CX-4945 effectively reverses the aberrant phosphorylation of Tau, amyloid precursor protein (APP) and presenilin 1 (PS1) in mammalian cells. To our surprise, feeding with CX-4945 significantly restored the neurological and phenotypic defects induced by the overexpression of minibrain, an ortholog of human DYRK1A, in the Drosophila model. Moreover, oral administration of CX-4945 acutely suppressed Tau hyperphosphorylation in the hippocampus of DYRK1A-overexpressing mice. Our research results demonstrate that CX-4945 is a potent DYRK1A inhibitor and also suggest that it has therapeutic potential for DYRK1A-associated diseases.DYRK1A is important in neuronal development and function, and its excessive activity is considered a significant pathogenic factor in Down syndrome and Alzheimer's disease. Thus, inhibition of DYRK1A has been suggested to be a new strategy to modify the disease. Very few compounds, however, have been reported to act as inhibitors, and their potential clinical uses require further evaluation. Here, we newly identify CX-4945, the safety of which has been already proven in the clinical setting, as a potent inhibitor of DYRK1A that acts in an ATP-competitive manner. The inhibitory potency of CX-4945 on DYRK1A (IC Editors' choice:In vivo validation of a potent DYRK1A inhibitor, with proven clinical safety, using Down-syndrome- and Alzheimer's-disease-like models. Dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) is a serine/threonine kinase important to brain development. DYRK1A plays a variety of functional roles within the adult central nervous system . The sigDYRK1A gene in the DSCR , a representative neurodegenerative disease . At the Ryoo et al., 2008Despite substantial efforts to develop potent and selective inhibitors of DYRK1A, only a few are currently available, and their potential clinical use remains to be tested further . Extensiin vitro, in mammalian cells and even in living organisms. Moreover, we pharmacologically validated the use of CX-4945 in vivo in model organisms by observing the effective rescue of neurological and phenotypic defects in a DS-like Drosophila model, and the significant suppression of Tau phosphorylation in the hippocampus of DS-like mice. As a potent inhibitor of DYRK1A with proven safety in clinical trials, CX-4945 will be a valuable tool in DYRK1A-related basic research and in the development of therapeutic drugs for DYRK1A-associated diseases, such as DS and AD.Here, we report CX-4945 as a novel inhibitor of DYRK1A with a high potency. Its strong inhibitory effect on DYRK1A has been extensively confirmed 50=∼3-10 nM) of Cdc2-like kinases (Clks), which regulate alternative splicing . We found that CX-4945 potently inhibited the activity of all DYRK-family proteins  and a molecule currently in phase 1b and phase 2 clinical trials for cancer treatment, is a potent inhibitor , which are well-known potent DYRK1A inhibitors . Similarly, CX-4945 strongly inhibited the phosphorylation of APP and PS1, which are also well-known substrates of DYRK1A and are crucial for amyloid plaque formation in DS and AD  signaling is related to the development of a DS- and AD-like phenotype, and because DYRK1A plays an important role in this signaling pathway , we examThe effect of CX-4945 on NFAT signaling was also examined with an alternative assay that measures NFATc1-mediated transcriptional activation, utilizing a firefly luciferase reporter driven by the NFAT response element (NRE-Luc). Treatment with IM and phorbol 12-myristate 13-acetate (PMA) induced a dramatic increase in luciferase activity (∼20 fold) as a result of the nuclear translocation of NFATc1 and the subsequent transcriptional activation B. In thiDrosophila melanogaster is a highly tractable genetic model organism that can be used to investigate the molecular mechanisms of human diseases. Basic biological and neurological properties are highly conserved between humans and Drosophila, and nearly 75% of human disease-related genes, including DRYK1A, have a functional homolog in the Drosophila genome. Based on these advantages, Drosophila has been frequently utilized as a powerful in vivo model for the pharmacological validation of therapeutic drug candidates.Drosophila gene minibrain (mnb) is a well-studied ortholog of human DYRK1A. Similar to DYRK1A in humans, mnb is highly expressed in neural tissues in Drosophila, and mutations in mnb cause phenotypic defects in neuroblast proliferation and brain development. Similarly, the tissue-specific overexpression of mnb also induces various phenotypic and neurological defects in peripheral tissue, such as wings, as well as in the central nervous system (CNS) structure, recapitulating DS-like phenotypes in the Drosophila system  with a penetration of 90% . Surprisingly, feeding newly hatched larvae with CX-4945 for 2 weeks considerably improved the wing defect. This phenotypic rescue was seen when using each of the applied doses  and was even apparent with nanomolar concentrations of CX-4945 (10-100 nM), whereas much higher concentrations of harmine (10-25 μM) were required to achieve a similar effect . This result is consistent with our other observations that CX-4945 has stronger inhibitory effects compared to harmine . Moreover, these results suggest that 100 nM of CX-4945 is an optimal concentration for further examination in an mnb-overexpressing Drosophila system.In order to validate the pharmacological use of CX-4945 in an 0% Fig. S. SurprisDrosophila model, mnb was overexpressed in eye tissue with or without the human Tau protein, using the UAS-Gal4 system. The expression of human Tau in Drosophila retinal tissue (GMR>Tau) produced a small rough eye phenotype  or with mnb (GMR>mnb+Tau) were significantly mitigated by feeding newly hatched larvae with 100 nM of CX-4945 -gal4::UAS-synGFP (#6923), UAS-Tau  and all other stocks and balancers were obtained from the Bloomington Stock Center . UAS-mnb transgenic flies have been described in our previous reports (Drosophila mnb (CG 42273) was subcloned into the pUAS vector, and UAS-mnb transgenic flies were obtained by using the P-element-mediated germline transformation. For defining optimal concentration of chemical inhibitors, 50 embryos of each genotype were raised on drug-containing food  and allowed to develop to adulthood (viability assay and wing vein quantification assay). Experiments were repeated at least three times. For analyzing the eye phenotype, newly eclosed flies were collected and allowed to mate for 2-3 days and then transferred to medium containing 100 nM CX-4945 or DMSO as a control. Flies were exposed to the drug until adult stage, and the eye phenotypes of newly eclosed male were photographed using the Olympus SZ60 binocular microscope equipped with an eXcope K5 CCD system. Fly eye sizes were measured on multiple samples (n>10) from each genotype using the National Institutes of Health ImageJ software. Average eye size was presented as a normalized percentage of control eye size. reports ; the fult-test was used to analyze the effects of CX-4945 on eye phenotype and adult lethality.For analyzing the embryonic neurological phenotypes and lethality, newly eclosed virgin females or male flies were raised on standard medium containing 0.5% DMSO or 100 nM CX-4945 for 7 days before mating. CX4945-exposed virgins and males were allowed to mate in drug-containing medium. Embryos from CX-4945-fed parent flies were collected and staged at 25°C. They were dechorionated and fixed, and immunostaining was performed as described previously . The samDYRK1A gene were maintained as described previously (DYRK1A transgenic mice with three copies of the human eviously . Experimt-tests. *, ** and *** represent P<0.05, P<0.01 and P<0.001, respectively, which are considered statistically significant.Data were analyzed by two-tailed paired or unpaired Student's


In the retina, like in most other brain regions, developing neurons are arranged into distinct layers giving the mature tissue its stratified appearance. This process needs to be highly controlled and orchestrated, as neuronal layering defects lead to impaired retinal function. To achieve successful neuronal layering and lamination in the retina and beyond, three main developmental steps need to be executed: First, the correct type of neuron has to be generated at a precise developmental time. Second, as most retinal neurons are born away from the position at which they later function, newborn neurons have to move to their final layer within the developing tissue, a process also termed neuronal lamination. Third, these neurons need to connect to their correct synaptic partners. Here, we discuss neuronal migration and lamination in the vertebrate retina and summarize our knowledge on these aspects of retinal development. We give an overview of how lamination emerges and discuss the different modes of neuronal translocation that occur during retinogenesis and what we know about the cell biological machineries driving them. In addition, retinal mosaics and their importance for correct retinal function are examined. We close by stating the open questions and future directions in this exciting field. The vertebrate retina is the part of the central nervous system (CNS) responsible for detecting, preprocessing, and sending visual information to the brain Dowling, . In thisNeuronal lamination is a hallmark of retinas across vertebrate species  axis of the retina is key for constructing the laminar architecture and subsequently functional neuronal circuits within the visual system. As such, neuronal migration is crucial for correct retinal layering. Given this, understanding how neurons migrate during retinogenesis is important to understand lamination and circuit formation.ex vivo cultures and organotypic slices of the cerebral neocortex and the cerebellum of rodents. The phenomenon of neuronal migration in the cerebral neocortex has been reviewed in depth elsewhere  radial migration and (2) tangential migration Figures . This caIn the cerebral neocortex, radial migration is subdivided into two distinct modes: (a) somal translocation and (b) glial-guided migration Figure . At earlSome neurons that later build the cerebral cortex move away from their point of origin via non-radial routes to reach their target position using a tangential migration mode , while amacrine cells reside at the most-basal  regions of the INL Figures . A body In contrast to these assumptions, several studies over the past two decades provided evidence that horizontal cells, similar to amacrine cells, migrate substantial basal distances before returning to apical location where they later reside. First, analysis of the spatiotemporal pattern of horizontal cells showed that they are scattered along the radial axis of the developing retina in chicken  what guides distinct neurons that are born at similar locations and developmental stages, toward their differing layers and (b) how do neurons know when and where to stop migration.It is clear that the gene expression profile of each neuronal cell type influences the cell's journey to its final destination. In addition, we have some insights on factors secreted by different cell types that could be involved in different aspects of retinogenesis from axon pathfinding Li,  to laminContact-dependent cues could come from either cell-cell interaction or the interactions between cells and the extracellular matrix (ECM). The ECM has been shown to influence migratory phenomena in different contexts within the developing nervous system (Franco and Müller, So far, only a limited number of studies have attempted to study extracellular influence on retinal neuronal migration and lamination. One of these studies showed that knocking down Laminin α1, a core component of the basal lamina, using morpholino approaches, impairs the basal translocation of retinal ganglion cells and results in their ectopic localization and subsequently a tissue-wide lamination defect in the zebrafish embryo (Icha et al., In addition to ECM components, interactions between different cell types may also influence migratory behavior of neurons. For example, because of the sequential birth order in the retina one could speculate that the correct lamination of earlier born neurons could influence the subsequent layering events. However, this does not seem to be true in all cases. In the zebrafish lakritz mutant (Kay et al., in vivo reaggregate systems could provide a platform to investigate the nature of cell-cell and cell-ECM interactions required for retinal lamination. Moreover, manipulating the components of such in vivo aggregate systems could enable the simpler dissection of molecular and cellular mechanisms governing neuron migration, neurite stratification, and lamination within the retina.One re-emerging approach to study how retinal neurons interact with their neighboring cells and extracellular components are retinal reaggregate cultures. In early studies, reaggregate cultures were often used to understand how different tissues such as the chick retina are formed (Moscona and Moscona, Visual perception is achieved through a series of processing steps that enable the retina to assess different aspects of the visual information. So far, we discussed how genesis of retinal cell types and their correct positioning contribute to the lamination of the retina. While these steps are prerequisites for proper retina development, building functional circuits is the next fundamental step for processing visual information and thus retinal function (Galli-Resta, The highly ordered mosaic architecture in the vertebrate retina ensures that cell bodies of the same neuron-types, also called homotypic neurons are distributed in non-random arrays within their respective layer and at any given location in the retina Figure . For exaStudies in different species, ranging from fish to primates, have suggested that retinal mosaics emerge early during development, at times when neurogenesis, migration and lamination are still in course. In fact, cone photoreceptors, horizontal cells, and cholinergic amacrine cells across species form regular arrays even when layering and polarization is not yet complete (Larison and Bremiller, Initially, it was assumed that the position of retinal neurons is fixed along their radial axis from their birth site. This led to the hypothesis that the spatial regularity of the retinal mosaics is due to fate determination mechanisms that act around the time of neuronal birth (Reese and Galli-Resta, Some effort has been made to understand what drives tangential dispersion during mosaic assembly and maintenance. One proposed mechanism is that either diffusible signals or contact-mediated interactions between homotypic neurons account for tangential dispersion of neurons and subsequently mosaic regularity of neurons (Reese and Galli-Resta, Despite some progress, the exact mechanisms by which the homotypic neurons that form mosaic arrays reliably recognize each other while migrating in the pool of diverse neuron types in the vertebrate developing retina remain elusive. Besides tangential migration, programmed cell death and cell-fate lateral inhibition have been also proposed to contribute to building mosaic arrays within the developing retina (Reese and Keeley, Neuronal lamination requires the regulation of a well-orchestrated chain of events involving the generation of the right types and number of cells, positioning them at the correct places through migration and finally integrating them into their functional circuitries. Defects in the proper execution in any of these steps may result in neuronal layering defects and consequently impaired retinal function. Despite recent progress, we still lack an overarching picture of how different aspects of lamination from neuronal birth to final positioning occur at the cellular and tissue scales. This is partly due to the fact that most studies investigating neuronal lamination in the retina relied on fixed samples and conventional confocal microscopy that implies high phototoxicity leading to short observation times (Icha et al., in vivo needs to be investigated. In addition, the development of in vivo culture systems such as human organoids, is an exciting entry route to extract the conservation of diverse features of lamination. Remarkably, lamination is an organizational hallmark of many regions of the brain. As such, the retina as an outgrowth of the brain could provide a platform for a mechanistic understanding of neuronal migration and lamination in other laminated brain regions.It is known that the process of lamination is regulated by a combination of intrinsic and extrinsic influences during development. We highlighted the potential role of ECM in neuronal migration, mosaic formation and lamination which needs to get into the spotlight in future studies. Another important question is to what extent and how cell intrinsic and extrinsic cues interact and how they contribute to different aspects of lamination, from genesis to migration and mosaic formation, in the developing retina. As discussed in this review, communication between neurons could contribute to constructing the retinal layers and functional units, and the nature of such complex interactions All authors conceived and planned the review outline. RA and MR-M: wrote the initial manuscript. All authors read and edited the text and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer OM and handling Editor declared their shared affiliation.


On the other hand, TRF1 carrying a phosphomimic mutation of T271D is competent in not only binding to telomeric DNA but also inhibiting telomerase-mediated telomere lengthening. These results suggest that TRF1 phosphorylation on T271 negatively regulates telomerase-mediated telomere maintenance. We find that in telomerase-negative ALT cells, TRF1 carrying either a T271A or T271D mutation is able to promote C-circle production but fails to support APB formation. These results suggest that TRF1 phosphorylation on T271 is necessary for APB formation but dispensable for C-circle production. These results further imply that APB formation can be mechanistically separated from C-circle production.TRF1, a component of the shelterin complex, plays a key role in both telomerase-dependent telomere maintenance and alternative lengthening of telomeres, the latter also known as ALT. Characteristics of ALT cells include C-circles and ALT-associated PML bodies, referred to as APBs. The function of TRF1 is tightly regulated by post-translational modification including phosphorylation, however TRF1 phosphorylation sites have yet to be fully characterized. Here we report a novel TRF1 phosphorylation site threonine 271. We show that a nonphosphorylatable mutation of T271A impairs TRF1 binding to telomeric DNA  Most human somatic cells encounter an erosion of telomeric DNA every time they replicate their genome, in part due to an inability of DNA polymerases to fill in the gap left from removal of the last RNA primerALT cells carry several hallmarks that differentiate them from telomerase-expressing cellsMammalian telomeres are coated with a six-subunit protein complex, known as shelterin/telosome, which consists of TRF1, TRF2, TIN2, hRap1, TPP1 and POT1710101113in vivo. We show that in telomerase-positive cells, overexpression of TRF1 carrying a nonphosphorylatable mutation of T271A fails to inhibit telomere elongation whereas overexpression of TRF1 carrying a phosphomimic mutation of T271D is able to suppress telomere lengthening. We further demonstrate that TRF1 carrying a T271A mutation is defective in binding to telomeric DNA in vivo, which may at least in part account for the inability of TRF1-T271A to suppress telomerase-mediated telomere elongation. We find that in ALT cells, TRF1 carrying either a T271A or T271D mutation is able to support the production of C-circles, indicating that T271 is not involved in regulating the level of C-circles. On the other hand, TRF1 carrying a T271A mutation is deficient in supporting APB formation, indicative of the role of T271 phosphorylation in APB formation, suggesting that the role of TRF1 in supporting APB formation can be separated from its role in promoting C-circle production in ALT cells. Taken together, the work presented here suggests that TRF1 phosphorylation on T271 plays an important role in modulating its activity in both telomerease-expressing and ALT cells.In this report, we demonstrate that TRF1 is phosphorylated on threonine 271 in vivo, we raised an antibody specifically against a TRF1 peptide containing phosphorylated T271 . Depletion of TRF1 resulted in telomere elongation , in agre10in vivo, we performed analysis of chromatin immunoprecipitation (ChIP) using an anti-Myc antibody and lysates isolated from early passages of TRF1-depleted HeLaII cells stably expressing the vector alone, Myc-tagged wild type TRF1, Myc-tagged TRF1-T271A and Myc-tagged TRF1-T271D. Both Myc-tagged TRF1-T271A and Myc-tagged TRF1-T271D were expressed at a comparable level to Myc-tagged wild type TRF1  is found to match the consensus phosphorylation site (RXXS/T) of Chk1 and a related kinase Chk2, which has been reported to phosphorylate sheltein protein TRF2in vitro , depletion or inhibition of Chk1 or a related kinase Chk2 fails to affect T271 phosphorylation in vivo , arguing against the possibility that Chk1 and Chk2 are the kinases responsible for phosphorylating T271 in vivo. Therefore further investigation will be needed to identify the kinase(s) responsible for T271 phosphorylation in vivo.Bioinformatics analysis fails to reveal a clear candidate kinase responsible for T271 phosphorylation. Among numerous potential hits, T271  has been previously describedRabbit polyclonal anti-pT271 antibody was developed by Biosynthesis Inc. against a TRF1 peptide containing phosphorylated threonine 271 (CKVVESKRTR-pT-ITSQDKPS). Antibodies to TRF1Cells were grown in DMEM medium with 10% fetal bovine serum (FBS) for HeLaII186 cells). Two aliquots of 50 μl supernatant (corresponding to one-quarter of the amount of lysate used for IP) were used for quantifying the total telomeric DNA and were processed along with the IP samples at the step of reversing the crosslinks. Four-fifth of immunoprecipitated DNA was loaded on the dot blots whereas two inputs each containing 5% of total DNA were included to access the consistency of loading. The ratio of the signal from each ChIP relative to the average signal from the two input lanes was multiplied by 5%  and a factor of 1.25 (because four-fifths of the precipitated DNA was loaded for each ChIP), giving rise to the percentage of total telomeric DNA recovered from each ChIP. Detection of telomeric DNA was done with a radioactively-labelled 800-bp TTAGGG repeat-containgin fragment.Chromatin immunoprecipitation (ChIP) was carried out essentially as described16Immunofluorescence was performed essentially as describedScoring of APBs was done as describedRsaI and HinfI and loaded onto a 0.7% agarose gel in 0.5xTBE. Following electrophoresis, the gel was dried and then denatured. Blotting for telomeric fragments was carried out in gel using a radioactively-labeled single-stranded telomeric DNA probe. The average telomeric restriction fragment length was determined by PhosphorImager analysis using ImageQuant and MS excel.Telomere length analysis was carried out essentially as described1232P-end-labeled single strand (CCCTAA)3 probe under native conditions as describedC-circle amplification assays were performed essentially as described14Protein extracts and immunoblotting were performed as described16t-test was used to derive all P-values.A Student’s two-tailed How to cite this article: Ho, A. et al. TRF1 phosphorylation on T271 modulates telomerase-dependent telomere length maintenance as well as the formation of ALT-associated PML bodies. Sci. Rep.6, 36913; doi: 10.1038/srep36913 (2016).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


The Musashi family of RNA binding proteins act to promote stem cell self-renewal and oppose cell differentiation predominantly through translational repression of mRNAs encoding pro-differentiation factors and inhibitors of cell cycle progression. During tissue development and repair however, Musashi repressor function must be dynamically regulated to allow cell cycle exit and differentiation. The mechanism by which Musashi repressor function is attenuated has not been fully established. Our prior work indicated that the Musashi1 isoform undergoes site-specific regulatory phosphorylation. Here, we demonstrate that the canonical Musashi2 isoform is subject to similar regulated site-specific phosphorylation, converting Musashi2 from a repressor to an activator of target mRNA translation. We have also characterized a novel alternatively spliced, truncated isoform of human Musashi2 (variant 2) that lacks the sites of regulatory phosphorylation and fails to promote translation of target mRNAs. Consistent with a role in opposing cell cycle exit and differentiation, upregulation of Musashi2 variant 2 was observed in a number of cancers and overexpression of the Musashi2 variant 2 isoform promoted cell transformation. These findings indicate that alternately spliced isoforms of the Musashi protein family possess distinct functional and regulatory properties and suggest that differential expression of Musashi isoforms may influence cell fate decisions. This form of regulation of gene expression permits a rapid cellular response to changing external cues through repression or translation of specific pre-existing mRNAs. Target mRNA specificity is achieved through sequence-specific targeting of RNA binding proteins (RBPs) and/or miRNAs that modulate the stability and/or translation of the target mRNA. The mechanisms by which the function of RBPs are regulated are not well understood but are of increasing interest, as it has become evident that aberrant control of mRNA translation contributes to a range of pathologies, including neurological disease and cancer10.Targeted control of mRNA translation is gaining recognition as a key mechanism for regulation of cell cycle and cell fate transitions23. Msi is thought to promote stem cell self-renewal and opposes cell cycle arrest and cell differentiation by repressing the translation of key target mRNAs12. Identified mammalian targets of Msi-mediated repression include the mRNAs encoding Numb, a Notch signaling inhibitor; p21, an inhibitor of cyclin-dependent kinases; adenomatous polypopsis coli, a Wnt signaling inhibitor; doublecortin, a protein associated with neuronal migration and development; and Dnmt1, a DNA methylating enzyme responsible for maintenance of epigenetic marks28. The mechanism by which Msi target mRNAs are de-repressed during developmental processes or tissue repair to allow cell cycle exit and stem/progenitor cell differentiation is not fully understood29. The low level of Msi proteins in terminally differentiated, mature cells suggests that target de-repression could be mediated through simple degradation of Msi protein. However, it has been observed that de-repression of Msi target mRNAs precedes loss of Msi protein, suggesting that alternate mechanisms act to regulate Msi function30. Moreover, there is evidence that the Msi1 isoform can switch function to activate, rather than repress, translation of target mRNAs. Target mRNA activation was first shown in oocytes of the frog, Xenopus, where pro-differentiation signaling triggered Msi1-dependent translation of target mRNAs including Mos, a MAP kinase signaling activator; cyclin B5, Msi1 itself and Dnmt133. While subsequent studies have reported specific examples of Msi1- and Msi2-dependent translation of mammalian target mRNAs39, it is unclear the extent to which Msi1 and Msi2 are controlled by similar regulatory mechanisms during cell fate transitions.The two Musashi (Msi) RBP protein family members, Musashi1 (Msi1) and Musashi2 (Msi2), have been identified as mediators of both physiological and pathological stem cell self-renewal40. Consistent with possible shared mRNA targets, Msi1 and Msi2 have been shown to effectively substitute for each other in the control of Xenopus oocyte maturation, mammalian neuronal stem cell self-renewal, intestinal stem cell quiescence and colorectal cancer42. Despite these apparent similarities, several lines of evidence suggest differences between the Msi family members, in terms of expression patterns, as well as interaction with protein binding partners and function. While co-expressed in many tissues, Msi2 is selectively expressed in hematopoietic stem cells43. Mammalian Msi2 does not appear to interact with the Msi1-associated proteins Lin28 or GLD2 poly[A] polymerase45 and it has been reported that Msi2 opposes proliferation in pancreatic cells while Msi1 acts to promote proliferation46. Together, these observations suggest that Msi1 and Msi2 may be subject to both shared as well as isoform-specific regulatory mechanisms.The Msi1 and Msi2 proteins each contain two highly conserved N-terminal RNA recognition motifs (RRMs), and recent crosslinking studies indicate that they may regulate overlapping mRNA target cohortsXenopus oocytes, as well as during differentiation of mammalian cells in culture. We demonstrate that Msi2 phosphorylation is mediated by both Ringo/CDK signaling and p42 MAP kinase (ERK) signaling pathways and that mutational disruption of Msi2 phosphorylation abrogates stimulus-dependent target mRNA translational activation and Xenopus oocyte maturation. Msi2 phosphorylation results in the translational activation of previously characterized Msi1 target mRNAs. Importantly, we characterized an alternatively spliced, truncated isoform of human Msi2 (variant 2) that lacks the exon containing the two conserved sites of regulatory phosphorylation. Expression of Msi2 variant 2 fails to promote target mRNA translation and is non-permissive for maturation of Xenopus oocytes. Overexpression of the Msi2 variant 2 promotes growth and cellular transformation of NIH3T3 cells, consistent with the Msi2 variant 2 failing to promote de-repression and translation of target mRNAs that inhibit cell proliferation. Our results indicate that the canonical Msi1 and Msi2 proteins have shared regulatory mechanisms while highlighting an unexpected role for alternative splicing in Msi2 isoform-specific control.In this study, we characterized the regulatory control of the Msi2 protein. We report that Msi2 undergoes stimulus-dependent phosphorylation on two conserved serine residues during maturation of Xenopus Msi2 protein where serine 356 and 381 were changed to non-phosphorylatable alanine (Msi2 S356A/S381A) in oocytes in which the endogenous Msi1 and Msi2 had been functionally attenuated through use of Msi1/Msi2 antisense oligonucleotide injection. We tested the capacity of the mutant Msi2 S356A/S381A protein to rescue progesterone-stimulated oocyte maturation , the Msi2 S356A/S381A mutant was unable to rescue activation of these mRNAs, consistent with a failure to rescue oocyte GVBD  motifs that are homologous with the sites critical for control of Msi1 function Fig.  and 47. ion Fig. . In the ein Fig. . Both muein Fig. . Oocyte VBD Fig. . These rXenopus Msi2, we found phosphorylation of both S356 and S381 were significantly increased in a progesterone-dependent manner and occurred early, prior to GVBD   and an over-expressed, GST-tagged form of VBD Fig. . This eaB+) Fig. . The MsiB+) Fig. , was dueB+) Fig. . Since tB+) Fig. , suggest47. Here, we found that treatment of oocytes with the MAP kinase signaling inhibitor, UO126, abolished MAP kinase activation and dramatically reduced progesterone-stimulated phosphorylation of both S356 and S381 sites on Msi2  contributed to the regulatory phosphorylation of Msi2. Our prior analysis of Msi1 regulatory phosphorylation implicated the sequential action of the Ringo/CDK signaling and subsequent p42 mitogen activated protein (MAP) kinase pathwayssi2 Fig. . Howeverion Fig. , R-AS. Hion Fig. . These rion Fig. . We concXenopus Msi2 ortholog. Since equivalent levels of the Xenopus and mMsi2 proteins were used  protein in Msi1/2 antisense-injected oocytes also rescued progesterone-stimulated oocyte maturation Fig. , albeit sed Fig. , the difion Fig. . mMsi2 wThe ability of mammalian Msi2 to rescue endogenous mRNA polyadenylation, translational activation and oocyte maturation was due to an ability to interact directly with Msi1 target mRNAs. We utilized a biotinylated Mos wild-type UTR containing an intact Msi binding element (MBE), or a Mos UTR with a mutationally disrupted MBE . The 32D cell line undergoes differentiation after switching proliferating cells from a culture containing interleukin-3 (IL-3) to one containing granulocyte-colony stimulating factor (G-CSF)ion Fig. . This inion Fig. . This inIn addition to the canonical Msi2 (RefSeq Accession NM_138962), humans are predicted  were transfected with eGFP-tagged forms of the canonical human Msi2 or the Msi2 variant 2 or with the eGFP moiety alone and grown on non-adherent plates in the presence of selection for the expression plasmid. One day after transfection, cells were examined for eGFP fluorescence and we verified equivalent transfection efficiency of all Msi2 expression plasmids. After 10 days in culture, anchorage-independent colony growth was scored  via the Cancer RNA-seq Nexus web portalon Table . Of theson Table  and uteron Table  when comXenopus Msi2 function is mediated through both the Ringo/CDK and the MAP kinase signaling pathways. Although the regulatory phosphorylation sites reside within S/P motifs, it remains to be determined if Ringo/CDK and/or MAP kinase directly phosphorylate Msi2, or if intermediary kinases are involved. Physiologically, Msi2 phosphorylation is required for target mRNA de-repression and translational activation as a mutant form of Msi2 that cannot undergo regulatory phosphorylation is unable to mediate oocyte maturation. We observed a similar regulatory phosphorylation of mammalian Msi2 when ectopically expressed in Xenopus oocytes, as well as the endogenous mammalian Msi2 in murine 32D myoblastic cells and human SH-SY5Y neuroblastoma cells in response to extracellular signals that induce cell differentiation. These findings suggest that regulatory phosphorylation of Msi2 serves an evolutionarily conserved role in promoting cell cycle exit and cell differentiation. It will be interesting in the future to determine if the increase in Musashi2 regulatory phosphorylation is transient or if the phosphorylated Musashi2 species is sustained throughout the differentiation process.We demonstrate that canonical Msi2 function is controlled through site-specific regulatory phosphorylation. Regulatory control of Xenopus Msi2 interacting protein45. However, the interaction was observed to be independent of progesterone stimulation indicating that regulatory phosphorylation does not play a direct role in Msi2-GLD-2 association. The phosphorylation of Msi2 may serve an alternate role to facilitate activation of associated GLD2 function, or may effect a remodeling of other co-associated proteins independently of GLD2, to promote de-repression and translation of target mRNAs.The mechanism by which C-terminal regulatory S356 and S381 phosphorylation of Msi2 results in a functional switch to promote translation of target mRNAs is unknown. One potential mechanism could involve the modulation of target mRNA 3′ end polyadenylation, a key process for the translational activation of mRNAs. In support of this role, we have recently identified the Germline Development Defective-2 (GLD2) poly[A] polymerase as a In view of the apparent requirement for Msi2 phosphorylation to promote target mRNA translation, we were intrigued by the expression of an alternatively spliced form of Msi2 that lacks the sites of regulatory phosphorylation and is compromised in ability to switch from a repressor to an activator of target mRNA translation. The alternatively spliced Msi2 variant 2 is expressed more highly in SH-SY5Y tumor cells than in normal iPSCs suggesting an association between expression of Msi2 variant 2 and cell transformation Fig. . In this60, although the reported isoforms all retain the two sites of identified C-terminal regulatory phosphorylation. The murine Msi1 and Msi2, as well as the human Msi1 and Msi2 genes encode the two sites of regulatory phosphorylation within a single exon. In murine embryonic stem cells (mESCs), two alternatively spliced murine Msi2 isoforms (both retaining the sites of regulatory phosphorylation) possess non-redundant activities, presumably due to differences in their ability to interact with specific cellular co-factors60. The alternatively spliced variant 2 form of human Msi2 differs from these examples since it has distinct N- and C-terminal amino acid sequences from the canonical Msi2 isoform. Of note, the substitution of the N-terminal sequence does not always correlate with alternative splicing of the C-terminal domain, when examined across species. The human Msi2 variant 2 has replaced exons 1 and 2 with an alternative exon 1b, and exon 12 (which encodes both sites of regulatory phosphorylation), exon 13 and exon 14 with an alternative exon 11b that results in a truncated protein 33, GST-mMsi129, GST-mMsi245, SRα vBRaf56 have been previously described.Plasmids encoding GST Xenopus Msi262 was amplified from a cDNA plasmid provided by Dr. Martha Rebbert to include 5′ Cla1 and 3′ Xho1 restriction sites. The digested PCR fragment was cloned into pXen263 to generate an in-frame GST tagged Xenopus Msi2.pXen XeMsi2. pXen XeMsi2 S356A/S381A - Serines 356 and 381 of the XeMsi2 sequence were changed to alanine by site-directed mutagenesis , using pXen XeMsi2 as a template.pXen hMsi2. Human Msi2  was PCR amplified to include a 5′ Cla1 site and a 3′ BamH1 site and cloned into Cla1/BamH1 digested pXen2.pXen hMsi2 S278A/S303A, pXen mMsi1 S312A/S337A were generated by Quikchange site-directed mutagenesis using the wild-type plasmids as a template.pXen hMsi2v2. Human Msi2 variant 2  was PCR amplified to include a 5′ Cla1 site, and a 3′ Xho1 site and cloned into Cla1/Xho1 digested pXen2.hMsi2 eGFP and hMsi2v2 eGFP. hMsi2 and hMsi2v2 were PCR amplified to include a 5′ HindIII site and a 3′ BamH1 immediately prior to the STOP codon and cloned into HindIII/BamH1 digested pEGFP-N1 (Clontech) to generate an in-frame C-terminal eGFP fusion protein.All cloned inserts and introduced mutations were verified by DNA sequencing.64. Oocytes were induced to mature with 2 µg/ml progesterone65. The rate of germinal vesicle breakdown (GVBD) was scored morphologically by observing the appearance of a white spot on the animal pole. Because oocytes from different frogs mature at different rates, the culture times were standardized between experiments to the time taken for 50% of oocytes to undergo GVBD. mRNA for oocyte injection was made by in vitro transcription using SP6 RNA Polymerase (Promega), as previously described66. Typically 23ng RNA was injected into each oocyte45. Where indicated, oocytes were pre-treated with 50 μM MEK inhibitor, UO126 (Promega) or DMSO vehicle for 30 minutes. At least 40 oocytes were scored for each condition, and all experiments were routinely repeated on three separate occasions. Where indicated, when 50% of control oocytes had reached GVBD, oocytes were segregated into those that had not (−) or had (+) completed GVBD. For all protein and RNA extractions, pools of 5–10 oocytes were used for each sample point. In all cases, oocytes were lysed in 10 μl NP40 lysis buffer/oocyte, and 0.5 oocyte equivalents loaded per lane65. Animal protocols were approved by the UAMS Institutional Animal Care and Use committee, in accordance with Federal regulations.Xenopus oocytes were isolated and cultured as described previouslyin vitro transcribed/translated using TNT SP6-coupled Reticulocyte Lysate System (Promega) from pXen1 (for GST), or pXen plasmids encoding the indicated Msi protein. 5′ biotin-labeled RNA oligonucleotide probes were synthesized by Integrated DNA Technologies and corresponded to the last 50 nucleotides of the Xenopus Mos 3′ UTR (Accession NM_001088094), a mutant Mos 3′ UTR encoding a disruption of the consensus MBE33 or the last 90 nucleotides of the murine Pou1f1 3′ UTR which encodes a consensus MBE (Accession BC061213). Binding reactions, electrophoresis and probe detection were performed as described33.GST fusion proteins were 67. The increase in PCR product length (retarded mobility) is specifically due to extension of the poly[A] tail68. The primers for endogenous Mos, cyclin B5 and Msi1 mRNAs have been described previously33. Verification of PCR product identity was performed by DNA sequence analysis.cDNAs for polyadenylation assays were synthesized using RNA ligation-coupled PCR as described previously53. Msi AS injected oocytes failed to undergo progesterone-stimulated maturation (GVBD) in the absence of expressed Msi protein. No inhibition of progesterone-stimulated maturation was observed with scrambled control antisense oligonucleotides.Antisense oligodeoxynucleotides targeting endogenous Msi1 and Msi2 mRNAs, Ringo mRNA or a randomized control oligonucleotide sequence 5′-TAGAGAAGATAATCGTCATCTTA-3′ were employed as previously described54. SHSY5Y human neuroblastoma cells were cultured in DMEM/F12 medium with 10% fetal bovine serum (Thermo Fisher Scientific/Gibco). Human iPSCs were cultured in Essential 8 Medium (Thermo Fisher Scientific/Gibco), as per the supplier's protocol.The murine IL-3-dependent 32D hematopoietic cell line, human SH-SY5Y, and human induced pluripotent stem cells  were originally obtained from ATCC . 32D cells were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin , and 5 ng/mL recombinant murine IL-3 . For inducing 32D cell differentiation, cells were washed with HBSS to remove IL-3, and 500,000 cells were plated on Day 0 in RPMI 1640 containing 10% FBS, 1% penicillin-streptomycin, and 40 ng/mL recombinant murine G-CSF . At the indicated time points, cells were harvested, cytospun, and then Wright’s-stained  for analysis of nuclear morphology to determine the extent of granulocytic differentiationGapdh expression (Qiagen QuantiTect Primer Assay), and relative expression values were determined by the QuantStudio 12K Flex Software version 1.2.3 using the Δ-Δ-cycle threshold method69. Msi2 variant 2 sense primer: GAGGGTTTGGCTTTGTCACTTTTG; Msi2 variant 2 antisense: AATTATGTCTTGTGAAACCGGC. Sequencing of qPCR products confirmed the identity of the expressed Musashi2v2 isoform.Cell RNA was isolated using the Maxwell 16 LEV simplyRNA tissue kit  and 50 ng RNA was utilized with RNA-to-Ct Power SYBR PCR master mix with a QuantStudio 12K Flex system (Thermo Fisher Scientific/Applied Biosystems), as per suppliers protocol. Three independent experiments were performed and each sample was analyzed in triplicate. Samples were normalized to http://www.ebi.ac.uk/ena/data/view/ERP000546). To estimate abundances at the gene-level and isoform-level we used the Tophat (v2.0.12)70 and Cufflinks (v2.2.1)71 software tools. Quality control for the reads was implemented using the fastx-toolkit (http://hannonlab.cshl.edu/fastx_toolkit). The quality of the reads in all downloaded FASTQ files was controlled by omitting any read with median phred score less than 30 (roughly 5–10% of the reads were filtered out). Tophat aligned the single-end 75 bp reads to the UCSC hg19 genome model and Cufflinks quantified transcript abundances for the transcripts available in the UCSC hg19 annotations. Cuffnorm (part of Cufflinks) provides gene and isoform normalized abundances in fragments per kilobase of exon per million fragments mapped (FPKM). Since single-end reads were used, Cufflinks could not estimate the fragment length distribution, and instead used an approximate Gaussian distribution with default parameters. Expression levels (in FPKM) at the isoform-level for all samples are summarized in Table We used the publically available transcription profiling (RNA-Seq) of 16 individual human tissues (Illumina Human Body Map 2.0 dataset) to find the tissues where Msi1, Msi2 and Msi2 variant 2 transcripts are expressed . FASTQ files were downloaded from the EMBL-EBI European Nucleotide Archive  and that there were at least 10 samples . Of the 28 cancer types available, 12 fulfilled the criteria and were analyzed here for Msi2 isoform-specific expression, broken down by cancer stage sub-type, where available. Of note, the CRN employs UCSC transcript ID designations and Uniprot protein IDs. The transcript encoding the canonical form of Msi2  corresponds to the UCSC designation uc002iuz and UniProt designation Q96DH6-1. The Msi2 variant 2 transcript (NM_170721) corresponds to the UCSC designation uc002iva and UniProt designation Q96DH6-2. Differential expression between cancerous and normal adjacent tissue datasets were considered significant if the adjusted p value was <0.0157. Expression data for Breast Invasive Carcinoma, and Endometrial Carcinoma are shown in Table To examine the relative expression levels  of Msi2 and Msi2 variant 2 isoforms in normal and cancer cell types we utilized the Cancer RNA-Seq NexusNIH3T3 cells were transfected with constructs expressing the eGFP moiety alone, hMsi2-eGFP, hMsi2 variant2-eGFP, or vBraf using Lipofectamine 2000 (Thermo Fisher Scientific). Approximately 72 hours after transfection the cells were dissociated with trypsin, passed through a single-cell filter, and seeded onto 24-well ultra-low attachment plates. Visualization of eGFP expressing (fluorescent) cells verified equivalent initial transfection efficiency for each construct . Cells were cultured for 10 days in DMEM with 0.5% NBCS, with media supplemented every 3 days. After 10 days the numbers of colonies per well were scored. Experiments were performed with 4 replicates per experiment, and repeated on 3 separate occasions.Antisera for phosphorylation specific human Msi2 S278 and human Musashi2 S303 were generated by immunizing rabbits with either the peptide CGFPGANS(phospho)PGPVADL or CVGNYISAAS(phospho)PQPGSGF (Proteintech Group Inc.). The antibodies were purified over a peptide-affinity column and used for western blot analyses at 1:1000 dilution. Abcam antibodies to Msi1 were used at 1:1000. Sigma Tubulin antibodies were used at 1:20,000 and Cell Signaling GAPDH antibodies were used at 1:10,000. The phospho-specific MAP kinase antibody  was used at 1:1000 to detect activating phosphorylation at Thr202 and Tyr204. The antibody to detect total MAP kinase  was used at 1:1000. The GST antibody (Santa Cruz) was used at 1:1000.72 containing sodium vanadate and a protease inhibitor cocktail (Sigma). Where indicated, a portion of the lysate was immediately transferred to STAT-60 for RNA extraction65. Gel electrophoresis, transfer and antibody detection was as previously described45 except membranes were blocked with 1% bovine serum albumin (Sigma) in TBST for 60 min at room temperature. Experiments were repeated at least twice and representative images are shown.Oocytes were lysed in NP40 lysis bufferFor examination of phosphorylation, oocytes were lysed in a modified NP40 lysis buffer lacking sodium pyrophosphate, sodium vanadate or protease inhibitors and incubated without (mock) or with lambda protein phosphatase (NEB) for 30 minutes at 37 °C. Samples were then analyzed by western blotting.t-test when only two groups were compared. A probability of p < 0.05 was adopted for statistical significance.All quantitated data are presented as mean +/− SEM. Statistical significance was assessed by one-way analysis of variance (ANOVA) followed by the Bonferroni post hoc test or by Student's The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.Supplementary Data


Type 2A protein phosphatase (PP2A) enzymes are serine/threonine phosphatases which comprise a scaffold A subunit, a regulatory B subunit and a catalytic C subunit, and have been implicated in the dephosphorylation of multiple cardiac phosphoproteins. B subunits determine subcellular targeting, substrate specificity and catalytic activity, and can themselves be regulated by post-translational modifications. We explored potential β-adrenergic regulation of PP2A in cardiomyocytes through phosphorylation of the regulatory B subunit isoform B56δ.1AR subtype, and was dependent on PKA activity. The functional role of the phosphorylation was explored in ARVM transduced with adenoviruses expressing wild type (WT) or non-phosphorylatable (S573A) B56δ, fused to GFP at the N-terminus. C subunit expression was increased in ARVM expressing GFP-B56δ-WT or GFP-B56δ-S573A, both of which co-immunoprecipitated with endogenous C and A subunits. PP2A activity in cell lysates was increased in response to ISO in ARVM expressing GFP-B56δ-WT but not GFP-B56δ-S573A. Immunoblot analysis of the phosphoproteome in ARVM expressing GFP-B56δ-WT or GFP-B56δ-S573A with antibodies detecting (i) phospho-serine/threonine residues in distinct kinase substrate motifs or (ii) specific phosphorylated residues of functional importance in selected proteins revealed a comparable phosphorylation profile in the absence or presence of ISO stimulation.Phosphate affinity SDS-PAGE and immunoblot analysis revealed increased phosphorylation of B56δ in adult rat ventricular myocytes (ARVM) exposed to the β-adrenergic receptor (βAR) agonist isoprenaline (ISO). Phosphorylation of B56δ occurred at S573, primarily through stimulation of the βIn cardiomyocytes, βAR stimulation induces PKA-mediated phosphorylation of the PP2A regulatory subunit isoform B56δ at S573, which increases associated PP2A catalytic activity. This is likely to regulate the phosphorylation status of specific B56δ-PP2A substrates, which remain to be identified. •PP2A subunit B56δ is phosphorylated on β-adrenergic stimulation of cardiomyocytes.•Phosphorylation occurs at Ser573 and increases B56δ-PP2A catalytic activity.•1-adrenoceptor subtype and protein kinase A.Response is mediated by the β•Phosphorylated B56δ abundance is increased in pathological cardiac hypertrophy. PP2A holoenzymes comprise a 65-kDa scaffold  is localized in chromosome region 6p21.1 and gives rise to three splice variants that differ at the N-terminus Little information is available regarding B56δ expression, regulation and function in the heart. Mice lacking B56δ were the first mice with targeted disruption of a B56 subunit to be studied; however, their cardiac phenotype was not studied 1AR and is mediated by PKA, and (iii) B56δ phosphorylation at S573 is necessary for βAR-mediated stimulation of PP2A activity.In view of the importance of the Gs-adenylate cyclase-cAMP-PKA pathway in mediating cardiomyocyte responses to β-adrenergic receptor (βAR) stimulation 22.1C, PLB and phospho-S/T kinase substrate antibodies were from Cell Signaling Technology . GAPDH, H2B and B56γ antibodies were from Abcam . B56α and B56δ antibodies were from BD Biosciences (610615) and Bethyl (A301-100A), respectively. PP2AA and B55α antibodies were from Santa Cruz . GFP and α-Actinin antibodies were from Roche (11814460001) and Sigma (A7732), respectively. pS16 PLB and pS282 cMyBP-C antibodies were from Badrilla (A010-12AP) and Enzo Life Sciences (ALX-215-057-R050), respectively. The cMyBP-C antibody was a kind gift from Professor Mathias Gautel 6-benzoyl cAMP (BNZ), and okadaic acid were from Calbiochem . The pEGFP-C1 vector encoding PPP2R5D and cardiac tissue from littermate B56δ knock out (KO) and WT mice were kind gifts from Professor Veerle Janssens cTnI, pS23/24 cTnI, PP2A2.2PPP2R5D in a pEGFP-C1 vector using the QuikChange II Site-Directed Mutagenesis Kit (Stratagene). The adenoviral vectors expressing wild type (WT) B56δ (AdV.GFP-B56δ-WT) and mutated (S573A) B56δ (AdV.GFP-B56δ-S573A) were constructed using the AdEasy system To replace S573 with alanine, a single point mutation was introduced into human 2.3l-creatine, 5 mM carnitine, 5 mM taurine and 100 IU/ml penicillin/streptomycin, and were cultured in plastic 6-well plates pre-coated with laminin. Cells were maintained in a humidified incubator  for 2 h after which, the medium was replaced with fresh medium and cells were incubated overnight. Where indicated, cells were transduced with adenoviruses 2 h post-plating. AdV.GFP was used at MOI 30. AdV.GFP-B56δ-WT and AdV.GFP-B56δ-S573A were both used at MOI 100.ARVM were isolated from the hearts of adult male Wistar rats by collagenase-based enzymatic digestion, as previously described 2.4Unless otherwise stated, ARVM were incubated with vehicle or 10 nM ISO for 10 min. PRO (100 nM), CGP (100 nM), ICI (100 nM) or vehicle was added to the culture medium 10 min before ISO stimulation. H89 (10 μM), PKI (10 μM) or vehicle was added 30 min before ISO stimulation. Cells were exposed to BNZ (500 μM) or vehicle for 30 min, and to OA (0.1 or 1 μM) or vehicle for 60 min. Experiments were performed at 37 °C.2.5g for 30 min at 4 °C. Proteins in the soluble fraction (supernatant) were denatured in 3X Laemmli sample buffer. Proteins in the insoluble fraction (pellet) were resuspended in 1X Laemmli sample buffer.The subcellular fractionation method was adapted from methods described in previous publications 2.62. Prior to transfer of proteins to PVDF membranes, gels were incubated 15 min in transfer buffer containing 1 mM EDTA then 15 min in transfer buffer alone.Heat-denatured protein samples were resolved on Tris-glycine SDS-PAGE gels and transferred to PVDF membranes. Membranes were blocked in Tris-buffered saline with 0.1% Tween-20 (TBST) and 5% (w/v) non-fat milk. Incubation with primary antibodies was performed overnight at 4 °C and incubation with secondary antibodies was performed for 2 h at room temperature. Protein bands were visualized on chemiluminescence film following incubation of the membranes with ECL Western Blotting Detection Reagents . Signal intensity was quantified on a calibrated GS-800 densitometer (Bio-Rad). Phosphate affinity (PhosTag) SDS-PAGE was performed on Tris-glycine gels containing 50 μM acrylamide-pendent PhosTag™ and 100 μM MnCl2.7ARVM were prepared for imaging on a confocal florescence microscope as previously described 2.8GFP, GFP-B56δ-WT and GFP-B56δ-S573A were immunoprecipitated from ARVM lysates using a GFP antibody. Cells were harvested in ice-cold lysis buffer containing: 50 mM Tris (pH 8), 138 mM NaCl, 27 mM KCl, 1% (v/v) Triton-X100 and complete mini protease inhibitor. The cleared lysate containing 500 μg of protein was incubated with anti-GFP (4 μg) over-night at 4 °C. Immunocomplexes were collected using Protein G beads. After three washes in lysis buffer, these were resuspended in Laemmli buffer.2.9pNPP Protein Phosphatase Assay kit (AnaSpec), as recommended by the manufacturer. Cells expressing heterologous proteins were washed in phosphate-buffered saline and harvested in 1X lysis buffer with added 1% (v/v) Triton-X100 and protease inhibitors. Master mixes of whole cell lysates in assay buffer (40 mM Tris (pH 8.4), 34 mM MgCl2, 4 mM EDTA and 4 mM DTT) were prepared on ice. Incubation with 5 nM okadaic acid or DMSO was performed for 10 min. Assays were performed in triplicates, in wells of 96-well plates. Individual wells contained 10 μg of protein in 50 μl of master mix and 50 μl of pNPP solution. Absorbance at 405 nm was determined in a plate reader pre-heated to 30 °C. Measurements were recorded at 5 min intervals, for a total of 30 min. The amount of pNP produced was determined using the equation c = A ÷ (ɛ × l), where c is the concentration of pNP, A is the optical absorbance of pNP at 405 nm, ɛ is the molar extinction coefficient of pNP  and l is the path length.Protein phosphatase activity was determined using the SensoLyte 2.102 for subsequent analysis.Transverse aortic constriction (TAC) surgery was performed in adult mice by Professor Brian Cooley . Male C57Bl/6 mice at 9 weeks of age were anaesthetised by intraperitoneal injection of 100 mg/kg ketamine and 15 mg/ml xylazine. Constriction of the aortic arch was achieved by ligation of the aorta with 6-0 silk, using a 27.5-gauge spacer. Sham animals were subjected to the same surgical procedure, with no constrictive suture. Hearts were harvested 7 days after the surgical procedure and frozen in liquid N2.11t-test, or One-Way ANOVA followed by Tukey's multiple comparisons test. P < 0.05 was considered significant. In figure legends, n indicates the number of independent experiments.Quantitative data are presented as mean ± SEM. Differences between groups were analysed by unpaired 33.119 mouse heart .To explore the effect of βAR stimulation on B56δ phosphorylation in cardiomyocytes, cell lysates from unstimulated and ISO-stimulated ARVM were resolved by standard- and PhosTag SDS-PAGE. Immunoblot analysis was performed with a polyclonal antibody raised against a peptide sequence at the C-terminus (aa 552-602) of human B56δ, which is conserved in the rat and mouse protein A. When p3.21 or β2ARs, respectively. ISO-induced B56δ phosphorylation at S573 was abolished by CGP and partially attenuated by ICI (1ARs 6-benzoyl-cAMP (BNZ) We next investigated the signaling mechanisms that underlie ISO-induced B56δ phosphorylation at S573. The response was blocked by propranolol, a non-selective βAR antagonist, confirming a βAR-mediated mechanism A. To detd by ICI A. ReflecICI 1ARs A. Guided3.3We have shown previously that ISO stimulation in ARVM induces a change in the subcellular distribution of a different B56 isoform, B56α 3.4To study the functional importance of B56δ phosphorylation at S573, we constructed adenoviral vectors for heterologous expression of GFP-tagged WT or non-phosphorylatable (S573A) B56δ  in ARVM. Control cells were transduced with an adenovirus that expresses GFP alone (AdV.GFP). Comparable GFP protein expression was achieved following 18 h incubation with AdV.GFP at multiplicity of infection (MOI) 30 and AdV. GFP-B56δ-WT or AdV.GFP-B56δ-S573A at MOI 100 . Immunoblot analysis of the same samples with the B56δ antibody confirmed comparable expression of GFP-B56δ-WT and GFP-B56δ-S573A, and showed that, relative to cells expressing GFP alone, expression of endogenous B56δ was unchanged . To further characterize the adenoviral vectors, we determined protein levels of endogenous PP2A subunits in transduced cells. Expression of GFP-B56δ-WT and GFP-B56δ-S573A was associated with increased expression of C subunits, but no change in the expression of A subunits. Expression of other B subunit isoforms  was also unchanged A. To detS/T) residues in the RRXS/T motif. This antibody detected basal S/T phosphorylation in GFP-B56δ-WT and the signal was markedly increased following ISO stimulation . In contrast, the antibody did not detect GFP-B56δ-S573A in the absence or presence of ISO stimulation , indicating that in ARVM S573 is the only B56δ site that is phosphorylated.In the HEK293 cell line, PKA activation has been reported to induce B56δ phosphorylation not just at S573 but also at S88, albeit to a much smaller extent To explore whether ablation of the S573 phosphorylation site impacts on B56δ subcellular localization, we determined the distribution of GFP-B56δ-WT and GFP-B56δ-S573A in soluble and insoluble compartments of fractionated cells. As in earlier experiments B, the puStudies in vitro and in non-cardiac cells have shown that B56δ phosphorylation at S573 increases the activity of its associated C subunit 3.5S/T motif detected phosphorylation of GFP-B56δ-WT but not GFP-B56δ-S573A , confirming the ability of the method to reveal differential phosphorylation of S/T residues in abundantly expressed proteins. In complementary experiments, we determined the phosphorylation status of cTnI, cardiac myosin binding protein-C (cMyBP-C) and phospholamban (PLB), which are phosphorylated by PKA following βAR stimulation and play important roles in mediating inotropic and lusitropic responses. PhosTag SDS/PAGE-immunoblot analysis of cTnI showed that the abundance of non-phosphorylated (0P), mono-phosphorylated (1P) and bis-phosphorylated (2P) cTnI was comparable in cells expressing GFP-B56δ-WT and those expressing GFP-B56δ-S573A, in the absence or presence of ISO stimulation  that selectively inhibits PP2A 3.6Finally, to explore whether the phosphorylation and/or the expression of cardiac B56δ is altered in a pathophysiological setting, we examined these in a mouse model of pressure overload-induced cardiac hypertrophy. Seven days post-surgery, heart weight/tibia length was 10.1 ± 0.8 mg/mm in mice subjected to TAC and 7.7 ± 0.2 mg/mm in mice subjected to sham operation , reflecting cardiac hypertrophy in the former. The abundance of both pS573 B56δ and total B56δ was increased in the hearts of mice subjected to TAC relative to mice subjected to sham operation . These f41AR and activation of PKA, and (iii) βAR/PKA-mediated phosphorylation of B56δ at S573 increases PP2A activity.The present study has revealed for the first time in a cardiac context that (i) acute simulation of βARs induces a rapid and sustained phosphorylation of the PP2A regulatory subunit B56δ selectively at S573, (ii) this phosphorylation occurs principally through stimulation of the β1AR-mediated activation of protein kinases 1AR-mediated regulation of Ser/Thr phosphatases that serve an opposing role is restricted largely to type 1 phosphatase (PP1), whose activity is regulated through PKA-mediated phosphorylation of the PP1-specific inhibitor of protein phosphatase-1 (I-1) In healthy myocardium, catecholamines regulate a variety of cellular processes primarily by β1AR antagonist) and ICI (a selective β2AR antagonist) indicate that the ISO response is mediated primarily by the β1AR subtype (1AR is comparable to that of ICI for the β2AR 1AR) 1AR signaling by the former antagonist. On the basis of this observation and evidence that in ARVM the relative β1AR:β2AR abundance is 65:35 2AR activation and that its relative ineffectiveness in inhibiting ISO-induced B56δ phosphorylation arose from a minor role for this βAR subtype in mediating that response.Here, we provide robust new evidence that in ARVM the non-selective βAR agonist ISO induces phosphorylation of the PP2A regulatory subunit isoform B56δ selectively at S573, in a rapid and sustained manner . Our dat subtype A. In celThe data that we have obtained from complementary studies, in which we explored the role of PKA in mediating B56δ phosphorylation at S573, indicate that activation of this kinase is both necessary for the ISO-induced response and sufficient per se to induce such phosphorylation. Thus, ISO-induced B56δ phosphorylation was significantly attenuated by pre-treatment of ARVM with either H89 or PKI, which inhibit PKA through distinct mechanisms We have shown previously that βAR stimulation by ISO in ARVM alters the subcellular localisation of another PP2A regulatory subunit isoform, B56α C protein that accompanied the heterologous expression of GFP-tagged B56δ in either form  revealed the presence of PP2AA and PP2AC proteins in comparable abundance (C subunits) was increased in ARVM expressing GFP-B56δ-WT but not those expressing GFP-B56δ-S573A physiological role(s) of B56δ phosphorylation, our data from an exploratory analysis of mouse hearts with pressure overload-induced cardiac hypertrophy show that, while the relative phosphorylation of B56δ was unaltered, the absolute abundance of pS573 B56δ was increased significantly in that setting. This change may be a compensatory response to pressure overload-induced cardiac hypertrophy, or play a mechanistic role in its pathogenesis. Notwithstanding, the dynamic regulation of B56δ phosphorylation and expression in cardiac hypertrophy warrants investigation of the functional consequences of their targeted manipulation in appropriate models. In that context, it is interesting to note also that B56δ expression has been reported to be increased in dog hearts following myocardial infarction and in the setting of tachypacing-induced non-ischemic heart failure In conclusion, the present study has identified and characterised a novel mechanism that is likely to regulate a specific pool of PP2A activity in cardiomyocytes in response to βAR stimulation, through PKA-mediated phosphorylation of the PP2A regulatory subunit isoform B56δ at S573. The functional importance of this novel mechanism now requires the establishment of a model system that allows replacement of endogenous B56δ with a non-phosphorylatable (S573A) mutant (e.g. a knock-in mouse) and the application of broad-spectrum phosphoproteomics analyses, as well the examination of cardiac responses to (patho)physiological stimuli, in such a model.None.


Kinetic analysis reveals co-occupancy of the allosteric sites by TIH and l-histidine. Crystallographic and native ion-mobility mass spectrometry data show that the TIH-bound activated form of the enzyme closely resembles the inhibited l-histidine-bound closed conformation, revealing the uncoupling between ATP-PRT open and closed conformations and its functional state. These findings suggest that dynamic processes are responsible for ATP-PRT allosteric regulation and that similar mechanisms might also be found in other enzymes bearing a ferredoxin-like allosteric domain.ATP-phosphoribosyltransferase (ATP-PRT) is a hexameric enzyme in conformational equilibrium between an open and seemingly active state and a closed and presumably inhibited form. The structure-function relationship of allosteric regulation in this system is still not fully understood. Here, we develop a screening strategy for modulators of ATP-PRT and identify 3-(2-thienyl)- Active and inactive state ATP-phosphoribosyltransferases (ATP-PRTs) are believed to have different conformations. Here the authors show that in both states, ATP-PRT has a similar structural arrangement, suggesting that dynamic alterations are involved in ATP-PRT regulation by allosteric modulators. Feedback allosteric circuits are a direct, rapid and efficient mechanism of metabolic control. These feedback allosteric circuits contribute to metabolic homeostasis by translating changes in the concentration of metabolites to enzyme activity and ultimately cellular responses3. Protein domains with a ferredoxin-like (FL) βαββαβ topology have been widely co-opted throughout evolution for allosteric control. Particular FL domains have evolved to recognize and respond to one -or sometimes more- molecules of very distinct nature, including nucleotides4, heavy metals5, and various amino acids. Aspartate carbamoyltransferase (an FL domain containing enzyme) is one of such enzymes, where multiple nucleotides inhibit or activate catalysis. Elegant work describing the complex analysis of regulation by multiple effectors (physiologic ligands) has been carried out6.Allostery is the change in protein activity triggered by binding of a ligand (effector) at a site different from the active sitearabino-heptulosonate 7-phosphate synthase 7 and the bacterial and plant chorismate mutase/prephenate dehydratase 8. In branched amino acids biosynthesis, proteins regulated via FL domains are the bacterial and plant biosynthetic threonine ammonia-lyase (EC 4.2.1.19)9 and the bacterial acetohydroxyacid synthase isozyme III 10. Other examples are in Thr, Lys, Met and Ile biosynthesis, the bifunctional chloroplastic aspartokinase/homoserine dehydrogenase 2 , and in Ser biosynthesis, D-3-phosphoglycerate dehydrogenase types I and II . The long form of ATP-phosphoribosyltransferase , the enzyme that catalyses the first committed step of l-histidine (l-His) biosynthesis, is also feedback inhibited by l-His12  superfamily15. The FL domains of GlnB and ACT have no known evolutionary relationships and represent a superfold that has likely emerged by convergence16.Protein domain structure databases place the FL domain of ATP-PRT in the same superfamily as the cyclic-di-AMP receptor family, peptidase S54, and the GlnB proteinsT. maritima DAH7PS and PHGDH, the available structures show that effector binding triggers domain reorganization leading to occlusion of the active site18. In the case of ATP-PRT, an early study proposed that l-His binding caused a transition from dimer to hexamer19. However, more recent data show that both free and inhibited ATP-PRT are hexameric but that the l-His-bound form is more compact due to a slight rotation of the regulatory domains20. We now understand crystal structures as being the static snapshots of a continuum of conformations sampled by the enzyme. In this light, ATP-PRT can be described to exist in an equilibrium between a relaxed (R) hexamer and a tense (T) compact hexamer, with l-His binding preferentially to the R state and causing the population to shift to the T state22.Our understanding of the mechanisms of amino acid-binding FL domain-mediated allosteric inhibition comes mostly from the few available crystal structures of free vs. inhibited forms. For l-alanine) (TIH) as a non-essential activator of M. tuberculosis ATP-PRT. Detailed kinetic measurements and structural characterization of ATP-PRT in the presence of TIH and/or l-His allowed us to probe the mechanism of allosteric regulation in ATP-PRT with unexpected results. Activation and inhibition of ATP-PRT leads to the same overall conformational tightening of the hexamer. This decoupling of activity and conformation indicates that allosteric regulation of ATP-PRT, and perhaps other FL domain containing allosteric enzymes, is more complex than the simple open–closed active–inactive model.In an effort to better understand the allosteric regulation mechanism of ATP-PRT, we implemented a Compound Screening in the Presence of an Inhibitor (CoSPI) strategy Fig. . CoSPI mε290 = 3600 M−1 cm−1), we tested the effect of 11 commercially available l-His analogues, at a single concentration (1 mM), on the inhibition of ATP-PRT by l-His  increase in the reaction velocity   Fig. . TIH inc24, the presence of a modifier (either an inhibitor or an activator) might have an effect on the dissociation constant of the ternary enzyme-substrate-modifier complex, this effect is given by the constant α. In addition, the modifier can have an effect on kcat, which is described by the coefficient β. Non-essential activation is generally characterized by a ligand that can bind and activate an enzyme, not being essential for enzymatic activity. Values of α lower than 1 and values of β higher than 1 are usually observed. However, mixed-type systems where α and β are higher than 1 are also possible, as are systems where α and β are both lower than 125. We performed measurements of ATP-PRT activation by TIH at different concentrations of either of the substrates, ATP or PRPP. Coefficients α and β were consistent with non-essential activation in both cases  , ESPSA  and ESPSAI , where I is the inhibitor, SA is ATP and SP is PRPP, as illustrated in the model  Fig. . Increasion Fig. . Tertiarion Fig. . Three ddel Fig. . The rated Table . When vaed Table  and 8b, l-His , ATP-PRT is fully inhibited, showing no activity  100% of the activity is restored and further activation is achieved with higher concentrations of TIH. Figure l-His and TIH, at all concentrations tested. Simultaneous binding of TIH and l-His to the hexamer was confirmed through a competition saturation transfer difference (STD) NMR experiment27. In an STD NMR experiment, signals are observed only for ligand protons in contact with the protein. In the absence of l-His signals of the TIH protons are observed  as a search model. Crystals of the two complexes grew in identical crystallization conditions and to comparable resolution, thus being an ideal case for a direct and detailed comparison between structures ; however, it is still activated by TIH, in the presence or absence of l-His, with similar affinity to the wild type  conformation so far attributed to the inactive state of the enzyme 28 and C. jejuni ATP-PRT in complex with ATP (PDB code: 4YB7)20  and in mildly alkaline pH (9.0) conditions in which ATP-PRT is more active12 , characteristic of the T-state. At basic pH, unligated, l-His and TIH-bound ATP-PRT are all in the T-state  to investigate the equilibrium between the R- and T-states in the presence of either 30, activation31, or binding to the target protein32, while other screenings test for secondary ligands that only bind after the first ligand binds34. Despite their undisputed usefulness, traditional screens fail to detect compounds displaying less common or peculiar effects, such as influencing the efficacy or affinity of a known effector, or altering effector–enzyme cooperativity. In order to screen for these effects, we developed CoSPI, a targeted screen of small molecules. The screen is carried out in the presence of a known inhibitor, and consists of determining and evaluating the entire inhibition profile (IC50 curve) of the known inhibitor in the presence of a single fixed concentration of each of the compounds. Hill number (nH), Vmax and IC50 are then estimated from such curves and compared. Three limiting behaviours can potentially be observed using a CoSPI design  but has no effect on Km ; this is a different type of activation from most cases reported to date39. To the best of our knowledge, a fivefold increase in kcat is one of the highest increases reported for a small-molecule allosteric activator. Having a similar structure and judging by its effect on l-His IC50  was sufficient to fully activate ATP-PRT in the presence of l-His at a concentration ≈ twice Ki (36 μM). Under these conditions Vmax reached values close to those obtained in the presence of saturating concentration of TIH (absence of l-His), Va,max, justifying the increase in fold-activation values from 6 to 18-fold. To confirm this behaviour, ATP-PRT activity was tested at increasing concentrations of TIH in the presence of l-His at a concentration of 20 times Ki pLysS cells were purchased from Millipore. Complete EDTA-Free protease inhibitor was purchased from Roche.Buffers and pJ411::hisG and pJ411::ppa) 50 grams of cells were thawed on ice, resuspended in buffer A (20 mM triethanolamine (TEA) pH 7.8, 300 mM NaCl, and 50 mM imidazole containing lysozyme, Complete® protease inhibitor cocktail and DNase) and lysed by sonication. After centrifugation at 25000 g for 30 min, the soluble fraction was loaded on a 100 ml Ni-NTA column and protein eluted by a 0 to 100% gradient of buffer B . UV-Vis peak fractions were analysed by SDS-PAGE, performed on a PhastSystem . Fractions containing only ATP-PRT or PPase were pooled, dialyzed into 20 mM TEA (pH 7.8), concentrated, flash-frozen and stored at −80 ˚C. This protocol allowed the purification to homogeneity of 1 g of ATP-PRT and 0.5 g of PPase. ATP-PRT concentration was determined using absorbance at 280 nm . After the Ni-NTA column step, ATP-PRT protein for crystallization was further purified by gel filtration using a Sephadex S200 16/600 column and concentrated in the elution buffer  to a final concentration of 13 mg/ml before flash freezing.All steps of ATP-PRT and PPase purification were performed at 4 ˚C using an AKTA purifier 10 . Frozen BL21(DE3)pLysS , in the presence of PPase. Pyrophosphatase is essential for this assay, as the equilibrium constant lies towards formation of ATP and PRPP. A typical reaction mixture contained 50 mM Tris-HCl (pH 8.5), 7 mM MgCl2, 200 mM KCl, 3 mM ATP, 1.5 mM PRPP, 600 nM PPase and 450 nM ATP-PRT.Steady-state kinetic measurements were conducted at 25 °C ± 0.2 °C with a Shimadzu UV-2550 spectrophotometer equipped with dual-beam optics and a Peltier system for temperature control. Initial velocities for the forward reaction of ATP-PRT were measured by following the formation of PR-ATP . To determine the effect of each analogue on enzyme activity (in the absence of l-His), ATP-PRT activity was measured in the presence of saturating concentrations of substrates and metals, and variable concentrations of each analogue (up to 5 mM).To determine the effect of 11 commercially available analogues on To determine the steady-state activation parameters and patterns associated with TIH, ATP-PRT activity was studied in the presence of variable concentrations of one substrate, PRPP or ATP, fixed saturating concentrations of the co-substrate (ATP or PRPP) and metals, and several fixed concentrations of TIH.l-His and TIH, ATP-PRT activity was studied in the presence of variable concentrations of one substrate, PRPP or ATP, fixed saturating concentrations of the other substrate (ATP or PRPP) and metals, and several fixed concentrations of l-His. The experiment was repeated at several fixed concentrations of TIH.To determine the interaction between l-His, a concentration 20-fold higher than l-His Ki, which fully inhibited ATP-PRT. Increasing concentrations of TIH were then added to the reaction, up to 8 mM TIH, a concentration 4-fold higher than TIH KA.ATP-PRT activity was studied in the presence of 0.4 mM 1H frequency of 600 MHz. Saturation transfer difference (STD) spectra43 of 20 µM ATP-PRT in the presence of fixed concentration of TIH and variable concentrations of l-His were recorded in 50 mM sodium phosphate buffer (pH 8.0). Spectra with on-resonance pre-saturation of the enzyme at 0.8 ppm were recorded with a saturation time of 2 s. An off-resonance control spectrum was recorded with a saturation time of 2 s. Water suppression was achieved by excitation sculpting44. Data processing and spectral subtractions were performed using Bruker TopSpin 3.2 software.NMR spectra were recorded at 25 °C on a Bruker Avance-600 spectrometer equipped with a triple resonance cryo-probe and operating at a l-His or TIH were obtained by co-crystallization experiments at 18 °C using the sitting drop vapour diffusion method. Drops of 0.2 µl (protein stock:well solution 1:1) were set-up with the assistance of the Oryx protein crystallization robot. Protein concentration was 9.3 mg/ml whereas the ligands concentration was 100 µM and 5.5 mM for l-His and TIH, respectively. Crystals of ATP-PRT complexes with TIH and l-His were obtained from condition 4 of Classics Lite Suite crystallization screen by Qiagen (2.5 %(v/v) isopropanol, 1.0 M ammonium sulphate). Crystals of ATP-PRT in complex with TIH grown in the presence of ATP, although no positive density for ATP molecule could be located, were obtained from condition 71 of the Protein Complex Suite by Qiagen . Crystals were frozen adding 20 % (v/v) glycerol to the mother liquor for cryo-protection and stored in liquid nitrogen until data collection. Diffraction data were collected at beamline IO4 at the Diamond Light Source, UK. Data collection temperature was 100 K. Wavelength for both ATP-PRT/TIH and ATP/PRT/l-His datasets was 0.9282. Integration and scaling were performed using XDS and XSCALE via xia2 expert system for X-ray diffraction data processing46. Crystals belong to the rhombohedral space group H 3 2 with one molecule in the crystallographic asymmetric unit, which gives a solvent content of 54–56%. Subsequent data handling was carried out using the CCP4 software package47.All crystals of ATP-PRT complexes with either 42, using as a search model the coordinates of M. tuberculosis ATP-PRT complexed with l-His and AMP (PDB code 1NH8), but with the ligands and solvent molecules removed. Models were improved and refined alternating cycles of model correction by Coot43 and restrained refinement by Refmac44 and Phenix.Refine45. Figures were drawn using either Coot43 or PyMol46. For each structure, 284 amino acids of the native protein are visible in the molecule. Residues R26 to V40 and R184 to E194 could be fitted, but display higher flexibility as evidenced by less well-defined electron density. The bound ligands are clearly defined in both structures and refine well. A summary of the data collection and refinement statistics is given in Table l-His.Molecular replacement was carried out with PHASEROn the day of analysis, the buffer was exchanged to 100 mM ammonium acetate buffer  of specified pH, using micro Bio-Spin Chromatography columns  following the instructions specified by the manufacturer. The desalting procedure was performed four to five times to achieve desired sample quality. pH of the buffer was adjusted with ammonia supplied by VWR International Ltd (UK). Solution pH readings were taken using a pH metre (Jenway 3305). High purity water was obtained from an Arium 611 water purification unit  fitted with a 0.2 µm filter.−1, respectively, the IMS wave velocity was 617 m s−1, and the IMS wave height was 40 V. Nitrogen was the carrier gas. Data were acquired and processed with MassLynx software .The IM-MS data were acquired on a Synapt G2S HDMS . Samples were ionized using nano-elecrospray ionization (nESI) method, where protein species were charged and transferred from solution into the gas phase. nESI capillaries were prepared in-house from thin-walled borosilicate capillaries  using a Flaming/Brown P-97 micropipette puller . These were then filled with sample using micro-loading tips . A positive voltage was applied to the solution via 0.125 mm platinum wire  inserted into the capillary. Gentle conditions were applied to preserve the native-like structure: capillary voltage 1.6 kV, sampling cone 99 V, source temperature 20 °C, trap collision energy 5.6 V, and transfer collision energy 2 V. The pressure of the backing region was 8.3 mbar. For IM-MS, the helium cell and the IMS gas flows were 180 and 90 ml minV is the maximal velocity, S is the substrate concentration, and K is the Michaelis constant for the substrate (Km). Inhibition data obtained under saturating concentrations of substrates and metals, and variable concentration of l-His, were fit to Eq. v0 is the uninhibited velocity, I is the l-His concentration, IC50 is the concentration of l-His necessary to give 50% inhibition and nH is the Hill number. Activation data obtained under saturating concentrations of substrates and metals and variable concentration of TIH were fit to Eq. 49vamax is the maximal velocity at maximum activation, A is the concentration of the activator TIH and AC50 is the concentration of TIH necessary to increase the velocity by 50%. Activation data showing linear, non-essential activation pattern in a double-reciprocal plot were fit to Eq. 39KA is the activator dissociation constant, α is the constant that denotes the modification of the substrate and activator dissociation constants in the presence of each other and β is the constant of the modification of the catalytic constant. Inhibition data showing linear, uncompetitive patterns in double-reciprocal plots were fit to Eq. Ki is the dissociation constant for the enzyme-inhibitor complex. Inhibition data showing linear, noncompetitive patterns in double-reciprocal plots were fit to Eq. Steady-state data were fitted using the nonlinear, least-squares, curve-fitting programs of Sigma-Plot for Windows, version 11.0. Individual saturation curves were fit to Eq. entclass1pt{minima50y0 is the value of slope or intercept in the absence of activator and y∞ is the value of slope or intercept in the presence of infinite concentration of activator.Tertiary replots of the slopes and intercepts against activator concentration were fit to Eq. M. tuberculosis ATP-PRT in complex with l-His and TIH have been deposited into the Protein Data Bank under the accession codes 5LHU and 5LHT. The UniProt accession codes P9WMN1 and P9WI55 for M. tuberculosis ATP-PRT and PPiase, respectively were used in this study. All other data are available from the corresponding author on reasonable request.The atomic coordinates and the structure factors for Supplementary Information


The movies are made publicly available to enable critical or extended analyzes.The visualization data (8 movies) presented in this article are related to the research article entitled “Freezing and glass transitions upon cooling and warming and ice/freeze-concentration-solution morphology of emulsified aqueous citric acid”   Specifications TableValue of the data•The movies provide visual insights into the physical chemistry of freezing dispersed aqueous solutions and can be used by other researches who work with freezing phenomenon in fields ranging from life sciences and biotechnology to geophysics and high-altitude ice clouds.•in-situ using OC-M and can be compared to freezing results obtained with other techniques, for example, confocal fluorescence microscopy as well as to results obtained by computer simulations.The movies demonstrating the freezing process were recorded •Since the movies are first of their kind, they can be used/give an impetus in/for the development of further experiments in different fields of science and technology where freezing phenomena play important role.12O drops. These movies also demonstrate how the ice/FCS morphology of frozen drops changes with decreasing drop size.The movies presented in this data article provide the visualization evidence of a freeze-induced phase separation (FIPS) into pure ice and a freeze-concentrated solution (FCS) which occurs during the freezing of micrometer-scaled CA/H22O/oil-surfactant-matrix of 1/10 by volume were subjected to magnetic stirring at different speeds in order to obtain CA/H2O drops of different size distributions in-situ observation of FIPS and ice/FCS morphology by applying OC-M We prepared 10–60 wt % CA solutions by mixing >99% anhydrous citric acid (Merck) with the corresponding amount of ultrapure water. For emulsion preparation we used an oil-surfactant matrix consisted of 80 wt % halocarbon 0.8 oil  and 20 wt % lanolin (Sigma Aldrich). CA/H