Drosophila rely on cyclic regulation of the period (per) and timeless (tim) clock genes. The molecular cycle requires rhythmic phosphorylation of PER and TIM proteins, which is mediated by several kinases and phosphatases such as Protein Phosphatase-2A (PP2A) and Protein Phosphatase-1 (PP1). Here, we used mass spectrometry to identify 35 “phospho-occupied” serine/threonine residues within PER, 24 of which are specifically regulated by PP1/PP2A. We found that cell culture assays were not good predictors of protein function in flies and so we generated per transgenes carrying phosphorylation site mutations and tested for rescue of the 01per arrhythmic phenotype. Surprisingly, most transgenes restore wild type rhythms despite carrying mutations in several phosphorylation sites. One particular transgene, in which T610 and S613 are mutated to alanine, restores daily rhythmicity, but dramatically lengthens the period to ∼30 hrs. Interestingly, the single S613A mutation extends the period by 2–3 hours, while the single T610A mutation has a minimal effect, suggesting these phospho-residues cooperate to control period length. Conservation of S613 from flies to humans suggests that it possesses a critical clock function, and mutational analysis of residues surrounding T610/S613 implicates the entire region in determining circadian period. Biochemical and immunohistochemical data indicate defects in overall phosphorylation and altered timely degradation of PER carrying the double or single S613A mutation(s). The PER-T610A/S613A mutant also alters CLK phosphorylation and CLK-mediated output. Lastly, we show that a mutation at a previously identified site, S596, is largely epistatic to S613A, suggesting that S613 negatively regulates phosphorylation at S596. Together these data establish functional significance for a new domain of PER, demonstrate that cooperativity between phosphorylation sites maintains PER function, and support a model in which specific phosphorylated regions regulate others to control circadian period.Circadian rhythms in  Drosophila melanogaster, the central molecular clock is regulated by transcriptional and translational feedback loops that drive rhythmic waves of gene expression. Additionally, the clock is controlled by post-translational modifications, including phosphorylation of a core negative regulator, PERIOD (PER). Using a proteomic approach, we identified two key phosphorylation sites within PER that cooperate to establish proper daily rhythms. Mutation of both residues results in a period of ∼30 hrs, whereas single mutant phenotypes are less severe suggesting these sites normally cooperate to control the pace of the clock. Biochemical and immunohistochemical data demonstrate that altered PER phosphorylation and changes in protein stability give rise to behavioral defects. Other mutations in the vicinity of the two sites above also alter circadian period, thereby confirming a new regulatory domain of PER. Our results also suggest that the phospho-residues identified in this study regulate phosphorylation in another important domain of PER. Together our data establish that cooperativity between neighboring phospho-sites as well as interactions between different phosphorylated domains of PER control its stability and overall circadian periodicity.Circadian rhythms coordinate an organism's activities with its environment to ensure appropriate physiology and behavior at the relevant times of day. In  Drosophila the major loop consists of the period (per) and timeless (tim) genes, which autoregulate their expression by inhibiting activity of their transcriptional activators, the CLOCK (CLK) and CYCLE (CYC) proteins at distinct times of day, thereby driving their own rhythmic expression as well as that of other clock-controlled genes Drosophila have been instrumental in not only identifying components of core clock machinery, but also for teasing apart more intricate regulatory mechanisms. Additionally, discovery of core clock genes in Drosophila ultimately led to identification of their human orthologs and the discovery that some of these genes are mutated in human circadian disorders Circadian rhythms, exhibited by a variety of organisms ranging from bacteria to plants and humans, help to establish daily temporal organization. These rhythms are typically generated by one or more feedback loops within core clock or “pacemaker” cells. In Levels of PER and TIM cycle over the course of the day with peak expression occurring in the mid- to late-night. TIM levels decline at the end of the night, and decrease rapidly following daybreak due to light-dependent degradation It is clear that cyclic phosphorylation of core clock proteins is critical for generating a functional clock. For example, temporal changes in the state of PER phosphorylation regulate various aspects of PER function including protein stability, subcellular localization, protein-protein interactions, and transcriptional repression Drosophila clockwork and help to control timing of phosphorylation, overall PER stability, and circadian periodicity. Importantly, S613 is conserved from flies to mammals and thus may have implications for human circadian disorders. Mutating T610 and S613 to alanine leads to a long daily period length due to a delay in PER protein degradation and changes in CLK phosphorylation and CLK-mediated output. Notably, it appears as though these two residues act cooperatively to modulate PER function. Additionally, our data suggest S613 impacts clock speed by regulating the well-known Per-Short domain.In this study, using a proteomic approach to identify phospho-occupied sites, we demonstrate that phospho-residues PER-T610, and to a larger extent PER-S613, act as key regulators of the Despite efforts to better understand the role of cyclic phosphorylation in regulating the circadian clock, the complete repertoire of phosphorylation residues within PER has not been identified. Additionally, how phosphorylation contributes to overall circadian regulation is still being investigated. While some phospho-sites have been determined We followed up the mass spectrometry analysis by mutating individual phospho-sites and testing them in cell culture assays. We first asked if any of the PP2A- or PP1-regulated sites are important for overall PER stability in cells. As shown in per-luciferase (per-luc) cell culture assay. In this assay, per-luc activity is induced by CLK, and the CLK activity can be inhibited by PER. We found that individually mutating S613, S826, S1080, or S1187 to alanine leads to significantly reduced repression in the per-luc assay  period length  are conserved in insects . Previou∼27 hrs; . Togetheper13.2 in lanes 2 and 5) and shows increased phosphorylation over the course of the day (per13.2 in lane 5), and is at its lowest levels at ZT20 (In an attempt to determine how the T610 and/or S613 mutation(s) affect the well-known cyclic phosphorylation profile of PER, we collected heads at discrete time points throughout the light∶dark cycle from flies carrying a wild type transgene or one containing each single or combined double mutations and performed western blots. Consistent with previously published data, we found that wild type PER is at its lowest levels and hypophosphorylated during the end of the light phase ∼ZT8-12,  [18]. Ac the day . Mutant  at ZT20 . Thus, pInterrogation of T610A and S613A single mutants revealed that defects observed in the phosphorylation state of the PER-T610A/S613A double mutant are mainly attributable to the mutation at S613  of PER throughout the day. We first optimized the PER antibody staining protocol and found that varying fixation times can greatly impact the reliability of the PER signal. Thus, for the PER antibody used in this study (UP1140), we incubated fly brains in freshly prepared 4% paraformaldehyde for no more than 15–20 minutes at room temperature. We found that incubation times approaching 45 minutes greatly reduce PER intensity and the results overall become less reliable.vs), which express the neuropeptide, pigment dispersing factor (PDF), while other dorsal neurons also help to modulate circadian behavior in response to light vs are the most important cells for driving rhythms in constant darkness, we monitored PER expression in these cells every two hours throughout a 24-hour cycle in both wild type and T610A/S613A mutant flies. As previously reported vs from ∼ZT2–ZT6  leads to a decrease in daily period length It is well documented that PER phosphorylation plays a pivotal role in setting the pace of the circadian clock, and more recently, progress has been made in teasing apart the intricacies of this regulation Therefore, how do the phospho-residues identified in this study, T610 and S613, impose regulation? Given the close proximity of T610 and S613 to the Per-Short domain, it was not unreasonable for us to imagine some sort of interaction between these two clusters of phospho-residues. Indeed, the epistasis data presented in in vivo. Overall, we believe our genetic epistasis experiments support our current view. Regardless, it is clear that the Per-SD domain is an important regulatory region, and appears to function upstream of or in parallel to previously identified functional domains of PER in controlling circadian period. Our findings suggest that a series of interactions between different domains of PER regulate its phosphorylation and its ultimate degradation to yield precise cycles of protein expression and of behavior.From the data presented in Act-per-HA transfected S2R+ cells were treated with vehicle (DMSO), 2 µM Tautomycin , or 50 nM Okadaic Acid (Biomol), along with MG132  for 4 hrs before harvest. Cells were then lysed and immunoprecipitated with anti-HA (Covance) antibody as described previously pProtein samples were excised from the Coomassie-blue gel and subjected to in-gel digestion with sequencing grade 12.5 ng/µl trypsin (Promega) in 50 mM ammonium bicarbonate pH 8.2 overnight at 37°C. Peptides were extracted with 50% acetonitrile (MeCN), 5% formic acid (FA) and then dried. Dried peptides were resuspended in 10 µL of 5%MeCN, 4% formic acid (FA), and 2 µL was loaded onto a pulled fused silica microcapillary column packed with C18 reverse-phase resin  using a Famos autosampler (LC Packings). Once loaded, the peptides were separated using an Agilent 1100 series binary pump across a 45 min linear gradient of 6–28% CH3CN in 0.125% HCOOH at a flow rate of 600 nL/min.Mass spectra were acquired in an LTQ-Orbitrap hybrid mass spectrometer (Thermo Fisher) over the entire run using ten MS/MS scans following each survey scan. Raw data were searched against the HA-Per amino acid sequence using Sequest software with no enzyme specificity and a 1.1 Da mass tolerance. The search parameters used for post-translational modification included a static modification of 57.02146 Da on cysteine (carboxyamidomethylation) and dynamic modifications of 15.99491 Da on methionine (oxidation) and 79.96633 Da on serine, threonine, and tyrosine (phosphorylation). The probability of correct phosphorylation site localization was determined for every site in each peptide using the AScore algorithm as described in Act-per-HA (a gift from S.M. Reppert) was used as a template for mutagenesis.Site-directed mutagenesis of PER phosphorylation sites was conducted using QuikChange  Site-Directed Mutagenesis Kits (Stratagene/Agilent). Primers were designed using Agilent's online primer design tool. p+ cells were cultured as described above but in 96-well plates. 48∼72 hr post-transfection, cells were lysed and processed using the Dual-Glo Luciferase Assay System (Promega) according to the manufacture's protocol using the Victor3V luminometer (PerkinElmer). Firefly luciferase (per-luc) activity was normalized to Renilla luciferase activity. For transfection of the cells in each of the 96-wells, 10 ng pAct-r-luc, 20 ng per-E-luc, 10 ng pAct-Clk-HA, and 5 ng WT and mutant pAct-per-HA were used.For the luciferase assay + cells were transfected with expression constructs using Cellfectin (Invitrogen) as described in the product manual. For the phosphatase inhibition assay, cells were co-transfected with indicated amount of pAct-Nipp1-V5 S2Rper13.2, per-T610A/S613A/S1187A, per-T610A/S613A, per-T610A, per-S613A, per-S1187A, per-P611A, per-P612A, per-Y614A, per-S596A, per-S589A, per-S596A/S613A, per-S589A/S613A, per-S596A/T610A/S613A, per-S589A/T610A/S613A. Males of these transgenic lines were crossed to yw, 01per females and circadian rhythms from male hemizygous progeny were analyzed in all experiments.The following transgenic lines were generated by BestGene, Inc : Bgl-II-per genomic fragment contained within the pSP73 vector was used as a template for mutagenesis. Following mutagenesis and sequence confirmation, the Bgl-II-per fragment was subcloned into the pCasPeR-per-13.2 (HA/His tagged) vector 1118w background by BestGene, Inc . Individual lines were tested for rescue of the 01per mutant phenotype using the traditional TriKinectics Drosophila Activity Monitor (DAM) setup. Data from each individual transgenic line are shown in the paper. For all rescue experiments, a fly was considered rhythmic if the FFT value was greater than 0.02.Site-directed mutagenesis of genomic per phosphorylation sites was conducted using QuikChange  Site-Directed Mutagenesis Kits (Stratagene/Agilent). Primers were designed using Agilent's online primer design tool . A Bgl-I01/Y +/−per transgene) were entrained to the appropriate LD/DL light schedule for a minimum of 3 full cycles prior to collection on day 6. At the indicated time points, fly heads were collected on dry ice and immediately homogenized on wet ice in 1× Lysis Buffer . For PER westerns, lysates were spun down, boiled, run on 6% Tris-Glycine gels , transferred to nitrocellulose membranes and probed with the following antibodies: anti-PER  or anti-HSP70 as loading controls. For CLK westerns, lysates were spun down, boiled, run on 3–8% Tris-Acetate gels , transferred to nitrocellulose membranes and probed with the following antibodies: anti-CLK  or anti-HSP70 as loading controls.2–3 day old male flies , and cDNAs were synthesized by using a high-capacity cDNA Reverse Transcription kit (Applied Biosystems). Quantitative real-time PCR was performed in an ABI prism 7100 using a SYBR Green kit (Applied Biosystems) with gene specific primers. The following primer pairs were designed according to 01/Y +/−per transgene) were entrained to the appropriate LD/DL light schedule for a minimum of 3 full cycles prior to dissections on day 6. At the indicated time points, fly heads of each genotype were gently opened and immediately exposed to 4% paraformaldehyde (in 1× PBS) for 15–20 minutes, followed by incubation in 1× PBST at room temperature for the duration of all dissections at each time point. Note, at each time point, there was no more than a 5–10 minute time delay between when the heads from the first and last genotypes initiated fixation. Dissected brains from each time point remained in 1× PBST at 4 degrees until all time points throughout the day were complete. At the completion of all dissections, brains were incubated with 1× PBST for a ∼30 minutes at room temperature, blocked with Dk-Block (5% Donkey Serum in 1× PBST) for 20 minutes, and incubated with primary antibody overnight in Dk-Block at 4 degrees. The following primary antibodies were used: Guinea Pig anti-PER  and Rabbit anti-PDF . The next day, following a 30-minute wash in 1× PBST at room temperature, brains were incubated with secondary antibodies for two hours in Dk-Block at room temperature. The following secondary antibodies were used at 1∶500: FITC Dk-anti-Guinea-Pig (1∶500) and Alexa555 Dk-anti-Rb. Samples were then washed for 30–60 minutes in 1× PBST at room temperature, cleared in 70% glycerol, mounted in VectaShield, and stored on the slides at 4 degrees.2–3 day old male flies Click here for additional data file.


Xenopus embryos aimed at identifying new regulators of MAPK/Erk, PI3K/Akt, BMP, and TGF-β/Nodal signalling pathways. Our gain-of-function screen is based on the identification of gene products that alter the phosphorylation state of key signalling molecules, which report the activation state of the pathways. In total, we have identified 20 new molecules that regulate the activity of one or more signalling pathways during early Xenopus development. This is the first time that such a functional screen has been performed, and the findings pave the way toward a more comprehensive understanding of the molecular mechanisms regulating the activity of important signalling pathways under normal and pathological conditions.Embryonic development requires exquisite regulation of several essential processes, such as patterning of tissues and organs, cell fate decisions, and morphogenesis. Intriguingly, these diverse processes are controlled by only a handful of signalling pathways, and mis-regulation in one or more of these pathways may result in a variety of congenital defects and diseases. Consequently, investigating how these signalling pathways are regulated at the molecular level is essential to understanding the mechanisms underlying vertebrate embryogenesis, as well as developing treatments for human diseases. Here, we designed and performed a large-scale gain-of-function screen in  During embryonic development, cells constantly receive and emit signals that determine their position, fate and migratory behaviour Drosophila, where both gain and loss of function screens are possible Drosophila led to the identification of many genes influencing FGF signalling Xenopus embryos provide a powerful system to investigate the role of growth factor signalling. Indeed this system has been instrumental in establishing much about what we know about the importance of various signalling pathways during early embryogenesis XenopusXenopus development Xenopus has also proven to be a very useful model in identifying novel factors that are important for early embryonic development Several gain and loss-of-function genetic screens have been performed in order to identify novel regulators of growth factors signalling during development. This approach has been particularly successful in Xenopus development. We then performed an in vivo large-scale gain-of-function screen aimed at identifying new molecules able to modulate the activity of the TGF-β/Nodal, BMP, PI3K/Akt, and FGF pathways during early vertebrate embryogenesis. Overall, 20 potential regulators have been identified out of 2,880 screened full-length clones isolated from egg, gastrula, and neurula stages. Finally, in situ hybridisation analyses have revealed that half of the putative modulators of growth factor signalling are regulated at the transcriptional level in time and space. Together, these results open new avenues of investigation in better understanding the regulation of signalling pathways during embryonic development.Here, we first characterised a set of antibodies to monitor the activation state of several signalling pathways, including the TGF-β/Nodal, BMP, MAPK/Erk, and PI3K/Akt pathways, which allowed us to analyse the temporal dynamics of these signalling pathways during early stages of All animal experiments were approved from the University of Manchester Animal Welfare Centre and were covered by a UK Home Office Project Licence.X. tropicalis full-length cDNA library (known as xt3: fl2) for the large-scale gain-of-function screen http://www.lifesciences.sourcebioscience.com). Briefly, each 96-well plate was subdivided into 12 pools by column, each containing 8 clones. The clones were individually cultured in 96-well deep plates and pooled for plasmid extraction. Plasmids were linearised using AscI, and capped mRNAs were synthesized in vitro using SP6 RNA polymerase (NEB). The quality of synthesised mRNA pools was checked by 1% agarose gel electrophoresis.We used of the Xenopus laevis eggs were artificially fertilised to ensure synchronized development. Embryos were treated from stage 6.5 until stage 10.5 with the indicated chemical inhibitors at the following concentrations: FGF Receptor inhibitor SU5402 (Sigma): 40 µM; PI3K inhibitor LY294002 : 50 µM; TGF-β Receptor inhibitor SB505124 (Sigma-Aldrich): 20 µM. DMSO was added at a final concentration of 1% (v/v), representing the highest concentration used as solvent for the inhibitors. For RNA pools, a total of 6.4 ng of mRNA was injected into each X. laevis embryo at the 1–2 cell stage. For subsequent de-convolution of single clones from pools, 800 pg of mRNA was injected. Treated/injected embryos were collected at stage 8, 10.5 and 14 from each pool. For each stage, 7 embryos were collected. Collected embryos were homogenized using PK buffer to extract proteins and the yolk was eliminated by centrifugation The equivalent of 1 embryo lysate was loaded onto 8% SDS-PAGE and after electrophoresis, proteins were transferred onto PVDF membrane. The following primary antibodies were used: anti-phospho-Akt S473 ; anti-phospho-Erk 1/2 T180/Y182 (Sigma-Aldrich #E7028), anti-phospho-Smad1/5/8 ; anti-phospho-Smad2 A5S (Millipore #05-093); anti-phospho-LRP6 , anti-Erk ; anti-Akt ; anti-Smad2 (BD Biosciences #610842); and anti-α-tubulin (Sigma Aldrich #T9026). All antibodies were used at 1∶1000 concentration except for anti-phospho-Erk 1/2  and anti-α-tubulin . Tris-buffered saline with 0.1% Tween 20 (TBST) and 5% milk was used for blocking, except for the anti-phospho-Smad1/5/8 where TBST with 5% BSA was used. Chemiluminescence detection was performed using HRP-conjugated anti-rabbit  or anti-mouse  antibody combined with Millipore Immobilon ECL reagent (#WBKLS0500).in situ hybridisation was performed using X. tropicalis embryos as described in vitro transcription using T7 RNA polymerase (Roche) and digoxigenin RNA labelling mix (Roche). Chromogenic detection was performed using BM purple AP substrate (Roche).Whole-mount Xenopus development, including the BMP, TGF-β/Nodal, MAPK/Erk, and PI3K/Akt signalling pathways. Based on the literature, we tested an anti-phospho-Smad1/5/8  and an anti-phospho-Erk antibody  wnt8 mRNA at the 1-cell stage, which is known to inhibit bmp4 expression We first identified a set of phospho-specific antibodies, which permit the monitoring of the activation state of several signalling pathways known to play major roles during early Having defined a set of antibodies allowing us to monitor the BMP, TGF-β/Nodal, MAPK/Erk, and PI3K/Akt signalling, we performed a time-course experiment to define the temporal dynamics of the activation state of these signalling pathways during early development. To this end, we collected embryos at stage 8 and 9 (blastula stages), 10, 10.25, 10.5, and 11 (gastrula stages), 12, 14, 20 (neurula stages) and 28 (early tadpole stage). After protein extraction, samples were analysed by Western blot assays to determine the phosphorylation status of the signalling molecules previously described . In accoThe time-course data of the various signalling pathways motivated us to design a large-scale gain-of-function screen aimed at identifying new modulators of the pathways during early embryonic development . For thein vitro transcribed mRNA in pools of eight from our full-length EST library, inject these pools into one to two stage embryos, and then identify pools that alter the phosphorylation state of phosphorylation state of any of the key signalling pathways via Western blot analyses. Once active pools have been identified, we would proceed by de-convoluting them in order to identify the single active clones contained within the positive pools  and a putative metalloprotease similar to hatching gland-like XheI protein (pool 12), to determine whether these two clones could alter the phosphorylation state of the signalling molecules in our screen. To simulate the same conditions of the full screen, we rearrayed the 12 clones together with their 7 corresponding neighbouring clones from their respective columns onto a new 96-well plate , TGas124h10, is on plate 044 at position H04 in the full-length clone library (information available at http://genomics.nimr.mrc.ac.uk). We therefore picked the eight clones in the “A04” column (from position A04 to H04) from plate 044, re-arrayed them into one column of a new 96-well plate, and extracted plasmid from these eight clones to make a single test pool. The same general strategy was used to generate the remaining eleven test pools used in the pilot screen  Xenopuscerberus strongly inhibited Smad2 phosphorylation but surprisingly had no effect on Smad1 phosphorylation  was identified as the sole regulator that had a dual effect of inhibiting Smad1/5/8 and activating Smad2  have orthologues with known function in other species, which suggests that the outcome of our screen may be applicable to other organisms.Having demonstrated the feasibility of our strategy, we then performed a screen on approximately a third of our full-length library (2880 of the 9216 clones) Xenopus . HoweverOf the 20 clones, only one showed an effect at the blastula stage, while all the others did not affect signalling until the gastrula stages . AlthougWe next analysed the category of molecules we had identified during the screen. The largest group  is comprised of signalling molecules, as expected . Howeverppp1r8 and fezf2, rhoa, and cdc42) had previously been shown to modulate Erk signalling F-box protein 43 , was able to induce a phosphorylation of Erk at the blastula stage. In Xenopus and mammalian models, Fbxo43 has been shown to mediate cytostatic arrest by inhibiting the anaphase-promoting complex (APC/C) rhoa and cdc42, seven other clones lead to the hyper-activation of Erk signalling at the gastrula stage (Transmembrane protein 209 (tmem209), also known as NET31, is a nuclear envelope protein. Elevated level of tmem209 promotes cell growth and human lung cancer Gem associated protein 2 (gemin2), also known as sip1, encodes a zinc finger/homeodomain containing protein and is highly expressed during early neural development gemin2 has been associated with motor neuron diseases Xenopus suggest that tbx6 is important for the formation of paraxial mesoderm and neural crest differentiation fgf8 expression, which might explain why tbx6 overexpression results in the activation of Erk foxh1 also caused an increased in Erk phosphorylation, suggesting a possible crosstalk between Nodal and MAPK/Erk signalling pathways. Fndc3a is a fibronectin-related protein, which is required for the adhesion between spermatids and Sertoli cells in testis, and mutations in the Fndc3a gene result in mouse infertility Eukaryotic translation initiation factor 3, subunit M (eif3m) is a translation initiation factor that is highly expressed in human cancer cell lines and colon cancer tissues and eif3m knockdown impairs cell proliferation protein phosphatase 1 regulatory subunit 8 (ppp1r8) was found to decrease Erk phosphorylation at stage 10.5. Ppp1r8 is an important regulatory subunit of the protein phosphatase 1 complex and its mis-expression could result in a global decrease in protein phosphorylation, including Erk Finally, more than half (55%) of the molecules identified in the screen modulated Erk activity, while a quarter modulated Akt (25%), and 15% of clones modulated either BMP or TGF-β/Nodal signalling. Only two clones showed multiple activities , the alpha catalytic subunit of cAMP-dependent protein kinase (PKA). Prkaca has been reported to be a crucial regulator in meiotic and mitotic arrest arginine-glutamic acid dipeptide (rere) gene, a member of the atrophin family of arginine-glutamic acid (RE) dipeptide repeat-containing proteins. Over-expression of such families of proteins triggers apoptosis, cytotoxicity, and neurodegeneration nuclear RNA export factor 1 (nxf1) gene, which belongs to a family of nuclear RNA export factor genes. Its protein product associates with NTF2-related export protein 1 to mediate transportation of nuclear mRNA into the cytoplasm ulk4 unc-51-like kinase 4, which is part of the ubiquitously expressed Ser/Thr-specific unc-51-like kinases family (ULKs). Mutations in the unc-51 gene family cause defects in axonal elongation and axonal structures in C. elegansULK4 has been associated with multiple myeloma, however its molecular mechanism remains unclear ppp1r8, which we described previously as the sole inhibitor of Erk found in the screen, also led to a decrease in Akt phosphorylation.We have also identified 5 clones that decreased Akt phosphorylation . These iWingless-type MMTV integration site family, member 8A (wnt8a), discussed previously. In addition, we discovered that overexpression of FEZ family zinc finger 2 (fezf2) also decreases Smad1 phosphorylation. Fezf2 has been shown to play an important role in forebrain, diencephalon, and olfactory placode development Fezf2 has been shown to regulate the differentiation of midbrain dopaminergic neurons, as well as axon projections between thalamus and cerebral cortex dipeptidase E (dpepe or pepE) is the last of this group. Dpepe retains strong sequence identity with bacterial PEPE gene, which cleaves N-terminal aspartyl peptides The screen also identified three genes modulating the phosphorylation level of Smad1/5/8 . One of Nodal-related 1 (xnr1), which had been identified for its ability in regulating left-right axis determination in XenopusDEAD (Asp-Glu-Ala-Asp) box polypeptide 55 (ddx55), a member of the DEAD box protein family characterized by the conserved motif Asp-Glu-Ala-Asp (DEAD) and a putative RNA helicases. The DEAD box protein family genes have been implicated in several cellular processes related to alteration of RNA secondary structure, and involved in diverse cellular functions including spermatogenesis, embryogenesis, cell growth, and division fezf2, mentioned previously as an inhibitor of Smad1/5/8 phosphorylation. We found that fezf2 overexpression led to an increase in Smad2 phosphorylation at gastrula stage. Notably, changes on the phosphorylation level of Smad1/5/8 and Smad2 caused by fezf2 mis-expression was similar to that caused by wnt8a mis-expression, suggesting that Fezf2 may promote canonical Wnt signalling.Finally, we identified 3 genes that change the phosphorylation level of Smad2 . The firin situ hybridisation. Half the Erk regulators (5 out of 11) and all of the BMP (3 out 3) and Nodal regulators (2 out 2) had regionalised expression patterns  together with the seven clones of the same column (grey dots). After that, the whole column (black and grey dots) were re-arrayed into one column of a new 96-well plate. Bacteria containing different clones were cultured individually and pooled together for subsequent plasmid extraction, linearization, and mRNA transcription to achieve 12 mRNA pools each containing 8 clones. mRNA pools were injected into X. laevis embryos at 1–2 cell stage and collected at specific stages as described in the main text. Collected embryos were homogenised and their protein contents extracted for subsequent Western blot analyses.(TIF)Click here for additional data file.


Drosophila morphogenetic protein Bicoid (Bcd) plays a transient or sustained role in activating its target genes to establish sharp expression boundaries during development.Morphogen molecules form concentration gradients to provide spatial information to cells in a developing embryo. Precisely how cells decode such information to form patterns with sharp boundaries remains an open question. For example, it remains controversial whether the Drosophila embryos.In this study, we describe a method to simultaneously detect Bcd and the nascent transcripts of its target genes in developing embryos. This method allows us to investigate the relationship between Bcd and the transcriptional status of individual copies of its target genes on distinct scales. We show that, on three scales analyzed concurrently—embryonic, nuclear and local, the actively-transcribing gene copies are associated with high Bcd concentrations. These results underscore the importance of Bcd as a sustained input for transcriptional decisions of individual copies of its target genes during development. We also show that the Bcd-dependent transcriptional decisions have a significantly higher noise than Bcd-dependent gene products, suggesting that, consistent with theoretical studies, time and/or space averaging reduces the noise of Bcd-activated transcriptional output. Finally, our analysis of an X-linked Bcd target gene reveals that Bcd-dependent transcription bursts at twice the frequency in males as in females, providing a mechanism for dosage compensation in early Drosophila embryos.Our study represents a first experimental uncovering of the actions of Bcd in controlling the actual transcriptional events while its positional information is decoded during development. It establishes a sustained role of Bcd in transcriptional decisions of individual copies of its target genes to generate sharp expression boundaries. It also provides an experimental evaluation of the effect of time and/or space averaging on Bcd-dependent transcriptional output, and establishes a dosage compensation mechanism in early  Regulation of transcription plays a pivotal role in many biological processes including pattern formation during embryonic development Drosophila embryos Bcd is a morphogenetic protein that forms a concentration gradient along the anterior-posterior (A-P) axis in early Here we describe experiments to simultaneously detect Bcd protein and the nascent transcripts of its native target genes inside the nuclei of developing embryos. We generate large experimental datasets to probe concurrently the input-output relationship between Bcd and the transcriptional status of its target genes on three distinct scales, embryonic , nuclear  and local . Our results reveal an association between transcriptional burst events and Bcd concentrations on all three scales. These results underscore a sustained role of Bcd in the transcriptional decisions of its target genes as embryonic development progresses. We also provide experimental evidence suggesting that, consistent with theoretical studies Drosophila embryos. This method combined fluorescence antibody staining to detect Bcd in situ hybridization (FISH) using intronic probes to detect target genes' nascent transcripts hunchback (hb) and orthodentical (otd), for our study. hb as distinct “intron dots” (green), and the nuclear envelope (red) in an embryo at early nuclear cycle 14. The nuclear marking allows us to extract data from within the nucleus, a location relevant to the input-output relationship in transcription. It also allows us to adjust our algorithms for detecting intron dots and to help estimate errors in such detections , the corresponding Bnuc, and normalized intensities of identified hb intron dots for all the captured nuclei from our experimental embryos.In addition to the hb intron dots within a bin of nuclei along the A-P axis as ρ = n/N, where n is the number of detected intron dots and N is the number of nuclei. To quantify the input-output relationship between the nuclear Bcd concentration and the nascent hb transcripts on the embryonic scale, we plot the number density of hb intron dots ρ either as a function of the normalized A-P position (x/L) (Bnuc) of the bins along the A-P axis   or as a A-P axis . The intposition  resemblehb intron dot data with Hb protein data, both obtained from flattened embryos at a comparable developmental time. Bnuc and x/L in a 3-D scatter plot (see hb intron data). In our analysis, we evaluated the following three specific properties that describe the target gene responses to the Bcd gradient: the boundary positions detected as either the intron dot density or nuclear Hb protein intensities, their respective Hill coefficient values and the noise levels.To investigate the relationship between gene transcriptional status and transcriptional products in response to the Bcd gradient in embryos, we compared our x/L where ρ is half maximal), 0.43±0.02 , is comparable to the boundary position detected by Hb protein, 0.44±0.01 . Second and importantly, the ρ profiles (ρ profiles (η = σ/ρ) is ∼80% at the anterior expression regions . Recent theoretical studies suggest that averaging of target gene products over space and/or time can reduce the noise of transcriptional responses of Bcd target genes Our comparative analysis reveals two results. First, the detected intron dot density boundary position . An analysis using data from individual embryos, thus eliminating between-embryo fluctuations in nuclear Bcd concentrations, revealed similar results  that transcends its morphogenetic role in instructing A-P patterning , differences in Bcd concentrations of individual nuclei within the bins represent noise that is encountered by the system. To determine whether such Bcd concentration noise may be propagated to the transcriptional status of its target genes, we compared the nuclei (within bins) that either have or lack intron dots, referred to as active or inactive nuclei, respectively.  results . Togethetterning . They de2 s−1While the diffusion constant of Bcd remains highly controversial, independent experiments support the existence of a specie of Bcd molecules that have a limited mobility inside the nucleus, with a diffusion coefficient in the order of ∼0.3 µmBnuc)? For our analysis, we plot histograms (Bpix inside the inactive nuclei (green), the active nuclei (blue), or the areas of the detected intron dot sites (red) at different A-P positions. These histogram data reveal two results that are consistent among different bins (x/L) in the anterior hb expression regions of the embryos. First, the peak locations of the probability density function  are higher for the active nuclei  than their inactive counterparts . The distributions of pixel intensities from the active and inactive nuclei are also distinguishable from each other . These findings are simply another way of expressing the results already presented in Bnuc is different between the active and inactive nuclei at given A-P positions.To extend our investigation of the sustained role of Bcd in transcriptional decisions of its target genes, we analyzed Bcd intensities at the detected intron dot sites. As a transcriptional activator, Bcd molecules are expected to bind specific DNA sites in target gene enhancers and take residence there for a period of time necessary for eliciting functional responses. We sought to ask a simple question that can further determine whether Bcd has a direct and causal relationship with the transcriptional status of its target genes in embryos: are the local Bcd concentrations  different from their corresponding nuclear environment  are distinguishable from each other . If the detected intron dot sites were randomly located  inside the active nuclei, one would have expected these two profiles to be similar to each other given that the total number of pixels inside the areas of all the detected intron dots within individual bins is sufficiently large . To further evaluate this point, we plot the Bpix probability ratio of intron dot sites to active nuclei  have significantly higher Bnuc than their inactive counterparts on average . Our results show that, despite a two-fold difference in the copy number of otd in males and females, the otd intron dot density as a function of either x/L or Bnuc is approximately the same . These results suggest that, for otd, dosage compensation operative during embryogenesis allows this X-linked gene to burst in transcription at twice the probability in males as in females.Since the same . In addiBnuc profile along the A-P axis) or not . These results suggest that the input-output relationship between Bcd and its target gene transcription, a relationship governed by the contributing chemical reactions, transcends the morphogenetic role of Bcd in A-P patterning. A comparison between our hb intron dot data and Hb protein data reveals that both the boundary steepness and the expression precision exhibit marked differences . If Bcd does indeed act as a direct and sustained input for transcriptional decisions, one would have expected this property. Given a finite number of transcription factor molecules available inside a nucleus Our large experimental datasets reveal that the Bcd intensities detected at the intron dot sites are higher than their nuclear environment  were constructed using PCR products amplified from Drosophila genomic DNA with the following primers: hb forward primer 5′-GCCATTACCAAGTGTCTCCATTTTG-3′; hb reverse primer 5′-AGAAAACAGAGAGAGTGGGGTTAGT-3′; otd forward primer 5′-AGATGTGCCATCCAGAGTACTAACT-3′; and otd reverse primer 5′-GAGGGCATTTCGCCTAATTAAACCA-3′. Both intronic probes were labeled with digoxigenin-11-dUTP (Roche Applied Sciences) and synthesized using the MAXIscript In vitro Transcription Kit (Ambion) according to manufacturer's instructions.Plasmids containing intronic sequences for 1118w embryos collected at 25°C were fixed, rehydrated in a stepwise manner to PBST , post-fixed in 10% formaldehyde for 20 minutes, and washed in the Hybridization buffer B  after being washed first in PBST. Embryos were then pre-hybridized for 1 hour in the Hybridization buffer A  at 60°C, followed by hybridization overnight at 60°C in pre-warmed Hybridization buffer A containing the probes. Hybridized embryos were washed in Hybridization buffer B at 60°C, followed by washing 5 times in PBST at room temperature and incubation in Roche blocking buffer for 30 minutes. Embryos were incubated overnight at 4°C in Roche blocking buffer containing mouse anti-digoxigenin monoclonal primary antibody  and rabbit anti-Bcd antibody , both of which had been presorbed. They were then washed in PBST 5 times at room temperature, incubated in PBST with Roche blocking buffer for 30 minutes, followed by incubation in Roche blocking buffer with Alexa Fluor 488-goat-anti-mouse secondary antibody  and Alexa Fluor 647-goat-anti-rabbit secondary antibody  for 1 hour at room temperature. Nuclear membrane was stained with Alexa Fluor 555-conjugated wheat germ agglutinin  for 30 minutes at room temperature. After washing in PBST, embryos were mounted, without bridge, in ProLong® Gold antifade reagent (Invitrogen) for Confocal imaging.FISH combined with protein staining on whole-mount embryos was performed as follows. 1–4 hour z-sections for each embryo. Pinhole size was set such that the optical slice was 1 µm for all channels. The detector gain for each channel was set by, using an embryo with the characteristic expression pattern, adjusting the gain until only several pixels were saturated. Amplifier offset was adjusted for each channel so that pixels away from the embryo were nearly zero. In our experiment, the imaging of different embryos did not require adjustments of the setting between embryos or between slides; thus different embryos analyzed in our study have the same conversion factors between the captured fluorescence intensities and the number of detected molecules. In our analysis, we used flattened embryos and captured on average 6 z-sections for each region of an embryo taken in 0.5 µm intervals, which effectively captured all the intron dots in each nucleus. The images were 8-bit  in depth and with 2048×2048 resolution . The captured z-stack images were projected and stitched together to generate the image data files of whole embryos for further analysis.Embryos were imaged on an inverted Zeiss LSM 510 Confocal microscope with plan-neofluar 40×1.3 oil objective. Embryos at early nuclear cycle 14 were chosen to be imaged according to the nuclear density and shape. We imaged embryos that were “flattened” to maximize the number of nuclei that could be captured by Confocal hb intron dot density (ρ) between two expression regions of the embryo (30%∼40% and 20%∼30% of embryo length) as a function of the pixel intensity threshold setting  the is 0.109 ; and 3) ron dots . FinallyFigure S1Establishing the threshold setting for detecting intron dots. (A) Shown is the difference in hb intron dot density ρ between two expression regions (x/L = 0.3∼0.4 and x/L = 0.2∼0.3) as a function of the pixel intensity threshold used in detecting the intron dots. In this test , the threshold of intron dot size is set at the optimized value of 4 (see hb intron dots per nucleus as a function of intensity threshold in the non-expression region of x/L = 0.55∼0.65. (C) Shown is the ratio of identified intron dots outside the nuclear regions to those inside the nuclear regions as a function of intensity threshold. Data shown are extracted from all nuclei identified from 14 embryos. (D) Shown is the percentage of nuclei with more than 2 hb intron dots out of all nuclei identified as a function of intensity threshold. Data were extracted from all nuclei identified from 14 embryos.of 4 see . The das(TIF)Click here for additional data file.Figure S2Single-embryo analysis. Using data extracted from single embryos, thus eliminating the between-embryo component from the extrinsic-like fluctuations, we analyzed the enrichment of Bcd concentration within hb transcriptionally-active nuclei and at the locations of nascent hb transcripts. Shown are two ratio profiles as a function of A-P position at the anterior expression region: the mean Bcd intensity at the intron dot locations over the mean Bcd intensity within active nuclei (red), and the mean Bcd intensity within inactive nuclei over the mean Bcd intensity within active nuclei (blue). Consistent with data from 14 wt embryos, active nuclei generally have more Bcd molecules than inactive nuclei in a single embryo, and the intron dot sites generally have higher Bcd intensities than the environment of the active nuclei in the same embryo.(TIF)Click here for additional data file.Figure S3Properties of detected otd intron dots. (A) Shown is a merged image of a wt embryo at early nuclear cycle 14 detecting the nuclear envelope (red), Bcd proteins (blue) and nascent otd transcripts as intron dots (green). Shown on the right is a magnified view of a section of the expression region. (B) Shown is the radial distribution of otd intron dots. Data shown here were extracted from one single wt embryo with 2,449 identified nuclei (with a mean diameter l = 5.88±0.83 µm) and 251 detected intron dots. There are several intron dots that are outside the illustrative mean nuclear boundary since all nuclei are not perfectly round , and each error bar is one standard deviation among the nuclei for a single embryo.(TIF)Click here for additional data file.


Saccharomyces cerevisiae MKP Msg5 binds the MAPK Slt2 within the cell wall integrity (CWI) pathway through a distinct motif (IYT). Here, we demonstrate that the IYT motif mediates binding of the Msg5 paralogue Sdp1 to Slt2 as well as of the MKP Pmp1 to its CWI MAPK counterpart Pmk1 in the evolutionarily distant yeast Schizosaccharomyces pombe. As a consequence, removal of the IYT site in Msg5, Sdp1 and Pmp1 reduces MAPK trapping caused by the overexpression of catalytically inactive versions of these phosphatases. Accordingly, an intact IYT site is necessary for inactive Sdp1 to prevent nuclear accumulation of Slt2. We also show that both Ile and Tyr but not Thr are essential for the functionality of the IYT motif. These results provide mechanistic insight into MKP-MAPK interplay and stress the relevance of this conserved non-canonical docking site in the regulation of the CWI pathway in fungi.Dual-specificity MAPK phosphatases (MKPs) are essential for the negative regulation of MAPK pathways. Similar to other MAPK-interacting proteins, most MKPs bind MAPKs through specific docking domains known as D-motifs. However, we found that the  MAPKs contain a highly conserved TXY motif in the kinase activation loop in which both Thr and Tyr residues must be phosphorylated to achieve kinase activation Saccharomyces cerevisiae has five MAPK signalling pathways involved in mating, pseudohyphal/invasive growth, osmoregulation, ascospore formation and cell wall integrity (CWI) S. cerevisiae Slt2 operate in other fungi, like Pmk1 in Schizosaccharomyces pombe. Not only MAPKs but many other components participating in fungal CWI pathways are conserved S. cerevisiae Msg5 and its paralogue Sdp1, both of which dephosphorylate the MAPK Slt2 S. pombe, the MKP Pmp1 is responsible for dephosphorylating the CWI MAPK Pmk1 All eukaryotic cells possess multiple MAPK pathways, which coordinately regulate many physiological processes. S. cerevisiae. We have recently shown that, whereas the interaction with the CD domains of the mating MAPKs Fus3 and Kss1 involves a canonical Msg5 D-domain, a novel motif (I102Y103T104) in Msg5 is mediating binding to the CWI MAPK Slt2 and its pseudokinase paralogue Mlp1 S. cerevisiae Msg5, we have studied the relevance of this motif for other yeast MKPs. Here we show that the IYT site is also involved in the interaction of Sdp1 to Slt2, and in Pmp1 binding to its partner MAPK Pmk1 in S. pombe, highlighting the importance of this motif in the interaction of MKPs with fungal CWI MAPKs.The MKP Msg5 is not only regulating the CWI but also the mating pathway in S. cerevisiae strains used in this work were DD1-2D (MATaura3 his2 trp1 leu2 ade1 msg5-1Δ::LEU2) MATa, ade2-101 trp1-63 leu2-1 ura3-52 his3-Δ200 lys2-801), BY4741  and Y00897  from Euroscarf . Yeast cultures were performed as reported previously The S. pombe strains employed were MI200 (+ pmk1-HA6H:ura4+h) + pmk1::KanRh) and MI212 (+ pmp1::KanR pmk1-HA6H:ura4+h) + pmp1::KanR pmp1-GST:leu1+ pmk1-HA6H:ura4+h), DP410 (+ pmp1::KanR pmp1(C158S)-GST:leu1+pmk1-HA6H:ura4+h), DP420 (+ pmp1::KanR pmp1(I10A Y11A T12A)-GST:leu1+ pmk1-HA6H:ura4+h), and DP430 (+ pmp1::KanR pmp1(I10A Y11A T12A C158S)-GST:leu1+ pmk1-HA6H:ura4+h) (this work). All strains are ade6-M216 leu1-32 ura4-D18. They were grown at 28°C in rich medium YES with 2% glucose, and supplemented with adenine, leucine, histidine or uracil , depending on specific requirements The S. cerevisiae cells, pEG(KG)-Mlp1 was constructed by PCR amplifying the corresponding MLP1 region using pET15b-Mlp1 GGATCCATGGCGACTGACACCGAGAG-35- TCGC (BamHI site is underlined) and GTCGACTTAGTTAACACCCTGAAATGAATTAGAG-35- TCGC . The BamHI-SalI digested PCR product was sub-cloned into pEG-KG  General DNA methods were performed using standard techniques. In order to express Mlp1 fused to GST in S. cerevisiae, the plasmid YCplac22Sdp1m was constructed. To this end, primers PROSdp1 AAGCTTGGTGGCCCAAAGTAACACTTC-35-CATG (HindIII site is underlined) and RSdp1 GGATCCCGGTACTTTTCTATAACTGTTGGC-35-TCGC (BamHI site is underlined) were used to amplify a fragment including the complete ORF and starting 1 Kb upstream of the initiation codon of SDP1. This fragment was cloned into pGEM-T to obtain pGEMT-PSDP1. The HindIII-BamHI fragment was inserted into pRS305myc HindIII-SacI DNA fragment from pRS305SDP1m carrying the 6MYC epitope was sub-cloned into YCplac22  to give YCplac22SDP1m. The YCplac22Sdp1IAYATAm plasmid was generated by site-directed mutagenesis of YCplac22SDP1m with the oligonucleotides FSDP1D 5-GTAGCCATAATCTCGCATGAACGCAGCTGCATCACCCACGCGAACACCGAAC-3 and RSDP1D 5- GTTCGGTGTTCGCGTGGGTGATGCAGCTGCGTTCATGCGAGATTATGGCTAC-3.To express Sdp1-Myc in IAYATA, the SDP1 ORF was PCR-amplified with primers FYSDP1 AAGCTTCTTGTAGCCATAATCTCGCATG-35-CATG (HindIII site is underlined) and RYSDP1 GAGCTCACTAGGTAATACGACTCACTATAG-35-TCGC (SacI site is underlined) using YCplac22Sdp1m and YCplac22Sdp1IAYATAm plasmids as templates and then the HindIII-SacI digested fragments were sub-cloned into pYES2 (2μ URA3 GAL1) (Invitrogen). pYES2SDP1C140Amyc and pYES2SDP1C140A-IAYATAmyc plasmids were generated by site-directed mutagenesis of pYES2SDP1myc and pYES2SDP1IAYATAmyc, respectively, following the method previously described 5- GAAGATACTGATACATGCCCAATGTGGGCTCTCGAGATCCGCAACG-3 and RDP1C 5- CGTTGCGGATCTCGAGAGCCCACATTGGGCATGTATCAGTATCTTC-3. pYES2SDP1IAmyc, pYES2SDP1YAmyc and pYES2SDP1TAmyc were obtained amplifying the SDP1 ORF with the reverse primer RYSDP1 and the following forward primers FYSDP1IAAAGCTTCTTGTAGCCATAATCTCGCATGAACGCATAC-35-, FYSDP1YAAAGCTTCTTGTAGCCATAATCTCGCATGAACGCATACACATCACCCACG-35-CATG and FYSDP1TAAAGCTTCTTGTAGCCATAATCTCGCATGAACATATACGCATC-35- (HindIII sites are underlined), respectively, using pYES2SDP1myc as template. The HindIII-SacI digested fragments were sub-cloned into pYES2.To overexpress Sdp1 and Sdp1IAYATA-GST were obtained using pETGEXCT plasmid SDP1 and IAYATASDP1 were amplified with upstream primers CCATGGCCATGAACATATACACATCACCCACG-35- CATG or CCATGGCCATGAACGCAGCCGCATCACC-35-CATG, respectively, (NcoI sites are underlined) and the downstream primer GAGCTCCGGTACTTTTCTATAACTGTTGGC-35-TCGC (SacI site is underlined). PCR products were sub-cloned into pGEM-T, and then the NcoI-SacI fragments carrying SDP1 and SDP1IAYATA from the generated plasmids were sub-cloned into NcoI-SacI sites of pETGEXCT. To produce recombinant Slt2 and Mlp1 His-tagged proteins, pET15B-derived plasmids pET15B-Slt2-His and pET15B-Mlp1-His were used as described previously Recombinant Sdp1-GST and Sdp1EcoRI-SacI fragment from pEG(KG) into YEp352 C319AGST expressing Msg5-GST and Msg5C319A-GST, respectively, were generated in two steps. Firstly, the BamHI fragment carrying the promoter and the corresponding ORFs of MSG5 from YCplac22MSG5m or YCplac22MSG5C319Am SmaI fragment from YEp352GST-bearing GST was sequentially cloned at the SmaI site. The YEp352MSG5C319A-IAYATAGST plasmid was obtained by site-directed mutagenesis on YEp352MSG5C319AGST using the mutagenic primers 5′-AGCGAGGCCTCTGCAGCTGCACTACCAACATCTTTGAAGAACCGAACTG-3′ and 5′-AGATGTTGGTAGTGCAGCTGCAGAGGCCTCGCTTCGCCTCATCGACAGC-3′.The YEp352GST plasmid was obtained by cloning the GST-encoding MLP1 transcriptional induction, the episomic vector YEp352, bearing an MLP1-GFP fusion (pMLP1-GFP) MLP1 promoter to the lacZ gene SLT2-GFP In order to determine +pmp1 ORF plus regulatory sequences were amplified by PCR using S. pombe genomic DNA as a template, employing the 5′-oligonucleotide Ppmp1X-F (TCTAGACTCCAGCATCACAGTCTTTTCCTTCTATAT), which hybridises at a position ∼700 bp upstream of the +pmp1 ATG start codon and contains an XbaI site (underlined), and the 3′-oligonucleotide Ppmp1B-R (GGATCCAGAAGCATCATTACTTATACTGCCGCTATAT), which hybridises at the 3′ end of pmp1+ ORF (except the stop codon) and incorporates a BamHI site. The resulting PCR fragment was digested with XbaI and BamHI and cloned into the integrative plasmid pIL-GST To construct the integrative plasmid pIL-pmp1:GST, the C158S):GST was obtained by site-directed mutagenesis employing pIL-pmp1:GST as template and the mutagenic oligonucleotides PpmpC158S-F (GTTTTAATTAATAGTCAAATGGGCA) and PpmpC158-R (TGCCCATTTGACTATTAATTAAAAC). To construct the plasmid pIL-pmp1(I10A Y11A T12A):GST, the mutagenic oligonucleotides used were PpmpAAA-F (ATGTCTCAAAAACTACCTCCCTTAAAAGCTGCCGCCTCCCAACTTCCCCTGGTTTC) and PpmpAAA-R (GAAACCAGGGGAAGTTGGGAGGCGGCAGCTTTTAAGGGAGGTAGTTTTTGAGACAT). The integrative plasmid pIL-pmp1(I10A Y11A T12A C158S):GST was prepared using pIL-pmp1(I10A Y11A T12A):GST as the template and the mutagenic oligonucleotides PpmpC158S-F and PpmpC158-R. In all cases, the mutated +pmp1 ORFs plus regulatory sequences were amplified by PCR with the oligonucleotides Ppmp1X-F and Ppmp1B-R, digested with XbaI and BamHI, and cloned into pIL-GST, as above. Finally, the resulting plasmids were digested at the unique NruI site within +leu1, and the linear fragments were transformed into strain MI212. Transformants +leu1 were obtained and the correct integration of the fusions was verified by both PCR and Western blot analysis.The integrative plasmid pIL-pmp1(E. coli strain Rosetta DE3 (Novagen). Cells were collected and lysed by sonication in PBS buffer containing 2 mM PMSF, 1 mM DTT, 1 mM EDTA, and 1 mg/ml Lysozyme in the presence of a protease-inhibitor cocktail (Roche). Extracts were clarified by centrifugation and then stored at −80°C. Budding and fission yeast extracts were obtained as previously described in Recombinant GST- or His-tagged proteins were expressed in the in vivo binding assays, cells were collected and lysed as above in lysis buffer lacking SDS and NP40. Yeast lysates were incubated with glutathione-Sepharose beads  for two hours. Beads were washed extensively with the same buffer, resuspended in SDS loading buffer, and proteins were analysed by SDS-PAGE and immunoblotting as described In vitro binding assays were performed by mixing E. coli extracts containing GST or GST-fused proteins with E. coli extracts bearing the corresponding His-tagged proteins, and then processed as above.For 2+-NTA-agarose beads (Qiagen) and glutathione-Sepharose beads , respectively, as described previously Purification of Pmk1-HA6H and Pmp1-GST fusions was performed with NiIn experiments carried out with budding yeast, immunodetection of Glucose-6-phosphate dehydrogenase (G6PDH) and Myc-tagged proteins was carried out using polyclonal anti-G6PDH (Sigma) and monoclonal 4A6 (Millipore) or 9E10 (Santa Cruz Biotechnology) antibodies, respectively. Polyclonal anti-phospho-p44/p42 MAPK (Thr202/Tyr204) , anti-GST (Santa Cruz Biotechnology) and anti-His antibodies (Sigma) were also used as described previously In experiments performed with fission yeast, dual phosphorylation in Pmk1 was detected with polyclonal anti-phospho-p42/p44 as above, whereas total Pmk1 was detected after incubation with mouse monoclonal anti-HA antibody .The immunoreactive bands were revealed with an anti-mouse-HRP-conjugated secondary antibody (Sigma) and the ECL system . GST fusions were detected with a goat anti-GST-HRP conjugated polyclonal antibody .β-Galactosidase activity was determined according to For Mlp1-GFP analysis, cells were collected, washed twice with PBS, and then analysed by flow cytometry in a Guava easyCyte flow cytometer, acquiring green fluorescence through a 488 excitation laser and a 525/30 BP emission filter (BFP). The marker was set using yeasts that do not express GFP.SLT2-GFP, exponentially growing cultures were centrifuged at 3000 r.p.m., washed once with water and observed. Cells were examined under an eclipse TE2000U microscope (Nikon) and digital images were acquired with an Orca C4742-95-12ER charge-coupled-device camera (Hamamatsu Photonics) and Aquacosmos Imaging Systems software.For the visualisation of GFP on live yeast cells containing pRS425-S. cerevisiae and Pmp1 from the evolutionarily distant fission yeast S. pombe . Therefore, in order to analyse the relevance of the IYT motif for Sdp1 function, we studied the effect caused by the expression of high doses of Sdp1 and Sdp1IAYATA in S. cerevisiae. To this end, we overproduced the Myc-tagged versions of the corresponding SDP1 alleles from the GAL1 promoter. Overexpression of a catalytically inactive form of Sdp1 has been shown to result in the accumulation of phosphorylated Slt2 C140A, which bears a mutation at the conserved cysteine that is known to abolish the catalytic activity within all PTPs, and the double mutant Sdp1C140A-IAYATA that is also affected in the IYT site. To easily analyse their down-regulatory impact on CWI signalling, the overexpression experiments were carried out in a gas1Δ mutant strain, which presents a constitutively activated CWI pathway due to the lack of beta-1,3-glucanosyltransferase activity, which is required for cell wall assembly RLM1. To this end, a reporter transcription assay based on the expression of lacZ under the control of the cell wall damage-inducible Rlm1-dependent MLP1 promoter was used gas1 mutant. Mutation of the IYT motif within Sdp1 did not result in any apparent differences with wild type Sdp1 regarding the level of phosphorylated Slt2. In contrast, MLP1 expression was significantly higher in comparison with that of cells expressing wild type Sdp1, indicating a reduced function of the mutant Sdp1IAYATA version on Slt2 activity. As expected, expression of Sdp1C140A caused an accumulation of phosphorylated Slt2  to Pmk1 resulted in abrogated function when the inactive phosphatase version also lacked the IYT motif (Pmp1C158S-IAYATA). Similar to the results obtained with both Msg5 and Sdp1 in budding yeast, expression of inactive Pmp1C158S induced a strong raise in the level of phosphorylated Pmk1  was responsible for binding to Slt2 and its paralogue Mlp1 in a CD-independent manner S. cerevisiae and Pmp1 from S. pombe precludes interaction with Slt2 and Mlp1, and with Pmk1, respectively. Second, substrate-trapping inactive versions of ScMsg5, ScSdp1 and SpPmp1 lacking this motif have a reduced ability to trap their target CWI MAPKs. Third, whereas inactive Sdp1 prevents Slt2 from concentrating in the yeast nucleus, this Sdp1 version lacking the IYT motif does not significantly alter Slt2 localisation. Fourth, the IYT sequence is present in the non-catalytic region of many fungal Msg5 orthologues. Although most of them belong to genera close to Saccharomyces, this motif is also born by the MKP Pmp1 of the evolutionarily distant yeast S. pombe. This suggests that this docking motif was present in ancestral MKPs and has probably been maintained through evolution in some MKPs for a specific purpose, namely the regulation of CWI MAPKs.It is well accepted that the specificity and efficiency of action of MAPK phosphatases (MKPs) on their cognate MAPKs are controlled by docking interactions. We found in a previous work that the interaction of the S. cerevisiae possesses two MKPs S. pombe MKP Pmp1. The short N-terminal regulatory region containing an IYT motif but lacking the typical D-domain in Pmp1 is the most likely reason for its exclusive targeting to the CWI-MAPK Pmk1.In contrast to other fungal species, S. cerevisiae Msg5 for binding to both Slt2 and Mlp1 Although the crystal structure of Sdp1 has been previously determined S. cerevisiae or Pmk1 in S. pombe, from the trapping complex, allowing signal propagation. In view of these results, the analysis of MAPK phosphorylation and specific transcriptional induction upon overexpression of a catalytically inactive MKP is a powerful approach for identification of target MAPKs of such MKP. This strategy can be especially useful for MKPs whose deletion or overexpression does not give rise to an evident phenotype, like Sdp1. Msg5 has proven to be a more potent phosphatase than Sdp1, controlling Slt2 phosphorylation both under basal and stimulating conditions sdp1Δ mutant strain analysed (data not shown). Moreover, recent large-scale analysis in S. cerevisiae comparing the impact of every single phosphatase gene deletion on either the global expression profile or the phosphoproteome also showed that the absence of Sdp1 affects gene transcription and protein phosphorylation to a much lesser extent tan Msg5 sdp1Δ mutants under cell wall stress conditions (data not shown), while an msg5Δ mutant is hypersensitive to this stress msg5Δ cells supports the idea that this phenotype is due to an up-regulated CWI pathway response.Mutations that reduce the catalytic activity of protein phosphatases without affecting their substrate affinity have been named “substrate-trapping”, due to their ability to form a stable enzyme-substrate complex In summary, our work provides evidence on the conservation of an interaction motif in MKPs for selective binding to CWI MAPKs. Furthermore, we unravel the essential residues within this MKP motif for binding to MAPKs. Since various MAPKs can be regulated by the same MKP and, conversely, several MKPs are able to dephosphorylate a particular MAPK, the existence of different mechanisms mediating binding of these proteins ensures accurate signal transduction. Our findings reveal the high flexibility of these proteins to differentially regulate structurally similar MAPKs.


Signaling through the T cell receptor (TCR) initiates adaptive immunity and its perturbation may results in autoimmunity. The plasma membrane scaffolding protein LAT acts as a central organizer of the TCR signaling machinery to activate many functional pathways. LAT-deficient mice develop an autoimmune syndrome but the mechanism of this pathology is unknown. In this work we have compared global dynamics of TCR signaling by MS-based quantitative phosphoproteomics in LAT-sufficient and LAT-defective Jurkat T cells. Surprisingly, we found that many TCR-induced phosphorylation events persist in the absence of LAT, despite ERK and PLCγ1 phosphorylation being repressed. Most importantly, the absence of LAT resulted in augmented and persistent tyrosine phosphorylation of CD3ζ and ZAP70. This indicates that LAT signaling hub is also implicated in negative feedback signals to modulate upstream phosphorylation events. Phosphorylation kinetics data resulting from this investigation is documented in a database (phosphoTCR) accessible online. The MS data have been deposited to the ProteomeXchange with identifier PXD000341. The T cell receptor (TCR) plays a central role in adoptive immunity through signaling processes that dictate T cell fate during development and upon exposure to antigen. Deregulation of the underlying signaling circuits may cause immundeficiency and autoimmune disorders. Signals triggered by TCR ligation with peptide-MHC complex are translated into intracellular phosphorylation events by Src and Syk family protein tyrosine kinases (PTK) LCK and ZAP70, respectively. Tyrosine phosphorylation of LAT (The Linker for Activation of T cell) allows the recruitment of signaling protein complexes that activate all major signaling pathways, thus regulating T cell functions LAT is indispensable for T cell maturation during thymus development Mass spectrometry (MS)-based quantitative phosphoproteomics is a powerful approach to decipher functional signaling networks Main experimental designs are schematically depicted in 0) and L-Lysine (K0) (CK Gas Products Ltd.), or L-Arginine-13C614N4 (R6)  and L-Lysine-12C614N2 -2H4 (K4) (Isotec) or L-Arginine-13C615N4- (R10) and L-Lysine-13C615N2 (K8)  at a final concentration of 0.29 mM L-Arginine and 0.219 mM L-Lysine and filter sterilized . Jurkat cells were grown at 37°C in a humidified 5% CO2-containing atmosphere for 5–7 cell doublings times in labeling media A, B or C, containing either light: R0, K0 (A) or medium: R4, K6 (B) or heavy: R8, K10 (C).Custom-made RPMI 1640 medium lacking L-Arginine and L-Lysine (Thermo Scientific) was supplemented with 10% dialyzed FCS (Gibco) and either L-Arginine , 1 mM Na3VO4, protease inhibitor cocktail (Roche), phosphatase inhibitor cocktail 1 and 2 (Sigma)). Lysates were cleared by centrifugation at 14,000×g for 5 min and the four samples of each time point were pooled. For each time series (TS1∶0 min (A), 0.5 min (B), 5 min (C) and TS2∶10 (A), 0.5 (B), 20 (C) min), the corresponding time points lysates were mixed in a 1∶1∶1 ratio of protein content (measured by the NanoDrop® ND-1000 UV-Vis Spectrophotometer). The two stimulation series had one common and two varying time points, resulting overall in five time points per experiment . Stimulation efficiency of each individual sample was determined by western blot using anti-phosphotyrosine (pY) . JCaM2.5 cells and JCaM2.5 cells stably transfected with LAT were stimulated with 10 µg/ml CD3 antibody (UCHT1) for varying times, cells lysed, and lysates subjected to total ZAP70 and ZAP-pY493 (Santa Cruz Biotech. Inc.) immunoblot.Two sets of 1×10Total protein in the two time series were precipitated overnight in 0.1 M ammonium acetate in chilled methanol. The next day, samples were centrifuged at 6000 g for 30 min and washed 3 times with chilled methanol and 2 times with chilled acetone. Total Protein pellet was re-suspended in 8 M urea, 15 mM Tris pH 8. Protein mixture was reduced in 10 mM DTT , 30 min 37°C and then alkylated with 50 mM IAA at 37°C in the dark.After dilution with 15 mM Tris, pH 8, to 2 M urea, trypsin was added in 1/100 enzyme/protein ratio and the samples were incubated overnight at 37°C.Tryptic peptide mixture was acidified prior to separation by strong cation exchange (SCX) chromatography as described elsewhere Titanium oxide enrichment was performed as described in details elsewhere 18 material  was packed into a spray emitter  using a high-pressure packing device . Mobile phase A consisted of 0.1% formic acid in water and B of 80% ACN, and 0.1% formic acid in water.LTQ-Orbitrap mass spectrometer (ThermoElectron) was coupled online to a nano-LC Ultimate 3000 (Dionex). To prepare the analytical column, C6 ions for maximum injection time of 500 ms. For MS2 scans, maximum filling was 1×104 ions with maximum injection time of 150 ms. Minimum signal was 1000 counts and normalized collision energy set to 35 with activation time of 30 ms. Dynamic exclusion was set to 60 seconds, repeat count 1, exclusion mass width relative to low and high was 20 ppm, expiration count and S/N threshold were 2 and 3.0, respectively, and multistage activation was enabled with neutral loss of phosphoric acid from 2–5 charge states.The 5 most intense peaks of the MS scan were selected for MS2 scans in the ion trap  from Mascot search engine in combination to raw, and protein sequence data files to perform identification, quantitation, integration of the results, statistical validation and estimation of FDR (set to 0.01%) and finally to write results in various output files (.txt). For phosphopeptides, the site assignment was scored by MaxQuant  was retrieved from the MaxQuant ‘evidence.txt’ file. SILAC ratios for the time-points in each sub-series were combined by normalizing to the common 0.5 min time point, present as the medium labeled sample (B). Phospho-peptides and sites were re-mapped to the complete Ensembl Human protein sequence database, to ensure complete representation of ambiguity in the site-to-protein mapping.To generate protein-protein interaction networks a custom perl script was used to map identified phospho-sites to interaction data from STRING-DB. For the set of all phospho-sites, protein mappings were assembled and reduced to gene-level identifiers. Interactions between the set of phospho-proteins with a STRING interaction score >0.7 were retrieved from STRING-DB. Topology of the phospho-protein networks was examined using custom Perl scripts. Random sampling of subset networks from both the phospho-proteins identified in this study and the set of all STRING interactions was performed. Distributions of edge/node number and network degree were obtained. Cytoscape2.8.1 software https://cellline.molbiol.ox.ac.uk/phos/cgi-bin/PhosphoTCR.cgi.A MySQL database was created to store the analysis results and a set of custom Perl-cgi scripts written to enable interaction with the data via a web interface. All MS/MS spectra used to generate the peptide data within the database are available as static images  via the PRIDE partner repository The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium . To determine a threshold above or below which a change in phosphopeptide abundance could be considered statistically significant, we computed a global P-value for fold changes from two data sets obtained from cells independently stimulated in identical conditions (S6). This procedure established a threshold of ±0.42 fold change over background with 95% confidence interval . In all experiments a total of 11,454 unique phosphorylation sites were identified in 2336 proteins. Phosphorylation kinetics data resulting from this investigation is documented in a database (phosphoTCR) that can be accessed online (see Methods). As expected, serine phosphorylation (pS) represents the largest fraction of identified sites (79%) followed by threonine  and tyrosine   and its LAT-deficient variant (JCam2.5) cell lines using SILAC approach and quantified relative changes in phosphopeptide abundance at 0.5, 5, 10 and 20 min after TCR stimulation  Fig. 1B. and Methods). Phosphorylation-specific networks in intact  n intact Fig. 2A To assess the specificity of the generated networks we computed the frequency of edges and average neighbors in subset interaction networks, sampled from the experimental data and randomly selected proteins. The number of edges Fig. 2C Although LAT deletion does not raze the global architecture of the TCR signaling networks, it reduces the global TCR-induced phosphorylation. In particular, the hub, through which LAT transduces the input signals, seems to disaggregate. This identifies LAT-dependent modules that could account for the onset of LAT-dependent autoimmunity and highlights alternative signaling routes .Fig. S2), a closer inspection of peptide-specific phosphorylation shows that kinetics of the pY492-93 (ZAP70) and pY111 (CD3ζ) last longer in the absence of LAT  except we now compare directly two cell lines for a single activation time point (0.5min). As previously reported for mice CD4+ T cells freshly deprived of LAT , anti-pY immunoblot) in tested Jurkat cell lines are similar except LAT (absent in JCam.2.5) and some differences in the intensity of a few protein bands. In particular a band at around 18 KDa, corresponding to the expected molecular weight of CD3ζ, is of higher intensity in the absence of LAT . These results corroborate the MS data  and possibly shed light on its underlying molecular mechanisms.Previous studies demonstrated that LSP is characterized by hyper-proliferation of freshly LAT-deprived peripheral CD4+ T cells and therefore LAT mutations causing partial loss of function might not be due to thymic selection altering TCR affinity threshold but rather to peripheral T cell malfunction However, here we provide quantitative data allowing more accurate comparison. Our data show that in the absence of LAT, protein phosphorylation persists not only on tyrosine but also serine and threonine and that some TCR-triggered signals are transduced independent of LAT.From a global perspective, LAT deletion decreases the overall level of phosphorylation and consequently interferes with phosphorylation-specific protein networking. However less stout, the outline of the signaling networks architecture in the absence of LAT remains comparable to the intact network. These results suggest that despite the disaggregation of LAT-dependent diversification module, some signals still propagate. One such group of proteins that contain GEF-GAP domains is forming a distinct network that seems unaffected in the absence of LAT.In agreement with previous studies, we show that LAT deficient cell lines are defective in ERK (Ras) and PLCγ1 (phosphatidylinositol) pathways Upstream of LAT, we found that the tyrosine phosphorylation of CD3ζ and ZAP70 was persistent in the absence of LAT. It seems therefore that unchecked phosphorylation of CD3ζ leads to further recruitment of ZAP-70 and its phosphorylation in turn escapes negative regulation.Fig. S3E). Its phosphorylation on S143 Different molecular mechanisms are reported to modulate activation of TCR and ZAP70 through dephosphorylation (PTPN22 and SHP1) and/or ubiquitin-induced degradation of signaling molecules (STS1/2) In conclusion our data indicates that: (1) TCR triggering induces phosphorylation-specific signaling networks organized in well-defined functional hubs, (2) perturbation of TCR singling by LAT deletion does not alter the global architecture of this network, (3) however, while modules through which LAT transduces the input signals seems to disaggregate, (4), signals still propagate to downstream components. (5) One such hub is formed by GEF-GAP domain containing proteins such as those involved in GTI-PIX-PAK pathway. (6) Unexpectedly, in perturbed modules, tyrosine phosphorylation of CD3ζ and ZAP70 last longer in the absence of LAT which indicates that LAT, in addition to its role in forward signaling, has negative regulatory function modulating upstream phosphorylation events possibly through phosphatase such as PTPN7. Our data open new avenues to study LAT-independent pathways and to establish further LAT-dependent nature of PTPs negative regulation of the TCR and ZAP-70 phosphorylation.Figure S1The workflow to measure kinetics of TCR-induced phosphoproteome. Jurkat cells were split in three sets, and each one grown in indicated SILAC media . Here we show details of the workflow only for a single series of time points, as the complementary time series was submitted to the same workflow (see see Methods). Resulted peptides mixture was submitted to strong cation exchange chromatography (SCX) followed by titanium oxide (TiO2) affinity enrichment of phosphopeptides and then analyzed by LC-MS/MS. The MS data were processed and analyzed using MaxQuant and CPFP. The results are documented in PhosphoTCR database (see Methods).(PDF)Click here for additional data file.Figure S2Dynamics of TCR-induced phosphorylation. (A) Clusters grouping phosphopeptides with similar kinetic profiles were generated using ProteinCenter software . (B) Kinases whose phosphorylation peaks at the indicated time points. The color code corresponds to different clusters. (C and D) Peptide specific profiles for some known and unexpected proteins in cluster 1 and 2. (E) Comparison of PTPN7 pS143 kinetics in LAT-efficient and -deficient cell lines.(PDF)Click here for additional data file.Figure S3LAT-dependent phosphorylation of ZAP70. Additional biological replicates for anti-pY293 ZAP70 and anti-ZAP70 immunoblots performed on indicated cell lysates Click here for additional data file.Figure S4Workflow for evaluating intra-experimental error and determining an activation p-value. Three sets of CL20 Jurkat T cells were labeled in culture media with distinct combinations of amino acid isotopomers. The three samples were stimulated using anti-CD3 antibodies for 0 min (k = 0), 0.5 min (k = 1), and 0.5 min (k = 2). The two 0.5 min stimulations are to serve as controls to determine the total intra-experimental error. After digestion of protein lysates with trypsin, the samples were split in two (split sample i = 1 or i = 2) before phosphopeptide enrichment, three LC-MS/MS injections, and data analysis are performed. This workflow produced two phosphoprylation test datasets (one from each split sample) that each included quantitative phosphorylation data on the three stimulation conditions. The red and blue segments delimit experimental steps for which distinct errors were estimated.(PDF)Click here for additional data file.Figure S5Intra-experimental error in phosphoproteomics experiments. (A) Comparison of the two split samples, which includes error contributions only from steps following sample splitting . (B) Comparison of the two 0.5 min stimulations, which includes error contribution from every step from initial culture . (C–D) Correlation between changes in phosphorylation and absolute intensity: shown are scatter plots of the absolute intensity versus the change in phosphorylation between (C) the split samples  and (D) the two 0.5 min stimulations .(PDF)Click here for additional data file.Figure S6Experimental error in independent biological replicates. (A) The histogram of the differences in the change of phosphorylation (γ = JR2-JR1) in peptides common to two biological replicates (JR1 and JR2). Dashed red lines indicate 95% confidence intervals based on total intra-experimental error  whilst grey dashed lines indicate 95% confidence intervals  based on inter-experimental error. (B) Scatter plots of JR1 vs. JR2 of the two biological replicates (correlation coefficient r = 0.54). We note that the correlation on the log2-transformed data is similar to published studies. Diagonal white line represents a slope of 1.(PDF)Click here for additional data file.File S1Supporting text. This file contains: additional information on the analysis of global dynamics of TCR-induced phosphorylation; methods to evaluate experimental error and define activation threshold; supporting references.(DOC)Click here for additional data file.


The present work concerns in what way a comparison between separate estimations of GFR based upon cystatin C (eGFRcystatin C and eGFRcreatinine was compared with measured GFR (iohexol clearance) and a combined cystatin C- and creatinine-based estimation of GFR in a Swedish-Caucasian cohort of 857 adult patients.The difference between eGFRcystatin C and eGFRcreatinine of ≥ 40% indicated a markedly reduced diagnostic performance of the combined cystatin C- and creatinine-based estimation of GFR.A difference between eGFR cystatin C and eGFRcreatinine can be used to evaluate the diagnostic performance of combined cystatin C- and creatinine-based estimations of GFR. If ‘threshold values’ for discordance are exceeded, it must be considered whether the clinical context requires the use of an invasive gold standard method to measure GFR. In some clinical contexts either creatinine or cystatin C are known to be invalidated as markers of GFR and in these situations the use of only the cystatin C- or the creatinine-based GFR estimate should be considered when the ‘threshold values’ are exceeded.Comparison of the agreement between eGFR However, the highest percentages of estimated GFR-values within ± 30% of measured GFR are obtained using GFR-prediction equations based upon both cystatin C and creatinine , missing plasma cystatin C values (n = 8) or technical assay errors (n = 5) leaving 406 subjects in the Malmö cohort. All procedures involving subjects and data were in accordance with the Helsinki Declaration of 1975 concerning ethical principles for medical research involving human subjects.The Lund population consisted of 451 patients  and the Malmö population of 425 patients of whom 19 patients were excluded because of missing plasma creatinine values (n = 857) are shown in n = 44).The characteristics of the two cohorts and the combined set  were administered intravenously in an antecubital vein. Iohexol clearance (referred to as ‘measured GFR') was calculated from plasma clearance of a single plasma sample of iohexol  drawn atace area .Plasma concentrations of creatinine were determined at Lund University Hospital by an enzymatic colorimetric assay on a Hitachi Modular P analyzer  and with a calibrator traceable to primary reference material with values assigned by isotope dilution mass spectrometry (IDMS) . At MalmPlasma cystatin C levels were determined by an automated particle-enhanced immunoturbidimetric method  using a creatinine) with age and gender [cystatin C) based on adults and including gender [2 body surface area.The Lund-Malmö creatinine-based equation (eGFRd gender  and the g gender  were selX − 0.0124 × age + 0.339 × ln(age) − 0.226  X = 4.62 − 0.0112 × pCr (if pCr < 150 μmol/L) X = 8.17 + 0.0005 × pCr − 1.07 × ln(pCr) (if pCr ≥ 150 μmol/L)GFR = e−1.686 × 0.948 , equivalent with GFR = e4.46 − 1.686 × ln (pCy) − 0.053 GFR = 86.49 × pCycystatin C+creatinine) were based on the arithmetic mean of eGFRcystatin C and eGFRcreatinine, which has proved as accurate as more complex equations [GFR estimates from the combined use of the two analytes . In the statistical testing we regarded cystatin C + creatinine − measured GFR| /measured GFR, and summarized as the percentage of estimates within 30% (P30) and 10% (P10) of measured GFR [agreement was reflected by the difference%, i.e. the absolute difference |eGFRcystatin C − eGFRcreatinine| expressed in percent relative to the arithmetic mean eGFR cystatin C + creatinine.|eGFRured GFR . The agrThe following analyses were made:s) were used to evaluate the overall association between accuracy (absolute percentage error) and agreement (difference%).Pearson's and Spearman's correlation coefficients  rounded to nearest integer and then categorized as < 10%, 10–19%, 20–29%, 30–39% and ≥40% difference. Fisher's exact test was used to evaluate differences in P30 and P10 across categories of agreement.The accuracy categorized as P30 and P10, respectively, using logistic regression with measured GFR and difference% as continuous covariates.Measured GFR is related to both accuracy and agreement and may thus confound the association between agreement and accuracy. To account for such confounding we modelled accuracy, i.e. Pcystatin C + creatinine, but not both eGFRcystatin C and eGFRcreatinin, were inaccurate according to P30 and P10, respectively. This index represents the upper limit of the improvement in accuracy that could be obtained if the most accurate eGFR  was consistently applied for each patient. Note that the sum of, e.g. P30 and the corresponding improvement index for P30 can never exceed 100%.We calculated an ‘improvement index', defined as the proportion of all GFR estimates for which eGFRThe improvement index depends on the accuracy, i.e. the potential for improvement is higher when accuracy is low. To account for such confounding in the association between agreement and the improvement index, we modelled the improvement index using logistic regression with inaccuracy (absolute percentage error) and difference% as continuous covariates.p<0.001 but rs = 0.05, p = 0.12), however, P30 was clearly decreased for differences between eGFRcystatin C and eGFRcreatinin exceeding a ‘threshold value’ of 40% (p = 0.02 when comparing P30 for 30–39 and >40% difference). The dip in accuracy when expressed as P10 seemed to occur already at 30–39% difference, but the statistical evidence for this dip was weak (p = 0.09 when comparing P10 for 20–29 and 30–39% difference). Measured GFR was noticeably lower for differences exceeding 40%, but the suggested inverse association between difference% and P30 remained clear when measured GFR was adjusted for using logistic regression (p = 0.001), whereas the association between difference% and P10 remained weaker (p = 0.05).The overall association between difference% and absolute percentage error was not consistent .The improvement index generally suggested higher potential for improvement in accuracy when the difference between eGFRiderable . This ascreatinine is reduced inter alia if a patient has an abnormally low or high muscle mass, recently ingested boiled meat or is treated with a drug that influences the tubular secretion of creatinine. In these clinical contexts the diagnostic performance of eGFRcystatin C is generally unaltered. However, the performance of eGFR cystatin C is impaired if a patient is treated with large doses of glucocorticoids and in this situation the performance of eGFRcreatinine is still acceptable. Although GFR-prediction equations based upon both cystatin C and creatinine (eGFRcystatin C + creatinine) generally are superior to GFR-prediction equations based upon either cystatin C (eGFRcystatin C) or creatinine (eGFRcreatinine) this may not be the case in these specific clinical contexts. Although these contexts may be easily recognized in some cases, they will not invariably be recognized. There may also be additional, not yet identified, clinical contexts invalidating either cystatin C or creatinine as useful markers for GFR. Comparing eGFR cystatin C and eGFRcreatinine might be helpful to identify both known and unknown causes when neither cystatin C nor creatinine are suitable as a marker for GFR [cystatin C and eGFRcreatinine is large enough to indicate such a condition. The present study based upon measured GFR and eGFRcystatin C and eGFRcreatinine in a patient cohort of 857 Swedish-Caucasian adult patients indicates that if the discordance is 40% or more, the diagnostic performance of eGFRcystatin C + creatinine is markedly reduced. Such discordance should initiate a more careful evaluation of the clinical context to disclose conditions invalidating either creatinine or cystatin C as a GFR marker. If such conditions are identified, GFR might be best estimated using a prediction equation based upon only the non-invalidated marker. If such conditions are not identified, it should be realized that the estimation of GFR is unreliable and that an invasive, gold standard, measurement of GFR might be required.The diagnostic performance of eGFR for GFR . To be acystatin C, eGFRcreatinine and eGFRcystatin C + creatinine. For other patient cohorts might contain proportionally more, or fewer patients, with conditions invalidating either cystatin C or creatinine as a GFR marker. The more patients with such conditions in the cohort, the greater the potential for improvement of eGFR by comparison of eGFRcystatin C and eGFRcreatinine. The equations for eGFRcystatin C, eGFRcreatinine and eGFRcystatin C + creatinine will also influence the ‘threshold value’ by being more or less sensitive for patient characteristics reducing the value of creatinine and cystatin C as markers for GFR.It should be realized, that the discordance value, the ‘threshold value', indicating requirement of a further evaluation of the clinical context to improve estimation of GFR, as presented in this work, is influenced by the actual patient cohort and by the equations used for estimating GFR, i.e. eGFR cystatin C + creatinine, but not both eGFRcystatin C and eGFRcreatinin, are inaccurate according to P30 or P10. This index will represent the upper limit of improvement in accuracy that could be obtained, if the most accurate eGFR  was consistently applied. In the present study the improvement index was 18.3 % for a discordance threshold of ≥40%, which means that if an accurate evaluation of the relevant clinical conditions could be performed for each patient the P30-value of 79.6% for eGFRcystatin C + creatinine, would theoretically increase to 79.6 + 18.3 = 97.9%. This ‘improvement index’ will, exactly like the ‘threshold value’ of discordance, also be influenced by the actual patient cohort and by the equations used for eGFR, i.e. eGFRcystatin C, eGFRcreatinine and eGFRcystatin C + creatinine and for the same reasons.It is possible to calculate an ‘improvement index', defined as the proportion of all GFR estimates for which eGFR