FHA must also interact with several other proteins to achieve full functionality in this process. [ABSTRACT FROM AUTHOR]