Kip1 is a cell cycle inhibitor that prevents cyclin dependent kinase (CDK)/cyclin complexes from phosphorylating their targets. p27Kip1 is a known tumor suppressor, as the germline loss of p27Kip1 results in sporadic pituitary formation in aged rodents, and its presence in human cancers is indicative of a poor prognosis. In addition to its role in cancer, loss of p27Kip1 results in regenerative phenotypes in some tissues and maintenance of stem cell pluripotency, suggesting that p27Kip1 inhibitors could be beneficial for tissue regeneration. Because p27Kip1 is an intrinsically disordered protein, identifying direct inhibitors of the p27Kip1 protein is difficult. Therefore, we pursued a high-throughput screening strategy to identify novel p27Kip1 transcriptional inhibitors. We utilized a luciferase reporter plasmid driven by the Kip1p27 promoter to transiently transfect HeLa cells and used cyclohexamide as a positive control for non-specific inhibition. We screened a “bioactive” library consisting of 8,904  compounds, of which 830 are Food and Drug Administration (FDA) approved. From this screen, we successfully identified 111 primary hits with inhibitory effect against the promoter of Kip1p27. These hits were further refined using a battery of secondary screens. Here we report four novel Kip1p27 transcriptional inhibitors, and further demonstrate that our most potent hit compound (IC50 = 200 nM) Alsterpaullone 2-cyanoethyl, inhibits Kip1p27 transcription by preventing FoxO3a from binding to the p27Kip1 promoter. This screen represents one of the first attempts to identify inhibitors of p27Kip1 and may prove useful for future tissue regeneration studies.p27 Kip1  is a member of the Cip/Kip family of cell cycle inhibitors which are characterized by their ability to bind and inhibit cyclin dependent kinases (CDK)/cyclin complexes, halting cell cycle progression in the G1 phase Kip1 has been associated with some forms of cancer in humans, and germline Kip1p27 deletion in mice results in sporadic pituitary tumors at old ages Kip1p27 are not usually causative of cancer, it is often dysregulated and associated with a poor prognosis Kip1 levels has not been the focus of previous studies. Despite this, recent experiments have cast a light on how p27Kip1 may antagonize stem cell pluripotency Kip1. Specifically, loss of p27Kip1 has been associated with regenerative phenotypes in spinal cord injuries Kip1 and its link to regeneration. Within the inner ear lies the organ of Corti, the sensory epithelial sheet which contains the sensory hair cells and their supporting cells. It was observed that p27Kip1 initiates its expression during embryonic development coinciding with the exit of these cells from the cell cycle Kip1 in these cells. In the postnatal mouse cochleae, removal of p27Kip1 from normally quiescent supporting cells forced these cells to re-enter the cell cycle Kip1 preceded conversion of supporting cells to sensory hair cells in vitroKip1inhibition therapy has been proposed for hearing restoration in mammals Kip1, may need to be developed to force proliferation to occur in older quiescent tissues. These observations led us to pursue screening strategies for reduction of p27Kip1.p27Kip1 belongs to a class of proteins called intrinsically disordered proteins (IDPs), which lack stable secondary and tertiary structure. IDPs represent extremely difficult targets for the development of small molecule inhibitors Kip1 for degradation are also problematic. Once p27Kip1 is excluded from the nucleus, it regulates cell migration and cancer metastasis Kip1 protein abundance and localization impede direct targeting of p27Kip1. Because of these observations, we decided to pursue a transcription-based approach to antagonize p27Kip1.p27Kip1p27 transcription is known to be regulated by the Forkhead box O (FoxO) family of transcription factors Kip1 locus, it has been demonstrated that acetylation of FoxO3a prevents it from binding to the Kip1p27 promoter Kip1p27 transcription.Kip1p27 transcription. After the assay was validated, we screened our “bioactive” library of 8,904  compounds and obtained 111 primary hits which inhibit Kip1p27 promoter activity. These initial hits were narrowed down to 4 hits though our intensive secondary screens, and we chose to focus on our most potent compound, Alsterpaullone, 2-cyanoethyl (A2CE), to understand how Kip1p27 transcription was modulated by this compound. Surprisingly, we discovered that known inhibitors of Sirtuin 2 (Sirt2), a deacetylase, mimicked A2CE effect on p27Kip1 transcription implicating Sirt2 deacetylation for the inhibitory effect of A2CE on Kip1p27 transcription inhibition. Since Sirtuin 2 removes acetyl groups and would promote FoxO3a binding to the Kip1p27 promoter, we analyzed this interaction using chromatin immunoprecipitation (ChIP) followed by quantitative real time PCR, and discovered that addition of A2CE prevented FoxO3a from binding to the Kip1p27 promoter.In this study, we chose to design a luciferase based cell assay and screen for small molecules which antagonize Kip1 screening assay and validated it by screening our “bioactive” library. Within this library, we discovered novel compounds that repress p27Kip1 transcription and mechanistically described how the most potent hit achieved this inhibition. In total, this screen represents a novel tool to address the repression of p27Kip1 and yields new compounds to achieve this inhibition.In this study, we established our p27All animal work conducted during the course of this study was approved by the Institutional Animal Care and Use Committee at St. Jude Children’s Research Hospital and was performed accordingly to NIH guidelines.2, 95% relative humidity. Transfections were carried out using lipofectamine LTX transfection reagent  following the manufacturers protocol.HeLa and HEK-293 cells were obtained from ATCC . 3T3-J2 (Swiss) cells were a gift from Dr. Richard Schlegel Kip1 promoter was excised, and cloned into BamH1 sites from pGL4.17 (Promega). Cloning was verified by both restriction enzyme digests and DNA sequencing. The non-specific plasmid (SV40-LacZ) was obtained from Addgene (#188116).Our original luciferase vector was obtained from Dr. Sakai 6 cells plated into a T75 flask  plate, followed by transfection utilizing 30 μL of transfection reagent and 5 μg of plasmid DNA gave optimal luciferase values.The number of cells transfected, amount of lipofectamine transfection reagent, and plasmid DNA were all optimized for maximal endogenous luciferase activity prior to screening. We found that 2x10Kip1-Luciferase plasmid were seeded into white, solid-bottom, tissue culture-treated, 384-well polystyrene plates  at a density of 5×103 cells per well in 25 μl media. Compounds including those previously described  2, 95% relative humidity followed by alamar blue cytotoxicity assay  and luminescence luciferase reporter activity assay  with SteadyLite HTS reagent  50 values with GraphPad Prism 6.01.HeLa cells transiently transfected with the p277 HeLa treated for 1 day with 10 μm A2CE or DMSO using the simple ChIP kit from Cell signaling (#9003). ChIP’s were quantitated by realtime PCR  using Syber Green (Biorad #170880). Results are displayed as percent enrichment compared to the input. Primers for the Kip1p27 promoter are from ChIP was performed on 5x10Kip1-luciferase transfections. Luminescence was quantified on either a GloMax Multi+ (Promega) or the Envision HTS microplate reader.Luminescence of LacZ was measured using the duel light luminescence kit from Life Technologies (#T1003) following the workflow diagrammed in Kip1 , or mouse p27Kip1 , combined with 18S  as the internal control.Cells were grown to ∼ 50% confluence at which time A2CE was added. 16–24 hours later cells were harvested in RNA stat 60  and mRNA harvested following the manufacturer’s protocol. The resulting mRNA was quantified , and further used to generate cDNA . qPCR was performed on 2ng of cDNA run in multiplex against human p272, 95% relative humidity). A2CE (250 nM-10 μM) was added after 24 hours, and explants were harvested after additional 24 hour incubation. Isolation and quantification of the mRNA was done the same as above.Organs of Corti were harvested from P1-P4 mice, plated on Matrigel , and grown in DMEM  or anti-β-actin antibody  in PBST overnight at 4°C. The membrane was washed three times with PBST followed by incubation in HRP-anti-mouse IgG or HRP-anti-rabbit IgG secondary antibodies diluted 1∶5000 in PBST for 1 hour at room temperature. After washing three times with PBST, the specific bands were developed on the films via using SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific). Densitometry was performed using ImageJ .HeLa cells were harvested and homogenized in RIPA buffer (10 mM Tris (pH 7.4), 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.1%SDS, 1% Triton-X100, 0.5% sodium deoxycholate, 10% glycerol, + protease and phosphatase inhibitors) and total protein was quantitated using a BCA assay . Samples were boiled for 5 min with SDS sample loading buffer prior to loading. Samples were run on 4–20% precast polyacrylamide gels  after which they were transferred to a polyvinylidene difluoride membranes . Membranes were blocked in 5% (w/v) fat-free milk–PBST (phosphate buffer with 0.05% Tween 20) for 1 hour at room temperature after which the membranes were incubated with rabbit anti-p27Kip1 protein was expressed in Escherichia Coli as 6xHis fusion constructs. The protein was purified by nickel affinity chromatography and then digested with thrombin to remove the fusion tag. Further purification by reverse-phase high performance liquid chromatography (HPLC) using a C4 column (Vydac) and 0.1% trifluoroacetic acid-containing water/acetonitrile solvent system yielded high purity protein. Protein concentration was determined by UV absorbance at 280 nm using a molar extinction coefficient of 15,470 M−1*cm−1.Full-length p27Statistics were performed using OriginLab 8.5. Where applicable, one-way ANOVAs with Bonferroni were run to test for mean difference, or a two sample T-test if only two conditions were compared.Kip1p27-luciferase plasmid transfected into HeLa cells. We have previously used the p27Kip1 plasmid Kip1p27 promoter into the BamHI site of the pGL4.17 commercially available luciferase backbone  of HeLa cells instead of the transient transfected reporter plasmid  normalized to the internal control, 18s  1-P4 cochleae were isolated and plated in matrigel. After 1 day of recovery the explants were treated with varying doses of A2CE. The following day whole cochlear mRNA was harvested, or whole explants were treated for alamar blue for viability. Kip1p27 mRNA after treatment with 10 μM A2CE, with no significant changes in viability  inhibitor , and A2CE (250 nM-10 μM) and did not observe any synergistic effect on luciferase luminescence at any dose. This implies that AGK2 and A2CE function in the same pathway.Although A2CE has no known effects on p27cription . To asceKip1p27, we treated HeLa cells transfected with the Kip1-luciferasep27 plasmid with piceatenol, a metabolite of resveratrol Kip1p27-luciferase by resveratrol , followed by qPCR. We observed a significant decrease in FoxO3a binding to the Kip1p27 promoter upon addition of 10 μm A2CE . This observation delineated the activity of kenpaullone from other members of this family of CDK inhibitors, and further supports the idea that A2CE can affect Kip1p27 transcription through Sirtuin inhibition, albeit by >2 orders of magnitude higher affinity than the modified paullones made in that study.The paullone analogs are all characterized as inhibitors of CDKs, and interestingly some are even used in the production of induced pluripotent stem cells Kip1p27 inhibition in this study. This could be due to Sirt2 playing a larger role in FoxO3a deacetylation in our system. Alternatively, since our primary screening procedure utilized transient transfection of HeLa cells, the primary location of our plasmid should be cytoplasmic. Since Sirt1 is described as primarily cytoplasmic, any deacetylation done by Sirt1 may not be captured in our primary screen. On face value, this may also raise the possibility that some of our primary hits are artificial, since we screened for inhibitors of plasmid based Kip1p27 expression. However, it is important to remember that our final secondary screen did not utilize plasmids but instead looked at endogenous Kip1p27 expression, demonstrating that all four of the hits we identified are not specific to plasmid based transcription, but represent true Kip1p27 inhibitors.Sirt1 and Sirt2 have both been described as affecting FoxO3a acetylation status Kip1p27 transcription. Our most potent hit, A2CE, yielded affinities in the sub-micro molar range, which encouraged us to identify the molecular mechanism through which A2CE repressed Kip1p27 transcription. We demonstrate that this inhibition occurs mainly through repression of FoxO3a binding to the Kip1p27 promoter. We further plan to characterize the effects of A2CE in regenerative systems, such as the inner ear, though local delivery, given our results in cochlear explant culture. Furthermore, since we have verified the ability of our assay to detect Kip1p27 inhibitors, we can pursue other larger unbiased libraries, or smaller specialized libraries focused on FoxO3A regulation, to further identify compounds which may yield p27Kip1 repressors with higher affinities. We are hopeful that our discoveries of Kip1p27 inhibitors will not only be useful for tissue regeneration, but that the assay itself may yield more potent regulators of Kip1p27.Use of luciferase-based assays for transcriptional screening has been around for over a decade, but to our knowledge, using this method to screen for small molecules that transcriptionally repress CDK inhibitors had not been attempted. We successfully screened our “bioactive” library and discovered four novel compounds which repress


We conclude that Ser-473 and Ser-489 are Km-regulating phosphorylation sites that are difficult to detect using current methods.Kinase homology domain (KHD) phosphorylation is required for activation of guanylyl cyclase (GC)-A and -B. Phosphopeptide mapping identified multiple phosphorylation sites in GC-A and GC-B, but these approaches have difficulty identifying sites in poorly detected peptides. Here, a functional screen was conducted to identify novel sites. Conserved serines or threonines in the KHDs of phosphorylated receptor GCs were mutated to alanine and tested for reduced hormone to detergent activity ratios. Mutation of Ser-489 in GC-B to alanine but not glutamate reduced the activity ratio to 60% of wild type (WT) levels. Similar results were observed with Ser-473, the homologous site in GC-A. Receptors containing glutamates for previously identified phosphorylation sites (GC-A-6E and GC-B-6E) were activated to ∼20% of WT levels but the additional glutamate substitution for S473 or S489 increased activity to near WT levels. Substrate-velocity assays indicated that GC-B-WT-S489E and GC-B-6E-S489E had lower Km values and that WT-GC-B-S489A, GC-B-6E and GC-B-6E-S489A had higher Km values than WT-GC-B. Homologous desensitization was enhanced when GC-A contained the S473E substitution, and GC-B-6E-S489E was resistant to inhibition by a calcium elevating treatment or protein kinase C activation – processes that dephosphorylate GC-B. Mass spectrometric detection of a synthetic phospho-Ser-473 containing peptide was 200–1300-fold less sensitive than other phosphorylated peptides and neither mass spectrometric nor  Natriuretic peptides are endogenous disulfide-linked endocrine and paracrine molecules, and recombinant forms of these peptides are drugs approved for the treatment of congestive heart failure Phosphorylation of the KHD is required for the activation of GC-A and GC-B and dephosphorylation reduces the GC activity of these receptors The role of phosphorylation of Ser-487 in regulation of GC-A is controversial. Mutation of this site to glutamate decreased activity but mutation to alanine had no effect. Schroter et al. found that a Ser-487-Glu mutant failed to desensitize, whereas our group found that receptors containing alanine or glutamate substitutions for Ser-487 desensitized similarly to WT-GC-A 32PO4 phosphopeptide mapping techniques or by mass spectrometry Although many phosphorylation sites have been found in GC-A and GC-B, several observations support the notion that undiscovered phosphorylation sites exist in these receptors. First, highly homologous, peptide-activated sea urchin GCs, which are also desensitized by dephosphorylation, contain 15 to 17 moles of phosphate/receptor The goal of this study was to identify undiscovered natriuretic peptide receptor phosphorylation sites using a functional screen that was independent of phosphopeptide purification or detection. A highly conserved juxtamembrane serine was identified in membrane GCs that are regulated by phosphorylation. Alanine but not glutamate substitutions for this residue in GC-A and GC-B reduced natriuretic peptide-dependent, but not detergent dependent, GC activity. Substrate-velocity assays indicated that GC-B activity losses associated with the alanine substitutions were due to large increases in the Michaelis constant and smaller decreases in the maximal velocity. Inhibition studies revealed that GC-A receptors containing a glutamate for Ser-473 exhibited enhanced homologous desensitization and that the dephosphorylation resistant receptor, GC-B-6E-S489E, was not inhibited by a calcium elevating treatment or protein kinase C activation.A. punctulata and S. purpuratus, as well as rat GC-C. The first six GCs are regulated by phosphorylation whereas GC-C lacks the phosphorylation sites identified in GC-A and GC-B To identify conserved serines or threonines, a multiple sequence alignment of the intracellular region of membrane GCs across several species was performed . The GCsTargeted residues were initially mutated to alanine in the rat GC-B cDNA and expression, glycosylation and phosphorylation of the receptors were verified by Western blotting . Three dGC activity was measured in membranes from 293 cells transfected with WT or mutant receptors in the presence of natriuretic peptide, ATP and magnesium-GTP or in the presence of 1% Triton X-100 and manganese-GTP. Analysis of previously identified phosphorylation sites in GC-A and GC-B under these conditions found that the alanine substitutions reduced natriuretic peptide-dependent activity but not detergent-dependent activity Since the mutation of Ser-489 to alanine was the only residue in our initial screen that changed the GC activity ratio without affecting processing, we focused on this residue. First, we characterized the degree of activity loss in fixed substrate concentration GC assays. The CNP-dependent to detergent-dependent GC ratio of S489A-GC-B was reduced to 60% of WT value . In contAlanine and glutamate substitutions for Ser-489 were also made in a receptor variant containing glutamic acid substitutions at the six previously identified phosphorylation sites (GC-B-6E) Since Ser-489 is conserved in all six phosphorylated GCs shown in To investigate whether the changes in ligand-stimulated activity associated with Ser-473 in GC-A was specific to that residue or simply a result of increased positive charge in that area of the protein, we mutated the residue N-terminal to Ser-473, Val-472, to alanine or glutamate. Membranes from cells transiently expressing WT or mutant receptors were then assayed for GC activity ratios . MutatioTo determine how the residue at 489 regulates the enzymatic activity of GC-B, we generated substrate-velocity curves for WT-GC-B, WT-GC-B-S489E, GC-B-6E and GC-B-6E-S489E for 9 minutes to determine maximal velocities and Michaelis constants for each enzyme . We founNext, we directly compared substrate-velocity profiles for WT-GC-B-S489E and WT-GC-B-S489A in a shorter five min assay and found that the single substitution of alanine for glutamate increased the Km five-fold and decreased Vmax almost two-fold . We alsoWhole 293T cells expressing WT-GC-A, WT-GC-A-S473A, WT-GC-A-S473E, GC-A-6E or GC-A-6E-S473E were incubated in the presence or absence of 1 µM ANP for 1 h at 37°C before membranes were prepared and assayed GC activity in the presence of ANP, ATP and magnesium-GTP or 1% Triton X-100 and manganese-GTP. The data were presented as the ratio of the hormone/detergent activities to control for differences in transfection efficiencies and activity losses resulting from GC-A degradation . Prior AA previous study showed that lysophosphatidic acid and hyperosmotic conditions inhibit CNP-dependent activation of GC-B in 3T3 and 293 cells by a process that involves calcium-dependent receptor dephosphorylation Despite the changes in activity observed when Ser-473 or Ser-489 were mutated, the GC results do not prove that this site is phosphorylated in cells. Thus, we attempted to obtain biochemical evidence of phosphorylation of GC-A at serine 473 and GC-B at serine 489 using mass spectrometric detection. Rat GC-A and GC-B were purified from HEK 293 cells by sequential immunoprecipitation-SDS-PAGE fractionation as previously described Additional experiments were performed where only the parent ion corresponding to the tryptic peptide containing phosphorylated serine 473 was selectively monitored . This was done to isolate a low intensity signal that may be “buried” among other peptides that ionize more efficiently. However, this approach also failed to yield a match to a tryptic peptide containing phosphorylated Ser-473 (data not shown).32PO4 labeled peptides isolated from tryptic digests of GC-A and GC-B from metabolically labeled cells as previously described 32PO4–labeled phosphopeptides were identified by autoradiography and the synthetic peptides were visualized by ninhydrin staining. However, no 32P-labeled material comigrated with the synthetic peptides (data not shown).We also attempted to identify phosphorylated versions of Ser-473 or Ser-489 from GC-A or GC-B, respectively, from receptors isolated from 293 or NIH3T3 cells by mixing synthetic versions of the peptides containing Ser-473 (ELVpSELWR) or Ser-489 (ELApSMLWR) with To determine if an intrinsic chemical property of this particular phosphopeptide hinders identification by mass spectrometry, we measured detection of synthetic phosphopeptides. Equimolar quantities of three peptides corresponding to tryptic peptides phosphorylated at Ser-473, Ser-487, and Ser-985 in GC-A  were mixed and subjected to nLC-MS-MS. The resulting ionized peptide signal intensity for each peptide is shown in By comparing sequences from several phosphorylated GCs, we identified a highly conserved serine nine residues past the membrane spanning regions of GC-A and GC-B. The conserved nature of this serine within a family of proteins that are phosphorylated on multiple serines is consistent with this residue being phosphorylated in both enzymes.The effect of the amino acid at position 473 in GC-A or position 489 in GC-B on enzymatic activity is striking. When these serines are phosphorylated as expected for most WT enzymes, the Km is around 0.1 mM, which is in the range of cellular GTP concentrations. However, when this single amino acid is mutated to alanine to mimic a constitutively dephosphorylated residue, the Km increases dramatically. Conversely, conversion to glutamate to mimic a constitutively phosphorylated residue “rescues” the enzyme. In fact, the Km for GC-B mutants containing glutamate substitutions for Ser-489 is slightly lower than the Km for the WT enzyme, which suggests that within the WT population most receptors are phosphorylated on Ser-489 but some receptors are dephosphorylated at Ser-489. The fact that the Michaelis constants for GC-B-6E, GC-B-6E-S489A and WT-GC-B-S489A are high but the Michaelis constants for GC-B-6E-S489E and WT-GC-B-S489E are similar to or lower than the WT enzyme strongly suggests that Ser-489 is phosphorylated on most WT-GC-B molecules isolated from 293 cells. Thus, we believe the functional evidence for phosphorylation of Ser-473 in GC-A and Ser-489 in GC-B is compelling. However, we cannot rule out the possibility that the changes in activity are simply coincidental.Previous studies on enzymatically dephosphorylated preparations of GC-A indicated that dephosphorylation reduced Vmax but not Km . Initial studies on GC-A-6E indicated that unlike the mutant containing alanine substitutions for these sites, this receptor was responsive to ANP 32PO4-tryptic phosphopeptide mapping approaches. However, this does not prove that these sites are not phosphorylated in physiologic systems since no current method of phosphorylation site identification is infallible. This is illustrated by the progression of phosphorylation site discovery in GC-A and GC-B. Metabolic labeling studies using 32P-orthophosphate in conjunction with phosphopeptide mapping techniques initially revealed six sites in GC-A and five sites in GC-B We were unable to obtain biochemical proof of phospho-Ser-473 or phospho-Ser-489 from cells using mass spectrometric or e.g. it preferentially adheres to the sample tube. Once injected into the instrument, the chemical properties may be such that it does not ionized well, and as a result, very little enters the detector. Alternatively, a poorly ionized peptide can easily be “buried” if several other peptides with higher intensities have overlapping retention times because the instrument preferentially picks the ions with the highest intensities to submit for tandem mass spectrometry. Unfortunately, the tryptic phosphopeptide corresponding to a phosphorylated Ser-473 suffers from poor detection issues. Thus, we were unable to validate that Ser-473 or Ser-489 are phosphorylated in GC-A or GC-B, respectively, possibly due to poor detection of the peptide by the current techniques.Similarly, mass spectrometry is not absolute in its detection of all endogenous phosphorylation sites. For example, we were unable to detect endogenous phosphorylation at serine 510 and threonine 513 in the human GC-A receptor using a mass spectrometric approach. There are several explanations for why an endogenous phosphorylation site may not be detected by a mass spectrometry approach. First, the chemistry of the tryptic phosphopeptide containing the site of interest may be such that it is lost during sample preparation prior to injection on the instrumentation, bona fide phosphorylation sites awaits chemical identification of peptides containing these sites from biological samples. However, the marked effect of the phosphomimetic mutations on the kinetic and desensitization properties of the enzymes provide compelling support for the hypothesis that Ser-489 in GC-B and Ser473 in GC-A are phosphorylated in physiologic systems.In conclusion, we identified a highly conserved serine in GC-A and GC-B that decrease natriuretic peptide-dependent GC activity when mutated to an alanine but not when mutated to glutamate as predicted for a phosphorylation site. We also found that the conversion of this highly conserved residue to a glutamate increases the activity of GC-A-6E and GC-B-6E to near WT levels. Importantly, we determined that that the phosphorylation of this single site dramatically reduced the Michaelis constant of GC-B and increased the magnitude of the homologous desensitization response for GC-A. Whether these residues are in vivo. The similarity of the kinetic parameters of GC-B-7E to WT-GC-B make the receptor containing the glutamate substitution for Ser-489 an excellent choice for future genetic replacement studies because this receptor is resistant to inhibition and has a Km that is similar to the WT receptor Finally, these studies are important in terms of designing receptor constructs for genetic “knock in” or “transgenic” experiments used to investigate the role of phosphorylation and dephosphorylation in the regulation of these receptors 32P]-α-GTP and 125I-cGMP radioimmunoassay kits were purchased from Perkin Elmer Life Sciences . PhosSelect affinity gel for immobilized metal affinity chromatography (IMAC), trypsin, creatine kinase, phorbol myristic acid and rat ANP and CNP were from Sigma-Aldrich . Protease inhibitor cocktail was from Roche Applied Bioscience . GTP, with either 5 mM MgCl2, 1 mM ATP, and 1 µM natriuretic peptide or 1% Triton X-100 with 5 mM MnCl2 being substituted for the MgCl2. Reactions were started by the addition of 0.08 ml of the above reagents to 50–200 µg of crude membrane protein suspended in 0.02 ml of phosphatase inhibitor buffer. Reactions were stopped by the addition of 0.5 ml 110 mM ZnOAc and 0.5 ml 110 mM NaCO3 on ice. 32P-cGMP was purified by alumina column chromatography and detected by the Cerenkov method in a gamma counter.Single substrate concentration GC assays were performed at 37°C for 5 min in a buffer containing 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid – pH 7.4, 50 mM NaCl, 0.1% BSA, 0.5 mM 1-methyl-3-isobutylxanthine, 1 mM GTP, 0.5 µM microcystin, 1 mM EDTA, and 1–2 µCi of [α-Substrate-velocity experiments were conducted for 5 or 9 min as previously described GC activities in membranes from transfected 293 cells were at least 50-fold higher than activities in membranes from untransfected cells, which indicated that the GC activity was due to the transfected enzyme. We previously demonstrated that GC-A and GC-B are regulated similarly in 293 cells as they are in endogenously expressing cells Receptor purification by immunoprecipitation, in-gel digestion, phosphopeptide separation and mass spectrometric detection were performed as we previously described Tryptic phosphopeptide mapping comigration studies were conducted as we previously described for GC-B All single substrate GC assays were evaluated using an unpaired t-test where p≤0.05 was considered significant. Values were presented as mean ± SEM. Vertical bars within the columns represent SEM. Brackets indicate t-test between the indicated values. Substrate-velocity curves were analyzed by non-linear regression using an a Michaelis-Menten model to determine Vmax and Km. Significant differences between nonlinear regression curves used to determine Km and Vmax values were determined using the extra sum of squares F test to generate p-values.Figure S1Effect of serine or threonine to alanine substitutions on the processing and expression of wild type and mutant forms of GC-B. 293 cells were transiently transfected with the indicated GC-B constructs and Western blotting of immunoprecipitated receptors assessed the expression and processing of the receptor. Immunocomplexes were fractionated on an 8% SDS reducing gel and blotted to an Immobilon membrane, which was probed with rabbit polyclonal antiserum 6328 against the C-terminus of GC-B. Detection was by horseradish peroxidase conjugated secondary antibody ECL. WT-GC-B consists of a thin, deglycosylated fastest migrating species, a partially glycosylated slower migrating species, and fully glycosylated and phosphorylated slowest migrating species (arrow) that is hormonally responsive. Mutation of Ser-647 caused the loss of the upper fully processed form of GC-B.(TIF)Click here for additional data file.


SLC6A5) are the second most common cause of human hyperekplexia.Inhibitory glycinergic neurotransmission is terminated by sodium and chloride-dependent plasma membrane glycine transporters (GlyTs). The mainly glial glycine transporter GlyT1 is primarily responsible for the completion of inhibitory neurotransmission and the neuronal glycine transporter GlyT2 mediates the reuptake of the neurotransmitter that is used to refill synaptic vesicles in the terminal, a fundamental role in the physiology and pathology of glycinergic neurotransmission. Indeed, inhibitory glycinergic neurotransmission is modulated by the exocytosis and endocytosis of GlyT2. We previously reported that constitutive and Protein Kinase C (PKC)-regulated endocytosis of GlyT2 is mediated by clathrin and that PKC accelerates GlyT2 endocytosis by increasing its ubiquitination. However, the role of ubiquitination in the constitutive endocytosis and turnover of this protein remains unexplored. Here, we show that ubiquitination of a C-terminus four lysine cluster of GlyT2 is required for constitutive endocytosis, sorting into the slow recycling pathway and turnover of the transporter. Ubiquitination negatively modulates the turnover of GlyT2, such that increased ubiquitination driven by PKC activation accelerates transporter degradation rate shortening its half-life while decreased ubiquitination increases transporter stability. Finally, ubiquitination of GlyT2 in neurons is highly responsive to the free pool of ubiquitin, suggesting that the deubiquitinating enzyme (DUB) ubiquitin C-terminal hydrolase-L1 (UCHL1), as the major regulator of neuronal ubiquitin homeostasis, indirectly modulates the turnover of GlyT2. Our results contribute to the elucidation of the mechanisms underlying the dynamic trafficking of this important neuronal protein which has pathological relevance since mutations in the GlyT2 gene ( Inhibitory glycine neurotransmission is terminated by specific transporters, GlyTs (GlyT1 and GlyT2), which mediate the reuptake of glycine from the synaptic cleft. GlyTs belong to the neurotransmitter:sodium symporter family (SLC6 gene family), which includes transporters for most of the neurotransmitters  in the central nervous system (CNS) Protein trafficking plays a fundamental role in the control of neuronal activity and it has been identified as a primary regulatory mechanism for several plasma membrane neurotransmitter transporters, providing a rapid means to modulate their activity Recent findings have demonstrated the importance of ubiquitination in the endocytosis of several membrane proteins, suggesting that the attached ubiquitin molecule may act as a platform for the recruitment of the clathrin-dependent endocytic machinery In the present study, we investigated the possible role of ubiquitination in the constitutive internalization of neuronal GlyT2 and its sorting to recycling and/or degradation pathways. Our results show that constitutive endocytosis of GlyT2 is dependent on the ubiquitination of the cytoplasmic C-terminal lysine cluster . The dynamic ubiquitination/deubiquitination process controls GlyT2 turnover through constitutive sorting mainly to the recycling pathway and targeting the transporter primarily to the degradation pathway via PKC-mediated ubiquitination. In neurons, the ubiquitination status of GlyT2 is highly responsive to the free ubiquitin pool, which is mainly controlled by UCHL1 deubiquitinase www.ebi.ac.uk.Male wistar rats were bred under standard conditions at the Centro de Biología Molecular Severo Ochoa. All animal work performed in this study was carried out in accordance with procedures approved in the Directive 86/609/EEC of the European Union with approval of the Ethics Committee of the Universidad Autónoma de Madrid. PYR-41 (4--benzoic acid ethyl ester), and inhibitors of UCH-L1 [LDN-57444 (LDN)] and ubiquitin C-terminal hydrolase-L3 (UCH-L3)  were purchased from Calbiochem . All other chemicals were purchased from Sigma-Aldrich. Antibodies against GlyT2 , dissociated with trypsin as described previously 2 in minimal essential medium supplemented with 10% fetal bovine serum. Transient expression was achieved using Lipofectamine™ 2000 (Invitrogen) following the manufacturer's instructions. Reproducible results were obtained with 50–60% confluent cells in a 60 mm dish using 6 μg of total DNA. The cells were incubated for 48 h at 37°C before the experiment was performed. Hippocampal neurons were obtained as described above. The cells (DIV10) were transfected with Effectene (Qiagen) following manufacturer's instructions using 10 μl of Effectene per 1 μg of DNA in a 12 mm dish.Madin – Darby canine kidney II (MDCK II) cells (American Type Culture Collection) were grown at 37°C and 5% COSubstitution mutants were generated with the QuikChange Site-Directed Mutagenesis kit (Stratagene), using the rat GlyT2 in pCDNA3 as described previously MDCK II cells and hippocampal neurons were grown on glass coverslips and transfected with the corresponding expression vectors as indicated above. Immunostaining was performed as described previously Brainstem and spinal cord primary neurons (100 µg) or MDCK II cells were lysed for 30 min at room temperature (RT) at a concentration of 1 mg of protein/ml in TN buffer  containing 0.25% Nonidet P-40 (NP-40), 50 mM N-ethylmaleimide and protease inhibitors (PIs: 0.4 mM phenylmethylsulfonyl fluoride [PMSF] + Sigma cocktail). Agarose-conjugated anti-multiubiquitin (12 μl) was added and incubated for 1 h at RT. The beads were collected by mild centrifugation and washed 3 times for 5 minutes with lysis buffer. Finally, the beads were pelleted and the ubiquitinated proteins were eluted in Laemmli buffer at 75°C for 10 min, resolved in sodium dodecyl Sulfate Polyacrylamide Gel electrophoresis (SDS/PAGE) gels (7.5%), detected in Western blots with enhanced chemiluminescence (ECL) and quantified on a GS-710 calibrated imaging densitometer (Bio-Rad).Surface proteins of transfected MDCK II cells or primary brainstem and spinal cord neurons (14 DIV) were washed with 1.0 ml of Phosphate Buffered Saline (PBS) at 4°C and incubated for 40 min at 4°C with 1 mg/ml of non-permeable sulfo-NHS-SS-biotin reagent (Thermo Fisher Scientific) in PBS. Cells were then washed 3 times with 1 ml of the same solution supplemented with 100 mM lysine and scraped in 50 mM Tris-HCl [pH 7.4], 150 mM NaCl (TN) buffer plus 0.4 mM phenylmethylsulfonylfluoride (PMSF) and protease inhibitor mixture (Sigma). Total proteins were solubilized for 30 min at 4°C in RIPA buffer . Streptavidin-agarose beads (40 μl per sample: Sigma) were added and incubated for 1 h at 4°C with agitation. Bead-bound biotinylated proteins (B) were eluted for 15 min at 70°C with Laemmli buffer . Total protein  and biotinylated protein  were run on a 7.5% SDS-polyacrylamide gel and after analyzed by Western blot with specific GlyT2 antibodies, the bands were visualized by ECL and quantified in the linear range on a GS-710 calibrated imaging densitometer (Bio-Rad) with Quantity One software. Calnexin immunoreactivity was used as a non-biotinylated protein control. The standard error of the mean (S.E.M) was calculated after densitometric analysis of at least three separate experiments.3H]-labelled glycine  diluted to a final glycine concentration of 10 μM, as described previously Transport assays in MDCK cells were performed at 37°C in PBS plus 10 mM glucose, containing 2 μCi/ml -glycine uptake assays depicted in 3H]-glycine uptake assays showed similar transport activity for 4KR mutant and wild-type GlyT2 . DUBs are fundamental in the regulation of protein ubiquitination, they influence in crucial functions in the nervous system and its dysfunction is involved in some neurodegenerative diseases Specific inhibition of UCHL1 with LDN-57444 and of UCHL3 with TCID diminished GlyT2 ubiquitination, which was more pronounced when UCHL1 was inhibited and when cells were exposed to these inhibitors for longer periods . The relWe previously demonstrated that GlyT2 is recycled between the cell surface and cell interior via constitutive and PKC-regulated clathrin-dependent endocytosis, resulting in the localization of a large proportion of the transporter in a subset of Rab11-positive endosomes in CNS nerve terminals under steady-state conditions In the present study, we sought to elucidate the putative role of ubiquitination in the constitutive internalization and sorting to recycling and/or degradation pathways of neuronal GlyT2. Our results demonstrate that constitutive GlyT2 endocytosis is dependent on ubiquitination, as indicated by the decreased ubiquitination and concomitant accumulation of GlyT2 at the cell surface following inhibition of E1 ubiquitin-activating enzyme. This enzyme is the first component of the multi-step enzymatic process  that mediates the transfer of ubiquitin to lysine residues on target proteins. Indeed, substituting the C-terminal cluster of lysine residues significantly inhibited GlyT2 internalization, pointing to the K751, K773, K787 and K791 residues as major targets of ubiquitination in constitutive GlyT2 endocytosis. Similarly, ubiquitination of cytosolic lysine clusters has been implicated in the constitutive endocytosis of the glutamate neurotransporter (GLT1) Moreover, the mutation of the C-terminal lysine cluster does not alter the neuronal localization of GlyT2 as indicated by the similar co-localization levels between 4KR mutant and GlyT2 wild type with the PDZ protein syntenin-1 and the SNARE protein component syntaxin1A in hippocampal neurons. Both proteins interact with GlyT2 and have been involved in the regulation of its presynaptic localization and trafficking In addition to promoting internalization, ubiquitination of neuronal membrane proteins can also dynamically control the post-endocytic sorting to the recycling or lysosomal degradation pathways The availability of ubiquitin to target proteins in distinct cellular pathways is essential for nervous system function and DUBs contribute to ubiquitin homeostasis by recycling ubiquitin from lysosomal and proteasomal substrates. The human genome encodes nearly 95 DUBs (SLC6A5) mutations associated with hyperekplexia It should be noted that ubiquitination is an important regulatory mechanism for inhibitory synaptic neurotransmission through control of the trafficking and turnover of glycine and GABA receptors


Prrxl1 displays a multiple band pattern on western-blots (WB), which is abrogated by incubation with a phosphatase. Our aim is to further characterize the nature and sites of Prrxl1 post-translational modifications and their impact on its function.Prrxl1 is a homeodomain transcription factor essential for the connectivity and survival of nociceptive neurons in the mouse embryo dorsal root ganglion (DRG) and spinal cord (SC). Prrxl13+ immobilized metal ion affinity chromatography resin, with affinity for phosphopeptides. Constructs corresponding to truncated versions of Prrxl1 were generated by molecular cloning, and their WB band pattern, transcriptional activity and DNA-binding activity were assessed by luciferase-reporter assays and by a modified DNA pull-down assay. CNBr chemical cleavage of recombinant protein was performed to enhance this analysis. Moreover, site-directed mutagenesis was performed for an evolutionarily conserved putative phosphorylation site which was subjected to the same analysis.Mice were dissected and nuclear extracts of DRG and dSC of mouse embryos  were prepared using a Triton-sucrose buffer and analysed by WB. This time-course analysis was further completed by 2D-electrophoresis (isoelectric focusing followed by SDS-PAGE) of embryonic and post-natal dSC extracts. Furthermore, nuclei of ND7/23 cells (a DRG-derived cell-line with nociceptive properties) and embryonic dSC were incubated with a GAPrrxl1 is highly phosphorylated and its phosphorylation state varies in a time and tissue specific manner. Phosphorylation seems to occur throughout the protein, but mainly in the N-terminus (encompassing a DNA-binding domain) and in the C-terminus (containing a conserved putative regulatory domain). Evolutionary conserved site analysis revealed a highly conserved phosphorylation site in the homedomain, whose mutation impairs transcriptional activation, but not DNA-binding. Luciferase reporter assays of truncated versions of Prrxl1 have identified the N-terminus as sufficient for the DNA-binding and transcriptional activity of Prrxl1.Altogether, our results show that Prrxl1 is mainly phosphorylated in the C-terminus and in the N-terminus. Moreover, these phosphorylations probably play a fundamental role in the regulation of Prrxl1 transcriptional activity in nociceptive neurons.


Gastroesophageal reflux disease (GERD) is the main etiological factor behind the recent rapid increase in the incidence of esophageal adenocarcinoma. During reflux, esophageal cells are exposed to bile at low pH resulting in cellular damage and inflammation, which are known to facilitate cancer development. In this study, we investigated the regulation of p73 isoform, ΔNp73α, in the reflux condition. Previous studies have reported that ΔNp73 exhibits anti-apoptotic and oncogenic properties through inhibition of p53 and p73 proteins. We found that direct exposure of esophageal cells to bile acids in an acidic environment alters the phosphorylation of ΔNp73, its subcellular localization and increases ΔNp73 protein levels. Upregulation of ΔNp73 was also observed in esophageal tissues collected from patients with GERD and Barrett’s metaplasia, a precancerous lesion in the esophagus associated with gastric reflux. c-Abl, p38 MAPK, and IKK protein kinases were identified to interact in the regulation of ΔNp73. Their inhibition with chemotherapeutic agents and siRNA suppresses ΔNp73. We also found that pro-inflammatory cytokines, IL-1β and TNFα, are potent inducers of ΔNp73α, which further enhance the bile acids/acid effect. Combined, our studies provide evidence that gastroesophageal reflux alters the regulation of oncogenic ΔNp73 isoform that may facilitate tumorigenic transformation of esophageal metaplastic epithelium. Esophageal adenocarcinoma (EA) is one of the fastest rising tumors in the United States and Western World, accounting for a 6-fold increase in incidence in the past three decades p53 is an important regulator of DNA damage response and a key tumor suppressor. Its inactivation predisposes Barrett’s epithelial cells to the genomic instability and facilitates progression to cancer Here we investigated the regulation of the ΔNp73 protein in conditions of gastroesophageal reflux.Human telomerase-immortalized CP-A (ATCC) and BAR-T1 . The following mammalian expression vectors were used: c-Abl (P242E/P249E)-pcDNA3 , IKKα (S176E/S180E)-pCMV and IKKβ (S177E/S181E)-pCMV2 , p38-pMT3 (Addgene), and MKK6 (S207E/T211E)-pcDNA3 (Addgene). siRNAs against IKKα, IKKβ and p38 were from Cell Signaling, c-Abl and control siRNAs were from Life Technologies. Cells were transfected with Lipofectamine 2000 (Life Technologies) following the manufacturer’s protocols.Cells were treated with bile acids cocktail (BA) consisting of a 20 µM equimolar mixture of glycocholic acid, taurocholic acid, glycodeoxycholic acid, glycochenodeoxycholic acid, and deoxycholic acid sodium salts ; total BA concentration was 100 µM. For cell treatment, BA cocktail was diluted in DMEM, pH 4.0 (BA/A); pH was adjusted with HCl. Human cytokines, TNFα and IL-1β were purchased from PeproTech. Cell survival was analyzed using MTT analysis as previously described Specific kinase inhibitors of c-Abl , IKK , p38 MAPK , and Aurora A  were added to growth medium at the indicated concentrations one hour before and immediately after treatment with BA/A.Antibodies for the following proteins were used: ΔNp73 (N-16), c-Abl (K-12) and phospho-p73 (Tyr99) from Santa Cruz Biotechnology; p73 from Bethyl Laboratories, phospho-c-Abl (Tyr412), phospho-serine (PSR-45) and FLAG-tag (M2) from Sigma-Aldrich; Phospho-IKKα/β (16A6), IKKα (#2682), IKK β (L570), phospho-Aurora A (Thr288) (C39D8), Aurora A (1G4), DYKDDDDK tag, β-actin (13E5), phospho-p38 MAPK (Thr180/Tyr182), and p38 MAPK (#9212) from Cell Signaling, p73 (Ab2) and phosphotyrosine (4G10) from Millipore. Immunoprecipitation was performed with ΔNp73 (N-16) antibody or Anti-FLAG M2 affinity gel (Sigma-Aldrich) following manufacturer’s protocol.After the Institutional Review Board’s approval, 16 specimens from patients with Barrett’s esophagus, 7 normal gastric and 5 normal esophageal specimens were collected at Vanderbilt University Medical Center, histologically verified and used for immunohistochemical staining for ΔNp73. Immunohistochemical staining of BE esophageal tissues was performed using ΔNp73-specific antibody (Imgenex) at 1∶200 dilution as previously described For indirect immunofluorescence staining, cells were grown to 50% confluency on chamber slides, treated with BA/A for 30 min and 7 hours after treatment were fixed in 1∶1 solution of methanol:acetone. The fixed cells were treated with 10% goat serum (Life Technologies) and incubated with the FLAG-tag(M2) antibody at 4°C for 16–18 hours and then with AlexaFluor 568-conjugated secondary antibody (Life Technologies) for 45 min at room temperature. Slides were photographed using Olympus BX41 fluorescent microscope (Olympus Co.).The use of all human pathology specimens for research was approved by the Institutional Review Board (IRB) of Vanderbilt University Medical Center . Since only de-identified tissues were included in this retrospective study, the IRB has waived requirements for informed consent.5′- TGTACGTCGGTGACCCCGCACG-3′ and 5′-TCGGTGTTGGAGGGGATGACA-3′. Data are presented as average ± SD.RNA was isolated using the RNeasy kit (Qiagen). Total RNA (1 µg) was reverse transcribed with the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Quantitative real-time PCR was performed using an iCycler thermal cycler and iQ SYBR Green Supermix (Bio-Rad). Levels of ΔNp73 mRNA were assessed using the following primers: t-test. Results are expressed as averages ± SE, if not specifically indicated. Results were considered significant at values of p<0.05.Statistical analysis was performed using the Student’s Expression of ΔNp73 was not previously assessed in patients with Barrett’s metaplasia. Therefore, we first analyzed the expression of ΔNp73 protein in patients with BE who were also diagnosed with GERD. Sixteen BE biopsies, 5 normal esophageal and 7 gastric specimens were analyzed by immunohistochemistry with ΔNp73-specific antibody. Since GERD-associated esophageal tumors overexpress ΔNp73 in vitro using previously published measurements of pH, concentration and composition of bile acids in the refluxate of GERD patients To investigate how ΔNp73 is regulated by gastroesophageal reflux, these conditions were recapitulated Since protein stabilization via phosphorylation has been demonstrated for all members of the p53 family, including p73, we first assessed the phosphorylation of ΔNp73 protein after BA/A treatment using pan-phosphoserine and pan-phosphotyrosine antibodies. Our analyses revealed that BA/A treatment increases phosphorylation of the ΔNp73 protein at tyrosine and serine residues suggesting that phosphorylation may play a role in the regulation of ΔNp73 . These fGiven that bile acids treatment can induce both cellular stress and proliferation, we also examined corresponding serine-threonine kinases p38 MAPK, IKKα/β and Aurora A To corroborate the role of IKKα/β and p38 kinases, these proteins were downregulated with specific siRNAs in SK-GT-4 cells, which were then treated with BA/A , 4C. We Considering that p38 and IKKβ are well-known mediators of inflammatory response and that inflammation plays a critical role in esophageal tumorigenesis, we explored their role in more detail. To induce these kinases, SK-GT-4 cells were treated with pro-inflammatory cytokines IL-1β (20 ng/ml) or TNFα (20 ng/ml) alone or in combination with BA/A and then analyzed for ΔNp73 protein expression at the indicated time points. Protein levels of ΔNp73 were strongly increased following treatment with both cytokines . MoreoveIn the present study, we investigated the regulation of oncogenic isoform of p73, ΔNp73, in precancerous conditions associated with gastroesophageal reflux and Barrett’s esophagus, which are known risk factors for the development of esophageal adenocarcinoma. We found that the ΔNp73 protein is strongly induced in Barrett’s epithelium of GERD patients, where this protein was primarily localized in the nuclei of epithelial cells. Approximately 30% of patients with Barrett’s metaplasia were found to have an increased expression of the ΔNp73 protein.We and others have previously reported that ΔNp73 functions as an oncogenic protein immortalizing primary murine cells and cooperating with other cellular oncogene in tumorigenic transformation in vitro. A short exposure of Barrett’s cells to acid and bile acids, which mimics a typical episode of gastroesophageal reflux, was found to induce the ΔNp73 protein and its translocation to the nucleus. The induction of ΔNp73 is mediated by posttranslational mechanisms through phosphorylation of ΔNp73 protein. Protein stabilization via phosphorylation is a common trait for all members of the p53 family. However, even for the most studied member of the family, p53, many questions remain about regulation of its protein stability We were able to recapitulate the induction of ΔNp73 in Barrett’s esophageal cells We identified p38 MAPK, IKKs and c-Abl kinases to be activated by BA/A and are involved in the regulation of ΔNp73 protein. Specifically, the ΔNp73 protein is phosphorylated at Tyr50 residue by c-Abl kinase. Identification of other phosphorylation sites requires additional studies and is currently precluded by a lack of phospho-specific antibodies.in vitro but also in the Barrett’s epithelium following reflux in vivoOur findings are consistent with previous studies showing that activation of p38 kinase contributes to an increase in proliferation and decrease in the apoptosis rate that were found in esophageal metaplastic cells exposed to reflux We found that inhibition of either p38, IKK or c-Abl kinases is sufficient to suppress ΔNp73 protein, suggesting that the combined phosphorylation at various serine/threonine/tyrosine residues are required for full stabilization of ΔNp73 protein. In addition, previous studies have demonstrated multiple functional interactions between p38, IKK and c-Abl kinases. c-Abl has been found to activate the p38 signaling pathway On a practical note, our studies imply that inhibition of upstream regulatory kinases by chemotherapeutic agents may be used as a plausible strategy for inhibition of ΔNp73 protein. Among them, c-Abl inhibitor Imatinib, which is employed for treatment of chronic myeloid leukemia, was found to efficiently inhibit ΔNp73 in esophageal cells exposed to BA/A.We also analyzed Aurora A kinase, which has been found to phosphorylate and induce p73 protein Another important finding directly related to the mechanism of ΔNp73 regulation is the role of cytokines. Pro-inflammatory cytokines, such as IL-1β and TNFα, are induced by reflux damage and contribute to esophageal tumorigenesis Figure S1Phosphorylation of ΔNp73 at serine and tyrosine residues is increased after BA/A treatment. AGS cell stably transfected with FLAG-tagged ΔNp73α plasmid were harvested 4 hours after BA/A treatment . The ΔNp73a protein was then immunoprecipitated with the M2-affinity gel and analyzed by Western blotting using either p-Ser or p-Tyr antibodies.(TIF)Click here for additional data file.


In particular, typical 2-Cys peroxiredoxins are the featured peroxidase enzymes that receive the electrons from NADPH by coupling with thioredoxin and thioredoxin reductase. These enzymes distribute throughout the cellular compartments and, therefore, are thought to be broad-range antioxidant defenders. However, recent evidence demonstrates that typical 2-Cys peroxiredoxins play key signal regulatory roles in the various signaling networks by interacting with or residing near a specific redox-sensitive molecule. These discoveries help reveal the redox signaling landscape in mammalian cells and may further provide a new paradigm of therapeutic approaches based on redox signaling.Mammalian cells have a well-defined set of antioxidant enzymes, which includes superoxide dismutases, catalase, glutathione peroxidases, and peroxiredoxins. Peroxiredoxins are the most recently identified family of antioxidant enzymes that catalyze the reduction reaction of peroxides, such as H CP) in the amino terminus of one subunit reacts with the hydroperoxides and the resulting CP sulfenic acid forms a disulfide linkage with the sulfhydryl group of resolving cysteine residue (CR) in carboxyl terminus of another subunit [2O2 reduction reaction through the formation of intramolecular disulfide linkage [2O2, which is continuously produced by the dismutation of superoxide anions formed as a result of a partial reduction of the dissolved oxygen molecules during mitochondrial respiration. Prx4 is in both endoplasmic reticulum (ER) and extracellular fluid. Recent studies indicate that Prx4 is involved in the oxidative protein-folding pathway by the reoxidation of protein disulfide isomerase [It is generally accepted that the cellular antioxidant enzymes belong to a group of the oxidoreductase enzymes maintaining the cellular redox homeostasis. However, the importance of antioxidant enzymes is given a spotlight after a paradigm shift of the cellular function of reactive oxygen species (ROS) from toxic respiratory by-products to a signaling second messenger. Peroxiredoxin (Prx) is a family of antioxidant enzymes exhibiting peroxidase activity which reduces the hydroperoxides to water in the presence of proper electron donors. Prxs are classified by the number of cysteine residues involved in the peroxidase activity: 2-Cys Prxs and 1-Cys Prx. The 2-Cys Prxs form a disulfide bond by reacting with the peroxides and the disulfide is reduced by thioredoxin which is coupled with thioredoxin reductase and NADPH. Therefore, 2-Cys Prxs are the first thioredoxin-dependent peroxidase enzymes [ subunit . In cont linkage . The 2-Csomerase , 6. The CP) and reactivated by sulfiredoxin-dependent reduction [in vitro enzyme reaction with high concentration of H2O2 and oxidatively-stressed cells, the CP-sulfenic acid at the active site of typical 2-Cys Prxs is overoxidized to sulfinic/sulfonic acids [CR) buries away in latent enzyme and then reacts with CP-sulfenic acid by local unfolding of the C-terminus [CP-sulfenic acid tolerates an additional reaction of the CP-sulfenic acid with the second molecule of H2O2. Consequently, the 2-Cys Prxs can be inactivated by overoxidation during the reaction cycle and, if the inactive enzymes are accumulated, the local H2O2 concentration may be raised (“Floodgate” hypothesis).Beside their primary function as antioxidant enzymes, the observation that 2-Cys Prxs peroxidase activity can be readily inhibited by overoxidation of the active site cysteine residue  and epidermal growth factor (EGF) signaling pathways [2O2-mediated reversible oxidation of PTPs has become an important regulatory mechanism controlling protein tyrosine phosphorylation [κB kinases α/β (IKKα/β) were shown to harbor a reactive cysteine between two serine residues, which are the dual phosphorylation sites critical for activation, in T-loop [2O2-mediated cysteine oxidation [2O2 level. In a live cell, the intracellular H2O2 level is determined by balancing the H2O2 generators  and antioxidants . Among cellular peroxidases, 2-Cys Prxs are the most abundant enzymes and versatile in the subcellular distribution. In particular, the evidence indicates that the two cytosolic forms, Prx1 and Prx2, are likely the key enzymes in the phosphorylation signaling pathway -α [2O2-treated cells, but not in PDGF-treated cells [2O2-treated cells and thus promoted the Ras- or ErbB2-drived cell transformation. Hence, it was proposed that Prx1 might contribute someway to the tumorigenesis. However, the function of Prx2 as a signal regulator was initially proposed by the differential regulation of TNF-α-induced MAP kinase activation [β only at two tyrosine sites (Y579 and Y857), which was not mimicked by addition of exogenous H2O2. Such selective regulation was achieved by the stimulation-dependent interaction of Prx2 and PDGFRβ proteins, which allowed the reactivation of a membrane-associated PTP. This is the first report showing the selective action of endogenous H2O2 distinguished from the exogenous source of H2O2. Recently, Prx2 was also shown to preserve the VEGFR2-dependent tyrosine phosphorylation in vascular endothelial cells by protecting the receptor from oxidative inactivation by both the endogenous and exogenous H2O2 [2O2 was not identified, it is an important finding that Prx2 functions upstream of the receptor tyrosine kinase whose activity is regulated by an oxidation-sensitive cysteine residue.Protein phosphorylation is one of the most important posttranslational modifications in the membrane receptor-mediated growth factor and cytokine signaling and as such modulates protein-protein interaction, enzyme activity, and protein stability and structure. Human genome encodes over 500 putative kinase genes and more than 150 protein phosphatases including dual-specificity phosphatases and protein tyrosine phosphatases (PTP). With the exception of the gical pH , which r in vivo , 18. Sinpathways , 20, therylation , 22. Recn T-loop –25. The xidation , 27. Suc pathway . The fired cells . Prx1 intivation . Also, Ptivation . In thisous H2O2 . This fu90-Pro-Arg-Lys), and among them the Prx1 and Prx2 were indeed phosphorylated by Cdk1/Cdc2 [in vitro, it was observed only in Prx1 in vivo using the mitotic arrested HeLa cells. However, its biological significance remains unsolved. Other studies also showed that the Prx1 threonine phosphorylation is mediated by serine/threonine kinase Mst1/2 [2O2 level. In contrast, the serine phosphorylation of Prx1 by a T-cell-originated protein kinase (TOPK) increased the peroxidase activity [32  in vitro and in human melanoma cells. It is noteworthy that the activated TOPK colocalized with Prx1 in nucleus, which is the first indication of nuclear Prx1. Later, both Prx1 and Prx2 were found in the nucleus and, particularly, Prx2 protects the cancer cell death against DNA damaging agents [The cytosolic 2-Cys Prxs are themselves linked to the phosphorylation networks as their activities are regulated by phosphorylation. Chang et al. reported that the 2-Cys Prxs contain the conserved CDK phosphorylation sequence  [There are several studies implicating the redox regulation of lysine acetylation network. One study showed that Hof HDAC2 . Anothermyocytes . Upon oxe (GSNO) . Interese (GSNO) .CR residue [2O2 [Many studies show that the 2-Cys Prx activity is regulated by acetylation . A recen residue . In the due [2O2 . It is nAlthough there is no evidence showing a direct regulatory role of 2-Cys Prxs in the lysine acetylation network, it will be interesting to investigate the mechanism of how the acetylation and deacetylation network is associated with 2-Cys Prxs in various subcellular compartments.ψm) [α receptor (TNFR)-1 and Fas (CD95), induces necroptosis in some cell types [α [α-induced necrosis in L929 cells [The role of ROS in cell death has been a long-standing issue because mitochondria are the key players in both apoptotic and necrotic cell death pathways. Indeed, mitochondria are the site where the electron transport takes place and leakage of the high energy electrons from the electron carrier complexes can combine with molecular oxygen to produce ROS . Higher ψm) . The invψm) –60. Contψm) . It has ll types , 63. Fortypes [α . Human Jtypes [α . Therefotypes [α . Furthertypes [α . The act29 cells . In addi29 cells , 70, was29 cells .The cell death studies by modulation of cellular antioxidant enzymes reveal a clear role of intracellular ROS in apoptosis. Particularly, the 2-Cys Prxs play a regulatory role in apoptotic, not necrotic, cell death . Prx1 wa2O2 [2O2 produced by Ero1, which is known as the main ER enzyme responsible for reoxidation of protein disulfide reductase [Despite the 2-Cys Prx being a sophisticated peroxidase enzyme with a high affinity to H2O2 , recent 2O2 . It was 2O2 . By this2O2 , 82. The2O2 . The fin2O2 . Another2O2 . In moto2O2 , 6. The eductase . This evin vitro activity assays showed that Prx2 is less active as a peroxidase enzyme than Prx1 [in vitro evidence that the 2-Cys Prxs is inactivated by overoxidation during the reaction cycle proportional to the enzyme activity [2O2 stress. Nonetheless, it turned out that Prx2 is more susceptible for overoxidation in the animal cells under H2O2 stress than Prx1 [2O2 stress in vivo. This discrepancy between in vitro and in vivo properties of Prx1 and Prx2 could be explained as a paradox: unlike the potential function assumed from the in vitro characterization, the Prx2 can be the real peroxidase enzyme in the cells while the Prx1 primarily functions as a redox regulator of diverse client proteins by interaction. This idea is supported to some extent because it was observed that Prx1 was more abundant in protein amount than was Prx2 in certain cell types like fibroblasts and HeLa cells.In contrast to the case of Prx1, the closest isoform Prx2 has barely been shown to directly interact with any protein. Actually, few reports are stating that Prx2 colocalizes and interacts with phospholipase D1 in phorbol ester-stimulated cells  as well han Prx1 . Given tactivity , it is chan Prx1 . The samPathway Studio software. We obtained the network relations for all four Prxs in the knowledge base of Pathway Studio, which were built by text-mining of literature texts. False positives or indirect relations were removed by inspecting the relevant sentences manually. In an effort to obtain a global picture of regulations by 2-Cys Prxs, we finally produced a network model among typical 2-Cys Prxs using the 2O2 is an important second messenger in a signaling network, the discovery of the 2-Cys Prx function related to signal transduction should provide clues necessary to understand redox signaling architecture and further solve medical problems in ROS-mediated chronic diseases.Four members of typical 2-Cys Prx subfamily are present in various cellular compartments, including cytosol, plasma membrane , nuclei, mitochondria, and endoplasmic reticulum. The majority of the abundant 2-Cys Prx enzymes primarily function as general antioxidant systems that maintain the intracellular ROS level within a safety zone in both normal and stressed cells. However, some part of the enzymes functions as the signal regulator at specific locations by modulating the local ROS change or by regulating the activity of the interacting/neighboring proteins in a redox-dependent manner. Since H


Pseudomonas syringae, which disable an overlapping range of kinases in Arabidopsis and Tomato. Both effectors target surface-localized receptor-kinases to avoid bacterial recognition. In turn, tomato has evolved an intracellular effector-recognition complex composed of the NB-LRR protein Prf and the Pto kinase. Structural analyses have shown that the most important interaction surface for AvrPto and AvrPtoB is the Pto P+1 loop. AvrPto is an inhibitor of Pto kinase activity, but paradoxically, this kinase activity is a prerequisite for defense activation by AvrPto. Here using biochemical approaches we show that disruption of Pto P+1 loop stimulates phosphorylation in trans, which is possible because the Pto/Prf complex is oligomeric. Both P+1 loop disruption and transphosphorylation are necessary for signalling. Thus, effector perturbation of one kinase molecule in the complex activates another. Hence, the Pto/Prf complex is a sophisticated molecular trap for effectors that target protein kinases, an essential aspect of the pathogen's virulence strategy. The data presented here give a clear view of why bacterial virulence and host recognition mechanisms are so often related and how the slowly evolving host is able to keep pace with the faster-evolving pathogen.The major virulence strategy of phytopathogenic bacteria is to secrete effector proteins into the host cell to target the immune machinery. AvrPto and AvrPtoB are two such effectors from  Pseudomonas syringae is a pathogen of many crop species and one of the model pathogens for studying plant and bacterial arms race coevolution. In the current model, plants perceive bacteria pathogens via plasma membrane receptors, and recognition leads to the activation of general defenses. In turn, bacteria inject proteins called effectors into the plant cell to prevent the activation of immune responses. AvrPto and AvrPtoB are two such proteins that inhibit multiple plant kinases. The tomato plant has reacted to these effectors by the evolution of a cytoplasmic resistance complex. This complex is compromised of two proteins, Prf and Pto kinase, and is capable of recognizing the effector proteins. How the Pto kinase is able to avoid inhibition by the effector proteins is currently unknown. Our data shows how the tomato plant utilizes dimerization of resistance proteins to gain advantage over the faster evolving bacterial pathogen. Here we illustrate that oligomerisation of Prf brings into proximity two Pto kinases allowing them to avoid inhibition by the effectors by transphosphorylation and to activate immune responses.The bacteria  Pseudomonas syringae, which are secreted directly into the host cell where they target a PRR complex composed of the receptor kinases FLAGELLIN SENSING 2 (FLS2) and BRI1-ASSOCIATED KINASE 1 (BAK1) that forms after perception of bacterial flagellin by FLS2 Plant immunity is innate and relies on two levels of pathogen perception, underpinned by different recognition strategies Plants have evolved a second layer of perception based on the presence of pathogen effectors within the host cell. Host resistance (R) proteins containing a central nucleotide-binding (NB) motif, of the STAND class, and C-terminal leucine rich repeats (LRRs) recognise specific effectors directly or indirectly, and induce strong defences leading to hypersensitive cell death response (HR). The responses induced by PRRs and NB-LRRs respectively have not been separated experimentally, and it is thought that these classes of proteins simply represent different points of entry to the same defence network The mechanisms by which accessory proteins communicate effector binding to the NB-LRR protein are unknown. For the described examples, each complex exists prior to effector interaction. Accessory proteins make contact with regions of the R proteins N-terminal to the NB domain The Pto/Prf complex recognises both the AvrPto and AvrPtoB effectors. The recognition event occurs through the accessory protein kinase Pto Here we show that Pto molecules transphosphorylate each other, but only when in complex with Prf, and only under conditions of complex activation. Pto was doubly phosphorylated within the kinase activation segment, and this was necessary but not sufficient for signalling. Full activation of the complex required additional disturbance of the P+1 loop, by mutation or by interaction with the effector. We derive a model for activation of the complex by effectors, and show how the oligomeric arrangement of the complex provides a trap for the effector that is very difficult for the pathogen to avoid.Nicotiana benthamiana by heterologous expression of its constituent components. In this system, co-expression of the tomato Pto and Prf proteins confers recognition of the effectors AvrPto and AvrPtoB leading to HR Agrobacterium tumefaciens to express Prf transiently as a genetic fusion with three C-terminal haemagglutinin epitopes (Prf-3HA) in stable transgenic 35S:Pto N. benthamiana plants K1128A) To elucidate the role of Pto kinase activity within the Prf complex, we reconstituted this complex in the model species N. benthamiana after heterologous expression of Prf -3HA, FLAG-tagged Pto, and effectors. After immunoprecipitation of Prf using anti-HA antibodies, we were unable to identify the putative Pto phosphorylation sites in these experiments for technical reasons. Subsequently, the total Pto protein comprising both the Prf-complexed and free forms were purified from N. benthamiana. Immunoprecipitated Pto was subjected to SDS-PAGE fractionation, in-gel tryptic digestion and mass spectrometric analysis. In three independent experiments, we identified peptides spanning over 80% of the Pto protein and found that residues Ser-198 and Thr-199 within the kinase activation segment peptide K/GTELDQTHLSTVVK were modified by phosphorylation  in all subsequent HR assays. Both single mutants induced cell death after AvrPto or AvrPtoB co-expression, comparable to wild-type Pto  detected both fast and slow migrating forms in the presence of Pto-FLAG and Prf-HA, but the slow form was again suppressed by the presence of prfK1128-HA , which mimics the negative charge of phosphorylation. Expression of ptoS198D/T199D did not induce effector-independent HR, but the mutant was able to respond to AvrPto and AvrPtoB (S198A/T199A variant described earlier (D164N/S198D/T199D) weakened but did not prevent recognition of AvrPto  abrogated its HR-inducing ability and resulted in a marked band shift of ptoS198A/T199A/L205D to a fast-migrating, not phosphorylated form, in comparison to ptoL205D . The cell death was estimated using images of cell death from three independent experiments using two leaves in each experiment. All pictures were taken two days post infiltration.Growth and transient expression conditions for N. benthamiana, leaves frozen in liquid nitrogen were added to extraction buffer, 150 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 5% glycerol (v/v), 10 mM dithiothreitol (DTT), 2% polyvinylpolypyrrolidone (PVPP), 1% plant protease inhibitor cocktail (Sigma), and 0.5 mM PMSF and homogenized with a Polytron. Protein extracts were centrifuged at 20,000 g for 20 min at 4°C. Supernatants were subjected to filtration through a 0.45 µm filter. Sepharose affinity matrices used were anti-FLAG M2 and anti-HA HA-7 (both Sigma). Extracts were mixed with affinity matrices as indicated for two hours at 4°C, with gentle rotation, in batch format. Affinity matrices were washed three times with an excess of extraction buffer. Proteins were stripped from the bead fraction by boiling in SDS loading buffer. Elution from anti-FLAG beads was performed by incubation with extraction buffer containing 200 µg/mL FLAG peptide (Sigma) for 10 min at 25°C with gentle mixing. To concentrate elutions, Strataclean (Stratagene) beads were added to bind proteins, and then pelleted by centrifugation subsequently the proteins were stripped from the beads by boiling in 1× SDS-PAGE loading buffer.For the analysis of protein accumulation from 2, 1 mM MnCl2, 1 mM DTT, 20 µM ATP, 183 kBq of γ[32P]-ATP (PerkinElmer Life Sciences) in a total volume of 30 µl. In Pto transphosphorylation assays, Pto-FLAG, ptoD164N-FLAG, ptoS198A-FLAG, ptoS199A-FLAG and ptoS198A/T199A-FLAG were transiently expressed in N. benthamiana and immunoprecipitated with anti-FLAG beads as described above. pti1K96N-His and GST-AvrPtoB were expressed and purified from Escherichia coli as previously K96N-His and 2 µg of GST-AvrPtoB were included in the kinase reaction mixture. All reactions were initiated by addition of the kinase mixture, incubated at 30°C for 20 min, and terminated by addition of SDS-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer and boiling for 10 min. Under these assay conditions, incorporation of radiolabel was found to be linear with time, the substrate and the enzyme concentration used. At the end of each assay, samples were loaded onto SDS-PAGE. Post electrophoresis, proteins were transferred onto polyvinylidene difluoride membranes and stained with Coomassie Brilliant Blue R-250. Subsequently, the membranes were subjected to autoradiography using a FUJI Film FLA5000 PhosphorImager . Relative autophosphorylation and transphosphorylation kinase activity was calculated as the ratio between incorporated radioactivity  and the amount of immunoprecipitated protein estimated using ImageJ based on Coomassie staining of the membranes and expressed as a percentage of Pto-FLAG relative autophosphorylation or transphosphorylation kinase activity.In vitro kinase assays were performed as described Co-immunoprecipitated protein complexes were separated by SDS-PAGE and gel slices were excised. Proteins were reduced with DTT and alkylated with iodoacetamide before in-gel trypsin (Promega) digestion overnight at 37°C. After digestion, the supernatant was moved to a clean tube and the gel pieces washed sequentially with 50% and 100% acetonitrile and the washes pooled with the supernatant. Volume and the organic content of the peptide solution were reduced by lypholisation and the peptides stored at −20°C until use. Peptides were dissolved in 0.5% formic acid immediately before analysis by LC MS/MS.m/z) 300 to 2000, minimum signal 1000, collision energy 35, 2 repeat hits, 60 sec exclusion. MS3 were triggered if the neutral loss of phosphoric acid (49 m/z for 2+ parent ions) was detected in the three most abundant ions on the preceding MS2. Collision energy for MS3 was 35. In all cases the mass spectrometer was operated in positive ion mode with a nano-spray source and a capillary temperature of 200°C, no sheath gas was employed and the source and focusing voltages were optimised for the transmission of angiotensin.LC-MS/MS analysis was performed using a LTQ-Orbitrap XL mass spectrometer (Thermo Scientific) and a nanoflow-HPLC system . Peptides were applied to a precolumn  connected to a self-packed C18 10-cm analytical column . Peptides were eluted by a gradient of 2 to 50% acetonitrile in 0.1% formic acid over 50 min at a flow rate of approximately 250 nL min-1. Data-dependent acquisition of MS/MS consisted of selection of the five most abundant ions in each cycle: MS mass-to-charge ratio (Peak lists (as .dat files) were prepared from raw data using extract_msn in BioWorks 3.3 (Thermo Electron Corp.) and collated using merge.pl (Matrix Science). The data generation parameters were: MW range 300.00–3500.00, threshold absolute 1000, group scan 10, minimum group 0, minimum ion cont 10, charge state auto  MS level auto. Peak lists were searched against SPtrEMBL, (containing 8385695 sequences) Taxonomy was restricted to Solaneae (7247 sequences) with the following variable modifications were allowed; oxidized methionine, phosphorylation on serine and threonine. Carbamidomethyl was specified as a fixed modification on cysteine residues. Precursor mass tolerance was 5 ppm, fragment tolerance 0.5 Da mass values were monoisotopic and two missed tryptic cleavages were allowed. Subsequently for proteins identified with >95% probability an ‘error tolerant’ search was preformed in Mascot with relaxed criteria for modifications and enzyme cleavage. Scaffold  was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 95.0% probability as specified by the Peptide Prophet algorithm In addition to the minimum PeptideProphet score provided by Scaffold, we manually evaluated the fragmentation spectra of all phosphorylated peptides of Pto to ensure that good b and y ion coverage was observed and that neutral loss of the phosphate (common in ion trap CID) supported the assigned phosphorylated residue. We used our PhosCalc algorithum to assist this interpretation . The peptides covering the activation loop (KGTELDQTHLSTVVK) were observed with and without a tryptic miscleavage of the N-terminal K, and single and double phosphorylation events were observed on both tryptic fragments. Furthermore, the single miscleaved peptide was observed in both 2+ and 3+ ionisation states, while most other peptides were predominantly 2+, thus providing abundant evidence of mass shifts and fragmentation patterns. This peptide contains four possible phosphorylation sites. We observed spectra, which supported single phosphorylation at T190, S198 or T199. Site S198 was most strongly supported and frequently observed. We also observed a doubly phosphorylated form of KGTELDQTHLSTVVK, again with and without the N-terminal miscleavage. Manual examination of the spectra supported phosphorylation at S198 and T199 in most cases, occasional spectra supported phosphorylation at T190 and either S198 or T199. It is important to note that even in cases where the exact site of modification remains ambiguous this does not detract from the double phosphorylation of the peptide as a whole, due to the clear difference in parent ion masses and overall fragmentation. Representative fragmentation spectra for the peptide (KGTELDQTHLSTVVK) are shown in Sequence data from this article can be found in the GenBank data library under the following accession numbers: Prf (U65391), Pto (DQ019170), avrPto (L20425), avrPtoB (Q8RSY1).Figure S1Inhibition of Pto/Prf–mediated signalling in trans. (A) The Pto/Prf ligand-independent hypersensitive cell death response (HR) in N. benthamiana is compromised by co-expression of the loss-of-function mutant prfK1128A. The indicated Prf-5Myc, prfK1128A-3HA, Cp-GFP and Rx-HA constructs were transiently expressed in stable transgenic 35S:Pto (no tag) N. benthamiana plants. As a control for specificity, prfK1128A was co-expressed with CP-GFP and Rx-HA. The picture was taken at three days post infiltration. Protein expression was confirmed by immunoblots (IB) with the antibodies indicated on the right. Coomassie Brilliant Blue (CBB) staining of the IB membrane verified equal protein loading. The experiment was repeated several times and typical results are shown. (B) AvrPto-induced hypersensitive cell death response is compromised by co-expression of the loss-of-function mutant prfK1128A. prfK1128A-3HA, Pto-Flag, ptoD164N-FLAG and AvrPto constructs were transiently expressed in stable transgenic ProPrf:Prf-5Myc N. benthamiana leaves as indicated and the tissue was stained with trypan blue 2 days post infiltration. The bar indicates 0.5 mm. Dead cells stain dark blue in this assay.(TIF)Click here for additional data file.Figure S2MS spectra of the Pto peptides 188–202 (GTELDQTHLSTVVK). (A) Spectra supporting single phosphorylation on S198 (Mascot score 85.96) (B) Spectra supporting single phosphorylation on T199 (Mascot score 49.23). (C) Spectra supporting double phosphorylation on S199 and T199 (Mascot score 49.33).(TIF)Click here for additional data file.Figure S3MS spectra of the Pto peptides 187–202 (KGTELDQTHLSTVVK). (A) Spectra supporting single phosphorylation on S198 (Mascot score 84.08) (B) Spectra supporting single phosphorylation on T199 (Mascot score 54.7). (C) Spectra supporting double phosphorylation on S199 and T199 (Mascot score 62.61).(TIF)Click here for additional data file.Figure S4Phosphorylation on Pto residues S198 and T199 is required for induction of cell death.  Relative Hypersensitive Response (HR) index was estimated from trypan blue staining of cell death in N. benthamiana leaves based on three independent experiments. Cell death stains dark blue in this qualitative assay and was estimated using ImageJ. The proteins were transiently expressed as indicated and pictures were taken two days post infiltration. Representative pictures are in (TIF)Click here for additional data file.Figure S5Inhibition of Prf CGF HR in trans. (A) The prfD1416V constitutive gain-of-function (CGF) phenotype in N. benthamiana is compromised by co-expression of the loss-of-function mutant prfK1128A. The indicated prfK1128A-3HA, prfD1416V-5Myc, Cp-GFP and Rx-HA constructs were transiently expressed in N. benthamiana plants. As a control for specificity, prfK1128A was co-expressed with CP-GFP and Rx-HA. The picture was taken at three days post infiltration. Protein expression was confirmed by immunoblots (IB) with the antibodies indicated on the right. Coomassie Brilliant Blue (CBB) staining of the IB membrane verified equal protein loading. The experiment was repeated several times and typical results are shown. (B) The K1128A and D1416V mutations do not affect the ability of Prf variants to dimerise. Transgenic 35S:Pto N. benthamiana leaves were transiently transformed with prfK1128A-5Myc alone, or in combination with Prf-3HA, prfD1416V-3HA, or prfK1128A-3HA. Infiltrated leaves were collected two days post infiltration for extraction of proteins. Tagged proteins were immunoprecipitated using anti-HA beads. Crude extract (input) and beads fractions were analysed by SDS-PAGE followed by immunoblotting with the antibodies indicated on the right. Equal protein loading was confirmed by CBB staining of the immunoblot membrane.(TIF)Click here for additional data file.Figure S6Mutants of S198 and T199 are kinase active forms of Pto. Kinase activity assays showing Pto autophosphorylation and transphosphorylation of Pti1K96N and AvrPtoB. Pto-FLAG, ptoD164N-FLAG, ptoS198A-FLAG, ptoT199A-FLAG, and ptoS198A/T199A-FLAG proteins were transiently expressed in N. benthamiana and immunoprecipitated with anti-FLAG M2 affinity matrix. pti1K96N-His and GST-AvrPtoB substrates were expressed and purified from Escherichia coli. Coomassie Brilliant Blue (CBB) staining of the immunoblot (IB) membranes verified that equal amounts of pti1K96N-His and GST-AvrPtoB were added in each assay. Relative autophosphorylation and transphosphorylation kinase activity was calculated as the ratio between incorporated radioactivity and the amount of immunoprecipitated protein estimated based on CBB staining of the IB membrane and expressed as a percentage of Pto-FLAG relative autophosphorylation or transphosphorylation kinase activity.(TIF)Click here for additional data file.Figure S7Double phosphorylation on S198 and T199 is necessary for signalling. (A) Trypan blue staining of cell death in N. benthamiana leaves. Pto-FLAG, pto mutant-FLAG and avrPtoBF479A constructs were transiently expressed in stable transgenic ProPrf:Prf-5Myc N. benthamiana leaves as indicated and the tissue was stained 24 or 48 hours post infiltration. The bar indicates 0.5 mm. Dead cells stain dark blue in this qualitative assay. Each row is derived from a single leaf, within which relative amounts of cell death were comparable, and is representative of three replicates. (B) Relative Hypersensitive Response (HR) index. Images of trypan blue staining of cell death in N. benthamiana leaves from three independent experiments were used to estimate the Relative HR index. The proteins were transiently expressed as indicated and pictures were taken as indicated in A. Each graph is derived from three leaves, within which relative amounts of cell death were comparable. Error bars are standard deviation.(TIF)Click here for additional data file.Figure S8he slower migrating from of Pto was suppressed in transD164N, but not by ptoS189A/T199A. by pto Prf-3HA, Pto-5Myc, Pto-FLAG, ptoD164N-FLAG, ptoS189A/T199A -FLAG and avrPtoBF479A constructs were transiently expressed in wild-type N. benthamiana as indicated. The E3 ligase mutant avrPtoBF479A was used instead of wild-type AvrPtoB to avoid ptoD164N degradation. Prf-3HA was immunoprecipitated (IP) using anti-HA antibodies. The relative abundance of slow- and fast-migrating forms of Pto-FLAG, ptoD164N-FLAG, ptoS189A/T199A -FLAG were quantified using anti-FLAG immunoblots (IB) (upper graph). Anti-Myc immunoblots (lower graph) were used for quantification of slow- and fast-migrating forms of Pto-5Myc in the presence of different Pto variants. Quantity One, Bio-Rad (adjusted volume = [CNT*mm2] data counts/mm2) was used for quantification of anti-FLAG and anti-Myc immunoblots. Error bars are standard deviation of relative abundance between the same samples in independent immunoblots, probed with the anti-FLAG or anti-Myc antibodies.T(TIF)Click here for additional data file.Figure S9Transphosphorylation is required for induction of cell death.  Relative Hypersensitive Response (HR) index was estimated from trypan blue staining of cell death in N. benthamiana leaves based on three independent experiments. Cell death stains dark blue in this qualitative assay and was estimated using ImageJ. The proteins were transiently expressed as indicated and pictures were taken two days post infiltration. Representative pictures are in (TIF)Click here for additional data file.Figure S10D164N compromises hypersensitive cell death response (HR) The kinase inactive ptoin trans. (A). The Hypersensitive Response (HR) triggered by AvrPto recognition is compromised by kinase-inactive ptoD164N. The indicated AvrPto, Pto-5Myc and ptoD164N-HA constructs were transiently expressed in stable transgenic ProPrf:Prf-5Myc N. benthamiana leaves. The picture was taken at three days post infiltration. Protein expression was confirmed by immunoblots (IB) with the antibodies indicated on the right. Coomassie Brilliant Blue (CBB) staining of the IB membrane verified equal protein loading. The experiment was repeated several times and typical results are shown. (B) The cell death phenotype triggered by the constitutive gain-of-function (CGF) mutant ptoL205D is compromised by the co-expression of kinase-inactive ptoD164N. The indicated ptoL205D-GFP, Pto-5Myc and ptoD164N-HA constructs were transiently expressed in stable transgenic ProPrf:Prf-5Myc N. benthamiana leaves. The picture was taken at three days post infiltration. Protein expression was confirmed by immunoblots with the antibodies indicated on the right. CBB staining of the IB membrane verified equal protein loading. The experiment was repeated several times and typical results are shown.(TIF)Click here for additional data file.Table S1Double phosphorylation of Pto peptide 187–202 and 1888-202 upon activation of signalling.(DOCX)Click here for additional data file.Table S2Double phosphorylation of Pto peptide 187–202 and 188–202 upon activation of signalling.(DOCX)Click here for additional data file.Table S3Summary of the different complex combinations examined, and their signalling outcomes.(DOCX)Click here for additional data file.