Cell polarization is essential for processes such as cell migration and asymmetric cell division. A common regulator of cell polarization in most eukaryotic cells is the conserved Rho GTPase, Cdc42. In budding yeast, Cdc42 is activated by a single guanine nucleotide exchange factor, Cdc24. The mechanistic details of Cdc24 activation at the onset of yeast cell polarization are unclear. Previous studies have suggested an important role for phosphorylation of Cdc24, which may regulate activity or function of the protein, representing a key step in the symmetry breaking process.in vivo phosphorylation sites. We first focus on sites matching consensus sequences for cyclin-dependent and p21-activated kinases, two kinase families that have been previously shown to phosphorylate Cdc24. Through site-directed mutagenesis, yeast genetics, and light and fluorescence microscopy, we show that nonphosphorylatable mutations of these consensus sites do not lead to any detectable consequences on growth rate, morphology, kinetics of polarization, or localization of the mutant protein. We do, however, observe a change in the mobility shift of mutant Cdc24 proteins on SDS-PAGE, suggesting that we have indeed perturbed its phosphorylation. Finally, we show that mutation of all identified phosphorylation sites does not cause observable defects in growth rate or morphology.Here, we directly ask whether multisite phosphorylation of Cdc24 plays a role in its regulation. We identify through mass spectrometry analysis over thirty putative We conclude that lack of phosphorylation on Cdc24 has no overt functional consequences in budding yeast. Yeast cell polarization may be more tightly regulated by inactivation of Cdc42 by GTPase activating proteins or by alternative methods of Cdc24 regulation, such as conformational changes or oligomerization. Cell polarization is the process by which cells establish asymmetry along a single axis and is essential for processes such as cell migration and asymmetric cell division The Cdc42 GTPase has been shown to regulate cell polarity in many organisms, including budding yeast in vitroin vivo.Polarization of yeast cells must occur at the proper time in the cell cycle, suggesting a potential regulatory role for the cyclin-dependent kinase (CDK) Cdc28 on Cdc24, which leads to timely activation of Cdc42. Interestingly, Cdc24 is sequestered in the nucleus during G1 by the CDK-inhibitor Far1 Δbud1 cells grow and divide at the same rate as wild type cells, although they bud in a random pattern per se. This may be due to redundant activation events mediated by the adaptor protein Bem1. In addition to possibly causing a conformational change by directly binding Cdc24, Bem1 also mediates the formation of a complex containing GTP-bound Cdc42, Cdc24, and the p21-activated kinase (PAK) Cla4, and enables Cla4 to phosphorylate Cdc24 GTP accumulation in vivo.Yeast cell polarity is also regulated in a spatial manner. Haploid yeast cells generally bud adjacent to the previous bud scar, which is a remnant from the previous cell division site. The Ras family GTPase Bud1/Rsr1 recruits Cdc24 to the bud site in order to establish polarized growth in vivo phosphorylation sites on Cdc24. Surprisingly, mutagenesis of these putative phosphorylation sites (including those matching consensus sequences for CDK or PAK family kinases and those not) has not resulted in any observable defects in cell polarization and growth by a variety of assays, suggesting that phosphorylation is largely dispensable for the regulation of Cdc24.There is precedent for regulation of Dbl homology GEFs by phosphorylation in vivo phosphorylation sites by mass spectrometry analysis. This was challenging because of the following factors: a) Cdc24 is present in low quantities In order to better understand how Cdc24 may be regulated by multisite phosphorylation and, in particular, how this phosphorylation contributes to the Bem1-dependent and actin-independent cell polarization, we set out to purify Cdc24 from yeast cells in order to identify the cdc28-13, a temperature-sensitive allele of CDC28, was performed to enrich for small-budded cells, and thus phosphorylated Cdc24. Initially, cell lysis was performed using a French press, but Cdc24 in these lysates was mostly insoluble. Solubility of Cdc24 was increased by lysing frozen yeast cells with a coffee grinder and increasing the salt concentration of the lysis buffer  method s buffer . ContamiCdc24-TAP was purified from 60 g of yeast cells , resolved by SDS-PAGE , and subAdjacent residues were grouped together to facilitate analysis . These single or clustered sites were then ranked based on their scores on NetPhos 2.0 in vivo function of the phosphorylation sites in Cdc24. Because of the large number of sites and because of previous work implicating CDK and PAK in Cdc24 regulation, we first examined the CDK and PAK sites. The different phosphorylation mutants are described in cdc24, were lost in the CDK mutants, i.e. they had reverted back to the wild type sequence. S245A was retained in the CDK-A/PAK-A mutant. The other mutant residues were successfully mutated, and these are denoted in We next used mutagenesis to study the The mutant strains were viable (see below), and we examined whether these mutations affected phosphorylation of Cdc24. Asynchronous cultures of the mutant strains were lysed and resolved by SDS-PAGE to assess the mobility shift of Cdc24, which was shown previously to be caused by phosphorylation CDC24 is an essential gene and the only known GEF for Cdc42 in yeast, we expected to see a severe growth phenotype if these mutations affect Cdc24 function. However, these mutations, including the CDK and PAK sites mutated individually or in combination, did not confer cell lethality or temperature-sensitivity, as all strains grew like the wild type control at all temperatures tested (Because s tested . Cell mos tested  and S2A.CDK-A/PAK-Acdc24 (RLY3088). Cell growth assayed by colony formation , but we noticed that Cdc24 protein in the Δbud1 strain had a significantly reduced mobility shift, suggesting an abnormal phosphorylation state  than the PH-A/linker-A mutant. Mobility shift may not be strictly caused by phosphorylation events, and it is possible that the additional mutations somehow affect the mobility of the protein. It is also possible that any compensatory phosphorylation on the 35A mutant may affect its mobility shift, more so than the compensatory phosphorylation on the PH-A/linker-A mutant. We conclude that we have perturbed most, if not all, of the native phosphorylation sites on Cdc24, but this perturbation did not affect the function of the protein in any obvious manner.We did, however, observe a decrease in the mobility shift of these mutants. Using the procedure described earlier, we quantified the mobility shift of the Cdc24 band from asynchronous cultures resolved by SDS-PAGE . The PH-in vitroin vivo significance of these phosphorylation events remained unclear. Here, we have mapped over thirty in vivo phosphorylation sites on Cdc24. However, to our surprise, mutation of sites matching the conserved consensus sequences for the CDK and PAK family kinases has not revealed any evidence for their functional importance. We have ruled out overt effects on temperature-sensitivity, cell growth, cell morphology, protein localization, and polarization kinetics in the presence and absence of actin. Our results on the putative CDK sites are consistent with a previous study reporting that mutation of these sites did not affect Cdc24 function in vivoIn the experiments described above, we investigated the potential role for Cdc24 phosphorylation in the establishment of cell polarity and polarized growth. These experiments were motivated by the suggestion that phosphorylation plays a key role in regulating the activity and/or localization of Cdc24 to achieve symmetry breaking in a cell cycle dependent manner. Although Cdc24 phosphorylation was abundantly observed in several studies, and two kinases, Cdc28 and Cla4, were shown to phosphorylate Cdc24 Δboi2 background) and impaired Boi1 localization Our results for multisite phosphorylation on Cdc24 differ from results of a study reporting multisite phosphorylation of Boi1, a protein that binds Bem1 and plays an essential but redundant role in cell polarization with Boi2. In that study, the authors were able to identify phenotypes resulting from perturbation of phosphorylation. Mutation of the 12 consensus CDK sites or the 29 mapped phosphorylation sites on Boi1 resulted in temperature sensitive growth  was transformed with these fragments. Transformants were replica-plated onto media lacking leucine or YPD media containing geneticin. Transformants that were Leu+ and GenS (indicating that the fragment had replaced the Δcdc24::KanMX locus) were sporulated and dissected. Integration of the LEU2 marker into the proper locus was confirmed by PCR. Point mutations that were successfully integrated were determined by sequencing of genomic DNA purified from the resulting strain. PCR-based GFP tagging was performed as previously described Yeast strains containing mutations of the CDK or PAK consensus sites (as described in CDC24 (RLY2775). The resulting strains were sporulated and dissected. Spores were identified that were both Leu+ and GenS, indicating that they carry the genomic deletion of Cdc24 (Δcdc24::KanMX), rescued by the mutant Cdc24 on the plasmid.Yeast strains containing the mutations of non-consensus sites  and 100–200 µl of 20% TCA were added to the pellets. The mixture was then vigorously vortexed for 7 min to lyse the cells. 400 µl of 5% TCA was added to the mixture, and all of the liquid was transferred to new tubes. The protein pellet was harvested by spinning for 10 min at 3000 rpm in a microcentrifuge. After removal of liquid, the pellet was resuspended in SDS-PAGE sample buffer and titrated with Tris base if necessary.Whole cell extracts for Western blot analysis were prepared by TCA precipitation. Cell pellets were harvested from 5–10 ml cultures grown to ODStandard methods were used for SDS-PAGE and Western blotting. Protein samples were resolved on 7.5% polyacrylamide gels, and proteins smaller than 50 kD were run off the gel in order to allow maximum separation of Cdc24 and its phosphorylated species (“smear”). Blots were developed using ECL or ECL Plus Reagents . For quantification, blots were scanned using the Typhoon 9400  and analyzed using ImageQuantTL.472-854 construct that was previously described Anti-Cdc24 antiserum was raised in rabbits  using an MBP-Cdc24cdc28-13 allele (RLY2194). The cdc28-13 strain was arrested in G1 at the restrictive temperature (37°C) for no more than 3 h and released into the cell cycle by cooling down the culture to 25°C using an ice water bath. Cells were monitored for bud formation and harvested about 1 h after release, when most cells were small-budded. Cells were washed once with harvest buffer ). Cell pellets were frozen in small strands in liquid nitrogen. Cell lysis was performed in a small coffee grinder (Krups), cooled with dry ice in order to keep yeast cells frozen. Lysed cells were resuspended in lysis buffer  and containing 1 mM PMSF). Extracts were immediately centrifuged at low speed (2430 g) in order to remove unlysed cells, then incubated on ice for 30 min. Extracts were then centrifuged at high speed  to remove nonsoluble components. Protein was precipitated using 40% ammonium sulfate, and the precipitate was stored at −80°C. Precipitate (which contained Cdc24-TAP) was resuspended in lysis buffer and centrifuged briefly to remove any undissolved precipitate. Extract was incubated with washed IgG Sepharose 6 Fast Flow beads  for 4 h at 4°C. Slurry was applied to Poly-Prep disposable columns (Bio-Rad), and beads were washed thoroughly with lysis buffer containing 0.1% NP-40. Beads were then washed with TEV buffer (harvest buffer without protease inhibitors), and incubated overnight at 4°C with purified GST-TEV (tobacco etch virus) protease to cleave off Cdc24. Beads were washed with small amount of lysis buffer, and the first two washes were pooled with the eluate. These were passed through Glutathione Sepharose 4B beads  to remove most of the GST-TEV protease. The flowthrough and first wash from the glutathione beads were pooled and precipitated by 20% TCA. The precipitate was resuspended in a small volume of SDS-PAGE sample buffer and resolved by SDS-PAGE on a 7.5% gel.Cells were synchronized by using a temperature-sensitive 2HPO4/20% isopropanol, pH 10.5 (with 4 M ammonia in ethanol), and the two combined extracts were acidified with 5% formic acid prior to drying by vacuum centrifugation. Finally, the dried extracts were desalted using STAGE tips as described Coomassie-stained bands corresponding to Cdc24 were excised from SDS-PAGE gels, destained to clarity with 50 mM ammonium bicarbonate/50% acetonitrile solution, and digested in-gel using established procedures The dried, desalted samples were resuspended in 6 µl of a 1% formic acid/2.5% acetonitrile solution immediately prior to sequencing analysis. Sequencing was performed by nanoscale microcapillary LC-MS/MS essentially as described 600 of 0.1 to make the starting cultures. Starting cultures were pipetted into the leftmost wells of a sterile 96-well plate, and serial dilutions of four-fold were made in the subsequent wells. After thorough mixing, a sterilized frogger was used to spot the array onto the desired number of agar plates. After the cells were allowed to grow for 2–3 d at the 23°C, 30°C, 35°C, or 37°C, or 7–13 d at 16°C, plates were imaged using a digital camera or scanner. Images of each plate were processed using Adobe Photoshop CS3. The contrast was increased using the Auto Levels command, in order to better display the yeast colonies against the dark background. The images were subsequently cropped to display only the strains that were relevant to the experiment.Overnight cultures were diluted to an ODYeast cells were fixed by adding formaldehyde to a final concentration of 5% and incubating at least 2 h at room temperature with gentle shaking. Cells were then washed once in PBS, before storing at 4°C in PBS. Before scoring for morphology, fixed cultures were briefly sonicated in order to separate clumps. In order to assess bud scar patterns, Calcofluor staining was performed essentially as described z-projections. MetaMorph software  was used to control both microscopes. Image J software  was used to process the images.Widefield imaging was performed on a Nikon Eclipse E1000 fluorescent microscope with a 100X Plan-Apo TIRF NA 1.45 oil objective. Samples were illuminated with a mercury lamp through a 3-cube GFP filter, and images were acquired using a Hamamatsu ORCA-ER camera. Confocal imaging was performed on a Zeiss Axiovert 200 M inverted microscope with a 100X αPlan-Fluar NA 1.45 oil objective, attached to a Yokogawa spinning disk confocal and Hamamatsu EM-CCD C9100 camera. Images of GFP-tagged phosphorylation site mutants were collected as a series of 13 optical sections, with a step size of 0.3 µm. The final images shown are maximum Cells were arrested for 3 h using 5 µg/ml α-factor. In order to determine budding kinetics, cells were released into the cell cycle by washing three times in sterile water before resuspending in fresh media. Samples were taken every 10 min and fixed with formaldehyde as described above. At each time point, 100 cells were scored for budding morphology . In order to determine polarization kinetics, all arrested cells were treated with 100 µM LatA  for 20 min to depolarize GFP-myc-Cdc42, and then released by washing three times in water before resuspending in fresh media with or without LatA. At each time point, >40 cells were scored for polarized localization of GFP-myc-Cdc42.7 cells of each strain were spread into an even layer onto YPD plates. A total of three plates were spread for each strain and grown for 3 d at 30°C to reach stationary phase. For each strain, 1 L of YEPD media was inoculated with the cells from two plates. Half of the culture was immediately harvested. All centrifugation steps were performed at 4°C. Each pellet was washed in buffer consisting of 50 mM HEPES pH 7.5, 0.1 M KCl, 3 mM MgCl2⋅6H2O, 1 mM EGTA, 1 mM DTT, supplemented with a protease inhibitor cocktail. The cells were frozen in liquid nitrogen as small pellets and stored at −80°C. The remaining culture was incubated at 30°C and harvested as above when most of the cells had become small-budded. Yeast extracts were prepared by grinding the frozen cell pellets with a mortar and pestle that had been cooled with liquid nitrogen. Lysed yeast powder was stored at −80°C until all samples had been lysed. The powder from each sample was resuspended in a small volume of the same buffer described above, but also including 0.2% Triton X-100 and 1 mM PMSF. Extracts were spun for 30 min at 100,000 g to remove unlysed cells and insoluble material. The amount of protein was quantified by the Bradford Assay (Bio-Rad), and extracts were adjusted so that they had the same protein concentration. Each pull-down reaction contained 20 µl of GST-CRIB beads  and 400–500 µl of yeast extract. Pull-downs were incubated at 4°C for 1 h with gentle shaking. After washing the beads 3–4 times in the same buffer, the beads were resuspended in SDS-PAGE sample buffer. All samples were boiled and spun briefly before loading onto an acrylamide gel.Bacteria expressing either a GST fusion construct containing the CRIB domain from Cla4 (RLB252) or just GST for control (RLB247) were lysed using a French Press (1700 psi), and the cleared lysate was loaded onto Glutathione Sepharose 4B beads . The beads were washed and stored at 4°C until use. Yeast strains expressing GFP-myc-CDC42 (pRL369) and either wild type Cdc24 (RLY2903) or mutant Cdc24 (RLY3072) were grown up overnight. 3×10Figure S1cdc28-13 cells (RLY434) were arrested at 37°C and synchronously released at 25°C. Samples of the culture were either fixed for morphology analysis or frozen for extract preparation every 10 min. Blots were probed with rabbit anti-Cdc24 antiserum. Faint band just above smear is a cross-reacting protein (*). B. Peak of mobility shift coincides with peak in the percentage of small-budded cells. At least 100 cells were scored for bud morphology at each timepoint. Percentage of small-budded cells was plotted over time. Budded cells present initially are likely because fixed cultures were not sonicated to separate cells before being scored. C. Comparison of Cdc24-TAP solubility in lysates prepared by either French press or manual grinding methods. Solubility was further increased by increasing salt concentration of lysis buffer. Cleared lysate (S1) was centrifuged at 10,000 g to give P2 and S2, and S2 was centrifuged at 100,000 g to give P3 and S3. Equal volumes of each fraction were loaded. Blots were probed with rabbit anti-Cdc24 antiserum. The top band of the doublet seen in some blots is a cross-reacting protein (*). D. Coomassie-stained gel showing final product of purification from RLY2194. Band at 75 kD is a heat-shock protein, and bands at 50 kD are GST and GST-TEV protease.Purification of Cdc24-TAP. A. Mobility shift of Cdc24 band changes during the cell cycle. (0.69 MB TIF)Click here for additional data file.Figure S2CDK-3A-GFP (RLY3098), Cdc24CDK-4A-GFP (RLY3066), Cdc24PAK-A-GFP (RLY3065), Cdc24CDK-DE-GFP (RLY3100), and Cdc24PAK-DE-GFP (RLY3101). Scale bar represents 5 µm. B. Confocal images of strains containing Cdc24-GFP , Cdc24linker-A-GFP (RLY3434), and Cdc24PH-A,linker-A-GFP (RLY3430). Scale bar represents 5 µm.Phosphorylation site mutants do not affect localization of Cdc24. A. Confocal images of strains containing Cdc24-GFP Click here for additional data file.Table S1Phosphorylated Cdc24 peptides(0.08 MB DOC)Click here for additional data file.Table S2Ranking of phosphorylated residues(0.01 MB PDF)Click here for additional data file.Table S3Scansite results of phosphorylated sites(0.06 MB PDF)Click here for additional data file.Table S4Plasmids used in this study(0.04 MB DOC)Click here for additional data file.Table S5Yeast strains used in this study(0.10 MB DOC)Click here for additional data file.


Both prostaglandin E2 and iloprost inhibited, in a concentration-dependent fashion, the lipopolysaccharide-induced generation of nitric oxide and tumour necrosis factor-α. The inhibitory effect of these prostanoids seems to be mediated by an increase of the second messenger cAMP since it was mimicked by dibutyryl cAMP and potentiated by the selective type IV phosphodiesterase inhibitor RO-20-1724. Our results suggest that the inhibition of nitric oxide release by prostaglandin E2 and iloprost in lipopolysaccharide-activated J774 macrophages may be secondary to the inhibition of tumour necrosis factor-α generation, which in turn is likely to be mediated by cAMP.The effect of prostaglandtn E


The fibroblast growth factor receptor (FGFR) interprets concentration gradients of FGF ligands and structural changes in the heparan sulfate (HS) co-receptor to generate different cellular responses. However, whether the FGFR generates different signals is not known.MAPK and so cannot stimulate DNA synthesis. Here we demonstrate that this is because in the absence of HS, FGF-2 fails to stimulate the phosphorylation of the adaptor FGFR substrate 2 (FRS2). In cells possessing the HS co-receptor, FGF-2 elicits a bell-shaped dose response: optimal concentrations stimulate DNA synthesis, but supramaximal concentrations (≥ 100 ng/mL) have little effect. At optimal concentrations (300 pg/mL) FGF-2 stimulates a sustained dual phosphorylation of p42/44 MAPK and tyrosine phosphorylation of FRS2. In contrast, 100 ng/mL FGF-2 only stimulates a transient early peak of p42/44 MAPK phosphorylation and fails to stimulate appreciably the phosphorylation of FRS2 on tyrosine.We have previously shown in rat mammary fibroblasts that in cells deficient in sulfation, and so in HS co-receptor, FGF-2 can only stimulate a transient phosphorylation of p42/44 These results suggest that the nature of the FGFR signal produced is determined by a combination of the HS co-receptor and the concentration of FGF ligand. Both the phosphorylation of the adaptor FRS2, the kinetics (sustained or transient) of phosphorylation of p42/44(MAPK) are varied, and so differing cellular responses are produced. MAPK represent a key pathway in cellular regulation by growth factors and their kinetics of activation can determine cell fate  thymidine  was added directly to the culture medium 18 h later for 1 h and radioactivity in DNA, precipitated with 5% (w/v) trichloroacetic acid, was measured by liquid scintillation counting.DNA synthesis assays were performed as described previously ,19. Ramasuc1 [2 in 9 cm diameter culture dishes (1.3 × 10 6 cells in 10 mL medium per dish) and then treated as for the DNA synthesis assay up to the addition of growth factors. At the times indicated, Rama 27 cells were washed twice with ice-cold Tris-buffered saline (TBS) contained 100 μM sodium vanadate and then lysed in 500 μL lysis buffer  glycerol, 1% (v/v) NP-40, 1 mM sodium vanadate, pH7.0) with one tablet of protease inhibitor mixture  per 10 mL. Culture dishes were gently rocked for 20 min at 4°C after addition of lysis buffer. Adherent material was then detached by scraping and the lysates were transferred to pre-cooled microcentrifuge tubes. Culture dishes were washed once with 200 μL of lysis buffer, which was combined with the lysate. Lysates were clarified by centrifugation at 12000 g for 10 min. The supernatants were then transferred to fresh microcentrifuge tubes containing 40 μL lysis buffer and p13 suc1 agarose suspension (3:1 v/v) and the slurry was incubated at 4°C on a rocker platform overnight. Agarose beads were collected by centrifugation at 12000 g for 5 min and washed by centrifugation three times with lysis buffer and once with water. Following resuspension of the final pellet in 60 μL electrophoresis sample buffer  SDS, 0.006% (w/v) bromophenol blue, 2% (v/v), β-mercaptoethanol, pH 6.8) with one tablet of protease inhibitor mixture per 10 mL, samples were boiled for 5 min.FRS2 binds the cyclin-dependent kinase substrate p13 suc1 , which psuc1 pull-down experiments. At the times indicated, Rama 27 cells were washed twice with ice-cold PBS and collected by scraping in 300 μL of 2× electrophoresis sample buffer and then boiled for 5 min.Rama 27 fibroblasts were cultured exactly as for p13 MAPK was detected using a monoclonal antibody directed against the doubly phosphorylated Thr 183/202Tyr 185/204 forms of these enzymes. This antibody does not cross-react, according to the manufacturer, with the corresponding doubly phosphorylated SAPK/JNK, p38 MAPKs or monophosphorylated p42/44 MAPK. The phosphorylation of FRS2 was detected using anti-phosphotyrosine 4G10 following P13 suc1 pulling down or by a polyclonal antibody directed against the phosphorylated FRS2-α (Tyr196). This antibody does not cross-react with unrelated tyrosine-phosphorylated proteins and it produced results equivalent to the pull down followed by Western blotting with 4G10. Where appropriate, the same membrane was re-probed with anti-actin or anti-FRS2 to show the level of loading. Each Western blot is one of at least three independent experiments.Polypeptides in samples containing identical amounts of protein were separated by electrophoresis on appropriate polyacrylamide gels in the presence of SDS and transferred to nitrocellulose membranes. Following blocking with TBS containing 0.05% (v/v) Tween 20 (TBS-Tween) and 5% (w/v) non-fat dry milk (blocking buffer) for 1 h, membranes were probed with appropriate primary antibodies overnight with gentle shaking at 4°C. After three washes with TBS-Tween, the membranes were incubated with the corresponding secondary peroxidase-conjugated polyclonal antibodies in blocking buffer for 1 h, washed three times in TBS-Tween, and bound peroxidase was detected with the SuperSignal chemiluminescent system  on Hyperfilm (Amersham Pharmacia Biotech). Activated p42/44 The authors declare that they have no competing interests.HZ performed the bulk of the experiments, with LD assisting. DGF and PSR conceived the idea for this study, HZ and DGF wrote the manuscript. All authors read and approved the submitted manuscript.


Several effects of leptin in the immune system rely on its capacity to modulate cytokine expression and apoptosis in the thymus. Surprisingly, some of these effects are dependent on signal transduction through the IRS1/PI3-kinase, but not on the activation of JAK2. Since all the well known effects of leptin in different cell types and tissues seem to be dependent on JAK2 activation, we hypothesized that, at least for the control of thymic function, another, unknown kinase could mediate the transduction of the leptin signal from the ObR towards the IRS1/PI3-kinase signaling cascade.Here, by employing immunoblot, real-time PCR and flow citometry we show that the tyrosine kinase, Fyn, is constitutively associated with the ObR in thymic cells. Following a leptin stimulus, Fyn undergoes an activating tyrosine phosphorylation and a transient association with IRS1. All these effects are independent of JAK2 activation and, upon Fyn inhibition, the signal transduction towards IRS1/PI3-kinase is abolished. In addition, the inhibition of Fyn significantly modifies the effects of leptin on thymic cytokine expression.Therefore, in the thymus, Fyn acts as a tyrosine kinase that transduces the leptin signal independently of JAK2 activation, and mediates some of the immunomodulatory effects of leptin in this tissue. Both sides of extreme nutritional dysfunction, i.e., malnutrition and obesity, are known to predispose to anomalous immune activities, which include immunodeficiency, increased predisposition to inflammatory and autoimmune diseases and development of certain types of cancer The mechanisms involved in leptin-dependent regulation of immune function include the capacity of leptin to inhibit thymic apoptosis and the modulation of thymic cytokine expression Here, we show that the tyrosine kinase Fyn, associates with the ObR and delivers a leptin-dependent immunomodulatory signal in the thymus of rodents.Escherichia coli (LPS) and propidium iodide (PI) were obtained from Sigma . SuperSignal West Pico Chemiluminescent Substrate was from Thermo Scientific . Ficoll-Paque™ PLUS was obtained from Becton Dickinson Biosciences , RPMI 1640 from Cultlab , and annexin V was purchased from the Laboratory of Immunobiology of the University of São Paulo . Chemicals for real-time PCR were from Invitrogen  and Applied Biosystems . All other chemicals were standard commercial products of reagent-grade quality. Antisense (5′ - CAC AGC CCA TTA TCC A - 3′) (FynAS) and scramble control (5′ - CAT CCAGTC ACT ACC A - 3′) (FynSCR) oligonucleotides specific for Fyn were produced by IDT . The oligonucleotide sequences were submitted to BLAST analyses (www.ncbi.nlm.nih.gov) and matched only for the Rattus norvegicus Fyn coding sequence (NCBI/NM 012755). Four peptides  were constructed based on different portions of the ObRb's intracellular region, to be used in a competition assay for the Fyn kinase against the receptor. These were purchased from Peptide Protein Research Ltd .Antibodies against JAK-2 (sc-278), Fyn (sc-16), Lck (sc-13), Src , pSTAT3 (sc-7993), SHP2 (sc-424), phosphotyrosine (sc-508), IRS-1 (sc-559), ObR (sc-8325), pERK (sc-7383), Bcl-2 (sc-492), Bax (sc-493), rabbit IgG-B (sc-2040), mouse IgG-B (sc-2039) and goat IgG-B (sc-2042) were from Santa Cruz Biotechnology . Antibodies against p-Src family Tyr416 (2101L) and p-Src family Tyr 527 (2105L) were from Cell Signaling Technology . Protein A Agarose and nitrocellulose paper  were from Amersham . Leptin, JAK inhibitor AG490 (tyrphostin B42) and Fyn inhibitor PP2 were from Calbiochem . Tris base, phenylmethyl-sulphonylfluoride, aprotinin, dithiothreitol, Triton X-100, Tween-20, glycerol, bovine serum albumin , lipopolysaccharide from ad libitum. All experiments involving animals were in accordance with the guidelines of the Brazilian College for Animal Experimentation (COBEA), and were approved by the University of Campinas Ethical Committee. Room temperature was maintained at 21–23°C with 12-h light/dark cycles. The animals were age-matched for individual experiments and randomly distributed into treatment or control groups with free access to a standard rodent chow  and tap water. For some experiments, we additionally employed 10 or 40 day-old male Wistar rats.Three-week old male Wistar rats and nine-week-old Lep/db (db/db) and C57BLKS/J mice were obtained from the University of Campinas Breeding Center. The Lep/db (db/db) mice were originally purchased from the Jackson Laboratory  and are currently established as a colony at the University of Campinas Breeding Center. Eight-month old Zucker rats were obtained from the Laboratory of Physiology of the Federal University of São Paulo (Brazil). The animals were allowed access to standard rodent chow and water −6M via the cava vein. The thymuses were extracted after different time intervals: 0, 5, 10, 15, 20 and/or 30 min. For experiments of Fyn inhibition with antisense oligonucleotide (FynAS) (or the scrambled antisense FynSCR), rats were treated with 400 µL of saline solution; 400 µL of leptin 10−6M; 400 µL of saline solution plus 2.0 nmol FynAS (or FynSCR), or with 400 µL of leptin 10−6M plus 2.0 nmol FynAS (or FynSCR) via intra-peritoneum (ip) during three consecutive days. For experiments of Fyn inhibition with the chemical inhibitor (PP2), rats were treated with 100 µl solution containing 2.5, 5 or 10 nM of PP2 via ip 30 min before the thymus' extraction. Five minutes before the extraction, rats were also treated with 100 µL of saline solution or with 100 µL of leptin 10−6M via the cava vein. For experiments of molecular associations using db/db mice, the animals were treated with 400 µL of saline solution or with 400 µL of leptin 10−6M via ip and the thymus was extracted after 15 minutes. For experiments of molecular associations using Zucker rats, the animals were treated with 100 µL of saline solution or with 100 µL of leptin 10−5M via ip during three consecutive days. The thymus was extracted on the following day. For the determination of gene expression by Real Time PCR, rats were treated with 100 µL of saline solution; 100 µL of lipopolysaccharide (LPS) 1 mg/mL; 150 µL of PP2 5 nM; 100 µL of leptin 31.2 µM or with different combinations of these treatments.For experiments of molecular associations and immunoblottings, rats were treated with 100 µL of saline solution or with 100 µL of leptin 10Rats or mice were anesthetized by ip injection of sodium thiopental (50 mg/kg body weight), and the thymus or hypothalamus were removed. The tissue was minced coarsely, and homogenized immediately in extraction buffer at 4°C with a Polytron PTA 20S generator  operated at maximum speed for 20 s. The extracts were centrifuged at 9,000 x g and 4°C in a Beckman 70.1 Ti rotor  for 20 min to remove insoluble material, and the supernatants were used for immunoprecipitation, for direct immunoblotting, as previously described Samples containing 1.0 mg total protein obtained from thymuses, as described above, were incubated with different concentrations of each one of the four peptides that were constructed to compete against the ObRb receptor for association with Fyn. The concentrations of the peptides employed were 0, 1, 10, 50 and 100 µg, and the incubations were performed overnight at 4°C under gentle rocking. The samples were used for immunoprecipitation with anti-ObR and immunoblotting with anti-Fyn, anti-JAK-2 or anti-SHP2.Rat thymuses were gently homogenized in a manual Dounce homogenizer. Thymocytes were overlaid onto a Ficoll-Paque™ PLUS layer, with density adjusted to 1.076 g/mL, and centrifuged at 1,000 x g at room temperature for 25 min. The interface cell layer containing thymocytes was recovered by Pasteur pipette, washed twice in PBS, and centrifuged at 500 x g for 10 min in vitro thymocyte cultures were obtained by seeding isolated thymus cells at a density of 105 or 106 cells/mL in RPMI 1640 in 1.5 cm2 plate wells in a humidified atmosphere (5% CO2 at 37°C). For determination of markers of apoptosis and cytokine expression, thymocytes were treated according to one of the following protocols: control; leptin 10−8M; PP2 10−8M; or, leptin 10−8M + PP2 10−8M. Apoptosis was evaluated after 23 hours using the Flow Cytometry method for all experiments.The The samples were analyzed in a FACSCalibur flow cytometer equipped with an argon laser and CellQuest software . Ten thousand events were acquired from each sample. The thymocyte populations were identified by their light-scattering characteristics, enclosed in electronic gates, and analyzed for the intensity of the fluorescent probe signal 5 or 106 cells were harvested at each time point, washed twice with PBS and resuspended in a binding buffer containing annexin V-FITC (1∶500). After 20 min of incubation at room temperature, thymocytes were centrifuged at 1,000 x g for 5 min and resuspended in binding buffer containing PI (1∶50). Apoptosis was quantified by FACS analysis as the number of annexin V-FITC positive and PI negative thymocytes as a percentage of the total number, while necrosis was quantified as the number of PI positive and annexin V-FITC negative thymocytes as a percentage of the total number of cells.Thymocytes were labelled with annexin-V, following the manufacturer's instructions Thymic total RNA was extracted using Trizol reagent , according to the manufacturer's recommendations. Real-time PCR analysis of gene expression was carried out in an ABI Prism 7500 sequence detection system (Applied Biosystems). The optimal concentration of cDNA and primers, as well as the maximum efficiency of amplification, were obtained through seven-point, 3-fold dilution curve analysis for each gene. Each PCR reaction contained 75 ng of reverse-transcribed cDNA. Primers were purchased from Applied Biosystems and were: TNFα, Rn99999017; IL-1β, Rn00580432; IL-6, Rn00561420; IL-10, Rn00563409; and GAPD, #4352338E, for rat. The PCR conditions were 2 min at 50°C; 10 min at 95°C, followed by 40 cycles at 95°C for 15 sec and 60°C for 60 sec. Real-time data were analyzed using the engine provided by Applied Biosystems.All numerical results are expressed as the means ± sem of the indicated number of experiments. The results of blots are presented as direct comparisons of bands in autoradiographs. The results of cell viability, estimated by annexin-V and propidium iodide staining, were analyzed by ANOVA and a post-hoc Tukey test. Level of significance was set at p<0.05.vs. Src and vs. Lck, n = 5) (−10M (100 µl leptin through the cava vein) and peaking at 10−6–10−8M. Therefore, in the remaining experiments, we concentrated our efforts on the characterization of leptin action through Fyn, only.Proteins of the Src family can mediate some of the effects of leptin in isolated and transfected cell systems , n = 5) . In addiThe highest expression of Fyn and ObR in the thymus of Wistar rats occurred at 21 days, according to immunoblot analysis of thymic protein extracts obtained from animals at 10, 21 and 40 days of life . TherefoFour peptides were designed to be used in binding-competition assays with the objective of defining the site of Fyn interaction with the ObR . As depivs. control, respectively)  . Secondlctively) .Leptin exerts a potent anti-apoptotic effect on thymic cells Leptin is known to modulate cytokine expression in the thymus Due to the classical hypothalamic actions of leptin in the control of food intake The ObR belongs to the IL-6-like, class 1 cytokine receptor family that contains an extracellular ligand-binding domain, a transmembrane domain and an intracellular signaling domain devoid of intrinsic catalytic activity Most actions of leptin were studied in the hypothalamus where this hormone/cytokine exerts potent anorexigenic/thermogenic effects A recent study has shown that, in an isolated cell system, proteins of the Src family can be activated in response to leptin in a JAK2-independent fashion 527Tyr residue and is constitutively associated at a low level with the ObR. Upon leptin treatment, Fyn undergoes a transient increase in association with the ObR, which coincides with the induction of tyrosine phosphorylation at the activating 416Tyr residue. Finally, the deactivating 527Tyr phosphorylation reinstalls and the levels of Fyn bound to the ObR return to basal levels. While tyrosine phosphorylated on the 416Tyr residue, and highly associated with the ObR, Fyn associates with IRS1 establishing a protein complex that may drive JAK2-independent signals.The dynamics of Fyn tyrosine phosphorylation, in response to leptin, were similar in timing to the activation of JAK2, although occurring faster than the engagement of the substrates of JAK2 and, therefore, suggesting that fyn activation exists as a parallel and independent phenomenon with regard to the classic JAK2 signaling pathway Using a peptide competition assay we mapped the transition of box1 to box 2 as the binding site for Fyn in the ObR. This site lies just below the JAK2 binding site and is approximately 70 residues up from the SHP2 binding site in vivo. With the antisense oligonucleotide approach, we reduced Fyn expression to approximately 40% of basal levels, while using the chemical inhibitor PP2 we virtually abolished leptin-induced Fyn activation. With these two methods we could then determine the role of Fyn in two important phenomena modulated by leptin in the thymus, apoptosis and cytokine expression In order to evaluate the role of Fyn in leptin actions in the thymus we used two distinct methods to reduce Fyn activity, The capacity of leptin to reduce the rate of apoptosis in the thymus has been evaluated in several studies When we evaluated the role of Fyn in leptin-inhibited apoptosis in the thymus, we found no significant changes in the expression of Bax and Bcl-2 and also in the number of apoptotic cells, as determined by flow cytometry. These experiments were repeated a number of times with variations in the doses and times of treatment with the respective inhibitors of Fyn and, consistently, we could detect no changes in the rate of apoptosis inhibited by leptin. Therefore, we believe that Fyn plays no important role in this process.ob gene are immunodeficient and present a defective production of cytokines such as IL-2, TNF-α, IFN-γ and IL-1β Regarding the effects of leptin in the modulation of cytokine production, studies have shown that it can induce the expression of inflammatory cytokines that play a role in the development of autoimmune diseases As a whole, this study identifies a novel tyrosine kinase that is capable of associating and transducing the leptin signal, independently of JAK2. This signaling pathway plays an immunomodulatory role in the thymus. As Fyn also responds to leptin in the hypothalamus, efforts to define its role in the neural action of leptin are required.


PepT1, an intestinal epithelial apical di/tripeptide transporter, is normally expressed in the small intestine and induced in colon during chronic inflammation. This study aimed at investigating PepT1 regulation by butyrate, a short-chain fatty acid produced by commensal bacteria and accumulated inside inflamed colonocyte.We found that butyrate treatment of human intestinal epithelial Caco2-BBE cells increased human PepT1 (hPepT1) promoter activity in a dose- and time-dependent manner, with maximal activity observed in cells treated with 5 mM butyrate for 24 h. Under this condition, hPepT1 promoter activity, mRNA and protein expression levels were increased as assessed by luciferase assay, real-time RT-PCR and Western blot, respectively. hPepT1 transport activity was accordingly increased by ∼2.5-fold. Butyrate did not alter hPepT1 mRNA half-life indicating that butyrate acts at the transcriptional level. Molecular analyses revealed that Cdx2 is the most important transcription factor for butyrate-induced increase of hPepT1 expression and activity in Caco2-BBE cells. Butyrate-activated Cdx2 binding to hPepT1 promoter was confirmed by gel shift and chromatin immunoprecipitation. Moreover, Caco2-BBE cells overexpressing Cdx2 exhibited greater hPepT1 expression level than wild-type cells. Finally, treatment of mice with 5 mM butyrate added to drinking water for 24 h increased colonic PepT1 mRNA and protein expression levels, as well as enhanced PepT1 transport activity in colonic apical membranes vesicles.Collectively, our results demonstrate that butyrate increases PepT1 expression and activity in colonic epithelial cells, which provides a new understanding of PepT1 regulation during chronic inflammation. Butyrate is a short-chain fatty acid (SCFA) produced in the colonic lumen by bacterial fermentation of carbohydrates and dietary fibers +/H+ exchanger (NHE3) Intestinal epithelial cells absorb small dietary peptides by the action of apical membrane peptide transporters. A cDNA encoding an apical membrane protein possessing this peptide transport capability has been cloned from humans and designated hPepT1 In the present study, we investigated the possibility that butyrate could induce colonic PepT1 expression and the underlying molecular mechanisms.in vitro studies.To test the response of the hPepT1 promoter to SCFAs, human intestinal epithelial Caco2-BBE cells were transfected with a reporter construct encoding the putative proximal promoter region of hPepT1 gene (722 bp) previously cloned in our laboratory . Furthervs lane 1). However, in the presence of AcD, hPepT1 mRNA was strongly reduced (lane 2) and the butyrate-induced increase of hPepT1 mRNA was suppressed (lane 4 vs lane 3). In agreement with this result, we found by real-time RT-PCR that hPepT1 mRNA levels in AcD-treated cells and AcD+ butyrate-treated cells were the same , but did not modify the Michaelis-Menten constant Km . These data exclude changes in the affinity of hPepT1 for peptides upon butyrate treatment.Since butyrate induces an increase of hPepT1 expression, we next examined the effect of butyrate on hPepT1 transport activity. hPepT1-mediated Glycine-Sarcosine uptake was measured in Caco2-BBE monolayers pre-treated with 5 mM of butyrate for 4, 8, 12 and 24 h. As shown in It was previously shown that protein kinase A (PKA) plays a permissive role in leptin-mediated stimulation of hPepT1 promoter activity Together, these results show that butyrate enhanced hPepT1-mediated transport events and intracellular signaling pathways like PKA may be involved.Butyrate is a potent histone deacetylase inhibitor and its suppression of histone deacetylation has been shown to lead to accumulation of multiacetylated forms of histone especially histone H4 +7 binding site reduced butyrate-induced activation of hPepT1 promoter by ∼30%, confirming the involvement of PKA. We have previously shown that the transcription factor Cdx2 plays an important role in leptin-mediated hPepT1 promoter activation −216 binding site did not affect hPepT1 promoter activation by butyrate.Since it is known that PKA activates the transcription factor CREB Together, these results demonstrate that the transcription factors Cdx2 and CREB have crucial roles in the activation of hPepT1 promoter by butyrate. Sp1 might also be involved, albeit to a lesser extent.−579 (−262 (vs Lane 1). However, the retardation complexes were not detected in the presence of labeled oligonucleotides containing point mutations at Cdx2−579 and Cdx2 −262 binding sites (Lane 3) or of a 200-fold molar excess of an unlabeled oligonucleotide competitor (Lane 4), indicating that the DNA binding of Cdx2 was sequence-specific. Furthermore, we performed Cdx2 supershift to confirm the binding of Cdx2 to hPepT1 promoter. As shown in To further confirm the importance of Cdx2 in the activation of hPepT1 promoter, we used electrophoretic mobility shift assays (EMSA) to characterize their binding to hPepT1 promoter at Cdx2−579  and Cdx279  to confirm that Cdx2 binds to hPepT1 promoter. Protein-DNA complexes from butyrate-stimulated or un-stimulated Caco2-BBE cells were cross-linked and immunoprecipitated with anti-Cdx2 antibodies. The precipitates were then PCR-amplified with primers specific to each of the Cdx2 binding sites . Our results showed that Cdx2 binds to its putative sites  in butyrate-stimulated cells but not in un-stimulated cells .in vivo role in butyrate induction of hPepT1 promoter activity.These results indicate that Cdx2 may have an To confirm the importance of Cdx2 in hPepT1 regulation, we generated a Caco2-BBE cell line over-expressing V5-tagged Cdx2 (Caco2-BBE/Cdx2) and investigated hPepT1 protein expression in this cell line. Immunoblot analysis using an anti-V5 antibody showed a ∼42 kDa band representing Cdx2 in Caco2-BBE/Cdx2, indicating that these cells over-express this transcription factor . Under tTogether, these results indicate that Cdx2 is important to hPepT1 expression under butyrate stimulation as well as at basal level.N-formyl-methionyl-leucyl-phenylalanine) We previously showed that hPepT1 transports pro-inflammatory bacterial peptides such as fMLP  N-formyl-methionyl-leucyl-phenylalanine (fMLP) in vivo perfusion of fMLP induces PepT1-mediated colonic inflammation in a short bowel model where PepT1 is expressed in colonic epithelial cells Our data demonstrated that butyrate, both 2 atmosphere and 90% humidity, and the medium was changed each 2 days. Cells were grown on 6-well plastic plates or on permeable supports .Caco2-BBE cells were grown in DMEM supplemented with 14 mM NaHCO3, 10% FBS, and penicillin/streptomycin . Caco2-BBE cells stably transfected with Cdx2-pcDNA3 plasmid SCFAs, Actinomycin D and fMLP were obtained from Sigma-Aldrich . H89 was purchased from Biosource .Guide for the Care of Use of Laboratory Animals from the US Public Health Service.Female C57BL/6 mice  used for this study were group-housed under a controlled temperature (25°C) and photoperiod (12:12-hours light-dark cycle), and allowed unrestricted access to standard diet and tap water. All animal procedures were approved by the Animal Care Committee of Emory University and were conducted in accordance to the www.cbrc.jp/) revealed that the cloned promoter region contained multiple putative biding sites for transcription factors. Site directed mutations at the putative CREB+7, Cdx2−579,−564,−262, AP1−216 and Sp1−199,−59,−33,−5 binding sites of the −634/+89 hPepT1 construct were introduced using the Quick Change XL Site Directed Mutagenesis Kit  with the following primers: hPepT1 promoter with mutated CREB+7, sense 5′-CAA CGG GGC CGG GCC TGG AAT TCA GGT CGG AGG AGT AG-3′ and antisense 5′-CTA CTC CTC CGA CCT GAA TTC CAG GCC CGG CCC CGT TG-3′; hPepT1 promoter with the first Cdx2−579 site mutated, sense 5′-GAA ATG TAG AAT CCC CCA GAG ATG CTT TCA AAG GTT-3′ and antisense 5′-AAC CTT TGA AAG CAT CTC TGG GGG ATT CTA CAT TTC-3′; hPepT1 promoter with the second Cdx2−564 site mutated, sense 5′-GGT TGA ATC TCA AAA TGA AGC CAC ACA CAC ACT CT-5′ and antisense 5′-AGA GTG TGT GTG TGG CTT CAT TTT GAG ATT CAA CC-3′; hPepT1 promoter with the third Cdx2−262 site mutated, sense 5′- AAC CTC CCC TTA GAC TTC TTC GAA ATG CAC ATT CTG G 3′ and antisense 5′-CCA GAA TGT GCA TTT CGA AGA AGT CTA AGG GGA GGT T-3′; hPepT1 promoter with the AP1−216 site mutated, sense 5′-AGC CCC GAC CTC CTG AGT CTG CTG GCC GGG GGG TGG-3′ and antisense 5′-CCA CCC CCC GGC CAG CAG ACT CAG GAG GTC GGG GCT-3′; hPepT1 promoter with the first Sp1−199 site mutated, sense 5′-CAG CTG GCC GGG GGG TTG GGC CTG GGA ATC CGC GTT-3′ and antisense 5′-AAC GCG GAT TCC CAG GCC CCA CCC CCC GGC CAG CTG-3′; hPepT1 promoter with the second Sp1−59 site mutated, sense 5′-CTC TGC TCC CCG CAG CAC CGT CCC CCG GGT GGA GCC-3′ and antisense 5′-GGC TCC ACC CGG GGG ACG GTG CTG CGG GGA GCA GAG-3′; hPepT1 promoter with the third Sp1−33 site mutated, sense 5′-TGG AGC CGG CGG CCC CTC CTC GCA GAG CTG GGG CTG-3′ and antisense 5′-CAG CCC CAG CTC TGC GAG GAG GGG CCG CCG GCT CCA-3′; hPepT1 promoter with the fourth Sp1−5 site mutated, 5′-GTA CCT GGG GCA ACG GGG ACG GGC CTG GAC GTC AGG-3′ and antisense 5′-CCT GAC GTC CAG GCC CGT CCC CGT TGC CCC AGG TAC-3′. The PCR conditions consisted of 95°C for 30 sec, followed by 12 cycles of 95°C for 30 sec, 55°C for 2 min and 68°C for 10 min. All mutants were confirmed by DNA sequencing.Analysis with the TFSEARCH prediction program (5′-GCC AUA GAC CUA CAG ACC Utt-3′) was purchased from Ambion  and transfected into Caco2-BBE cells using the siPORT NeoFX transfection reagent (Ambion). 48 hours after transfection, cells were treated with 5 mM butyrate for 24 h, harvested and analyzed by Western blot.Cdx2 siRNA  and 2 µg of the relevant hPepT1 promoter construct using 10 µg/ml lipofectin (Invitrogen). After stimulation, the resulting luminescence was measured for 10 s in a luminometer . Each luciferase activity was normalized based on the control Renilla luciferase activity. Extracts were analyzed in triplicate, and each experiment was performed for at least three times.2N-FRHRSKAYPKREHWC-COOH) and H2N-RLEKSNPYFMSGANSQKQN), corresponding to residues 248–261 and 689–708 of the deduced hPepT1 protein. The peptides were coupled to keyhole limpet haemocyanin, and used to raise two antibodies:  in rabbits via a standard 80-day immunization protocol. The reactivity of the resulting antisera against the appropriate peptide was tested by enzyme-linked immunosorbent assay (ELISA), and the antibodies were affinity purified against the synthetic peptide. Anti PepT1689–708 is specifically directed to human PepT1 and anti-PepT1 248–261 is directed to mouse and human PepT1 Based on a computerized predictive model for antigenicity and uniqueness, we designed two synthetic peptides  and reverse transcribed using Thermoscript™ RTPCR System (Invitrogen). The PCR was carried out in a final volume of 50 µl using 2 µl of RT first-strand cDNA product, 1 µM of each forward and reverse primer, and PCR SuperMix (Invitrogen). The primers used were: hPepT1 sense CtTarget−Cthousekeeping)treatment−(CtTarget−Cthousekeeping)nontreatment, and the final data were derived from 2−ΔΔCT. The primers used were as follows: hPepT1 sense 5′-CGT GCA CGT AGC ACT GTC CAT-3′ hPepT1 antisense 5′-GGC TTG ATT CCT CCT GTA CCA-3′; hGAPDH sense 5′-GTC GGA GTC AAC GGA TTT GG-3′ hGAPDH antisense 5′-AAG CTT CCC GTT CTC AGC CT-3′; mousePepT1 sense 5′-CGT GCA AGT AGCACTGTC CAT-3′; mousePepT1 antisense 5′-GGC TTG ATT CCT CCT GTA CCA-3′; mouse36B4 sense 5′-TCC AGG CTT TGG GCA TCA-3′ mouse36B4 antisense 5′-CTT TAT CAG CTG CAC ATC ACT CAG A-3′.Real-time PCR with iCycler sequence detection system (Bio-Rad) was used to assess PepT1 transcripts. Briefly, 50 ng of reverse-transcribed cDNA was amplified followed by 40 cycles of 95°C for 15 s and 60°C for 1 min, using 10 µM of gene-specific primers and the iQ SYBR Green Supermix (Biorad). The GAPDH or 36B4 expression levels were used as housekeeping genes, and fold-induction was calculated using the Ct method as follows: ΔΔCT = . Samples (5 µg) were incubated for 15 min at room temperature with a biotin-labeled double-stranded oligonucleotide  containing Cdx2−579, Cdx2−262 or CREB+7 binding site. Complexes were resolved by electrophoresis on 5% TBE gels in 0.5× TBE buffer. Gels were transferred to Biodyne B Pre-cut Modified Nylon Membranes (Pierce) and complexes were visualized using the Chemiluminescent Nucleic Acid Detection System (Pierce). The specificity of the complexes was analyzed by incubation with a 200-fold excess of unlabeled oligonucleotides as well as a mutated probe. Supershift assay was performed using 2 µg of Cdx2 antibody .Cdx2 or CREB-DNA binding activities were analyzed in cellular extracts prepared in totex buffer  for 1 h to arrest transcription were incubated with 5 mM butyrate for indicated times. The hPepT1 mRNA was quantified by real-time RT-PCR as described above. Northern blot analysis was performed using the North2South Complete Biotin Random Prime Labeling and Detection Kit  with 20 µg of template total RNAs and probe generated by RT-PCR with primers as followed: hPepT1sense 3, 1% SDS) and the protein-DNA cross-links were reversed by treatment with 200 mM NaCl at 65°C for 4 h. The DNA was purified by incubation with proteinase K at 45°C for 1 h, followed by phenol/chloroform extraction and ethanol precipitation. The Cdx2 promoter elements in the immunoprecipitates were detected by PCR using specific primers . The CREB+7 promoter elements were detected using specific primers: CREB+7 sense 5′-CTC GGG AGC ACG GAC CTC T-3′ CREB+7 antisense 5′-CCT GGC AGG TGC TAC TCC TC-3′.Acetyl-histone H4 and Cdx2 chromatin immunoprecipitation assay (ChIP) was performed using the ChIP assay kit . Briefly, after stimulation cells were fixed with 1% formaldehyde for 10 min at 37°C (protein to DNA cross-linking), resuspended in SDS lysis buffer for 10 min on ice. Cells were then sonicated to shear the DNA into 200–1000 bp fragments and centrifuged. The supernatant was diluted in ChIP dilution buffer and pre-cleared with protein A-agarose/salmon sperm DNA to reduce the nonspecific background. The samples were then immunoprecipitated with anti-acetyl-histone H4 or anti-Cdx2 antibodies overnight at 4°C. The complexes were collected in protein A-agarose/salmon sperm DNA slurry 1 h a 4°C, and then washed once each with the provided low salt, high salt, and LiCl wash buffers, and twice in TE buffer . The immunoprecipitated chromatin was eluted from the protein A using freshly prepared elution buffer (10 mM NaHCO+-10 mM HEPES (pH 7.4) in the basolateral compartment and with HBSS+-10 mM MES (pH 6.2) in the apical compartment for 15 min at 37°C. Apical and basolateral compartments were then incubated for 15 min at room temperature with HBSS+-10 mM MES (pH 6.2) containing 20 µM [14C]Gly-Sar±20 mM Gly-Leu and HBSS+-10 mM MES (pH 7.2), respectively. Cells were washed twice in ice-cold PBS, filters were cut, and cell-associated radioactivity was determined using a β-counter. The results, expressed as specific uptake of [14C]Gly-Sar mediated by hPepT1, are calculated as follows: (Uptake of [14C]Gly-Sar)−(Uptake of [14C]Gly-Sar + Gly-Leu).Caco2-BBE cells grown on filters for 15 days were treated with butyrate, washed and incubated with HBSS3H]KPV and the hPepT1-mediated specific uptakes of [3H]KPV were performed as described above.The kinetic parameters of peptide transport in Caco2-BBE monolayers were examined using different doses of Gly-Sar was performed for 10 s at room temperature using transport buffer  containing 300 µg of colonic apical membranes and 20 µM [14C]Gly-Sar±20 mM Gly-Leu, following by addition of ice-cold stop solution  and filtration. The filters were then washed twice with stop solution and the radioactivity was determined using a β-counter.Colonic apical membranes were prepared from mucosal scraping (n = 25 mice/condition) as previously described P<0.05 was considered significant.All evaluations were performed using SigmaPlot  and InStat v3.06  softwares, with data reported as means±S.E. Multiple groups were compared by ANOVA, using Tukey's post-hoc test.


Tau protein exists as six different isoforms that differ by the inclusion or exclusion of exons 2, 3 and 10. Exon 10 encodes a microtubule binding repeat, thereby resulting in three isoforms with three microtubule binding repeats (3R) and three isoforms that have four microtubule binding repeats (4R). In normal adult brain, the relative amounts of 3R tau and 4R tau are approximately equal. These relative protein levels are preserved in Alzheimer's disease, although in other neurodegenerative tauopathies such as progressive supranuclear palsy, corticobasal degeneration and Pick's disease, the ratio of 3R:4R is frequently altered. Because tau isoforms are not equally involved in these diseases, it is possible that they either have inherently unique characteristics owing to their primary structures or that post-translational modification, such as phosphorylation, differentially affects their properties.We have determined the effects of phosphorylation by a kinase widely believed to be involved in neurodegenerative processes, glycogen synthase kinase-3β (GSK-3β), on the microtubule binding and inducer-initiated polymerization of these isoforms in vitro. We have found that each isoform has a unique microtubule binding and polymerization profile that is altered by GSK-3β. GSK-3β phosphorylation had differential effects on the isoforms although there were similarities between isoforms and the effects were generally mild.These results indicate that tau phosphorylation by a single kinase can have isoform specific outcomes. The mild nature of these changes, however, makes it unlikely that differential effects of GSK-3β phosphorylation on the isoforms are causative in neurodegenerative disease. Instead, the inherent differences in the isoform interactions themselves and local conditions in the diseased cells are likely the major determinant of isoform involvement in various neurodegenerative disorders. Tauopathies, such as Alzheimer's disease (AD), are characterized by the abnormal aggregation of a hyperphosphorylated microtubule associated protein, tau. The aggregation and phosphorylation of tau correlates with neurodegenerative changes in these diseases (reviewed in ). It is Tau polymerization into filaments similar to those observed in neurodegenerative diseases can be modeled in vitro by the addition of inducer molecules such as arachidonic acid (ARA) and heparin (reviewed in ) in a coIn addition to being abnormally polymerized, tau has been shown to be hyperphosphorylated in AD pathology. "Hyperphosphorylated" is a loosely defined term used to describe abnormally or inappropriately phosphorylated tau in AD. Although there is not a strict definition, some properties of hyperphosphorylated tau include altered electrophoretic mobility, conformational changes, increased levels of phosphate incorporation, and site-specific inappropriate phosphorylation -10. WhilWe have found that there are fundamental differences in the functions of 3R and 4R isoforms. These differences are largely determined by the presence or absence of exon 10, with some contributions from exons 2 and 3. Phosphorylation by GSK-3β modulates the function of tau, but only mildly. The data suggest that phosphorylation alters all isoforms to approximately the same degree. These results suggest that the inherent properties of the isoforms are primarily responsible for their variable involvement in disease pathology, rather than the differential effects brought about by GSK-3β phosphorylation.E. coli  are classified by the presence or absence of N-terminal exons 2, 3, and C-terminal exon 10 (containing microtubule binding repeat 2) Figure . We expri Figure . The isoBecause the primary sequences of the tau isoforms are different, the various combinations of exons could influence their interactions with inducer molecules or cause conformational changes affecting tau-tau interactions during the polymerization process. The polymerization characteristics of each isoform was determined by incubating 2 μM protein with a range of ARA concentrations (0–150 μM) and monitoring the extent of polymerization with right angle laser light scattering , promotes strong disassociation (~10×) from microtubules  labeled ATP . Samples were filtered and washed to remove unincorporated γ-32P, then counted in a liquid scintillation counter (Packard 1600TR) [Tau protein was phosphorylated as detailed in . Briefly 1600TR) .2, 0.5 mM EGTA). Samples were incubated at room temperature for 30 minutes, and centrifuged at 23°C in 7 × 20 mm polycarbonate Beckman centrifuge tubes in a Beckman TLA 100 rotor at 100,000 × g for 5 minutes to sediment the microtubules and tau bound to microtubules. The pellets were resuspended in 1 × SDS sample buffer  and tau bound to microtubules were separated from the pelleted tubulin by electrophoresis. The SDS-polyacrylamide gels were scanned with a Hewlett Packard Scanjet 7400 C and the density of the tau contained in the pellet (bound tau) was determined using Adobe Photoshop and a tau standard curve on the same SDS-polyacrylamide gel. Isoforms 0N3R, 0N4R, 1N3R, and tubulin have similar electrophoretic mobility. In order to distinguish tau from tubulin bands, the protein were transferred to PVDF by standard western blot techniques and probed with tau 46.1 primary antibody (gift from Lester I. Binder) and goat anti-mouse horseradish peroxidase conjugated secondary (Pierce). Amersham ECL Plus Western Blotting Detection System was used to detect the secondary antibody . The PVDF membrane was visualized using the Kodak Image Station 4000R . Band intensities were determined by the histogram function of Adobe Photoshop. The amount of free tau left in solution was determined by subtracting the amount of tau in the bound fraction from the total amount of tau added to the reaction and normalized to the band intensity of tubulin. The concentrations of tau free in solution versus bound tau were plotted in GraphPad Prism 4 and fit to a noncooperative single-site binding equation: Free = (Bmax * Bound)/(kd + Bound). Binding reactions were performed in triplicate for all tau isoforms.Microtubule binding assays were performed using the commercially available Microtubule Binding Protein Assay Kit  using the manufacturer's protocol. Varying concentrations of tau (0 μM–10 μM) were mixed with a constant concentration of paclitaxel stabilized microtubules at a final concentration of 1.6 μM tubulin dimers and 20 μM paclitaxel in a 50 μL reaction with the general tubulin buffer supplied with the kit  before blocking, to remove the phosphate groups that interfere with tau 46.1 antibody binding. Images of gels and blots were collected and processed by methods described above, except that due to the large concentration of tubulin we were unable to normalize the data to the corresponding band. Due to variable data, 5 outliers were excluded from calculations based upon being more than twice as different as the corresponding trials. These points are 0N4Rp trial 3 1 μM and 2 μM, 1N3Rp trial 1 8 μM and trial 2 10 μM, and 2N3Rp trial 2 10 μM.Individual tau isoform phosphorylation reactions (92 μL), containing 18.11 μM final concentration of tau, 0.018 U GSK-3β per pmol of tau in buffer containing 40 mM HEPES, pH 7.64, 5 mM EGTA, 3 mM MgClThe authors declare that they have no competing interests.KV participated in the design of the study, performed the experiments described and drafted the manuscript. TCG conceived of the study, participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.Polymerization of 0N3R and 0N4R isoforms with 75 μM ARA into larger aggregates is seen, but infrequent. 0N3R  and 0N4R  isoforms exhibit filament elongation and clustering under non-phosphorylated conditions. Reactions from Figure Click here for file


N-linked sugars to an Asn-X-Cys motif in cystatin F and provide evidence that the mannose 6-phosphate sorting machinery is used to divert cystatin F from the secretory pathway and to mediate its uptake from extracellular pools. These studies identify a function for the oligosaccharides on cystatin F and raise the possibility that cystatin F might regulate proteases in transby secretion in an inactive form by one cell and subsequent internalization and activation by another cell.Cystatin F is a cysteine protease inhibitor that is selectively expressed in immune cells and unlike other cystatin family members is targeted to a significant extent to intracellular compartments. Initially made as an inactive glycosylated disulfide-linked dimer, cystatin F is converted to an active monomer by proteolytic cleavage following transport to the endosomal/lysosomal system. This active form of cystatin F targets cathepsin C/DPPI and probably other cathepsins in immune cells. We show that efficient targeting of cystatin F to the endocytic pathway is dependent not on its unique dimeric conformation but rather on its oligosaccharide chains. We demonstrate the unusual addition of  The cystatins are a family of naturally occurring cysteine protease inhibitors comprising at least 11 members . Some arThe cystatins are structurally well conserved and comprise a single α-helix associated with a 5-stranded β-sheet whose loops combine with residues from the N-terminus to form an ‘edge’ that engages the protease active site N-linked oligosaccharides. Finally, the amino acids present in the protease-binding loops and N-terminal region of cystatin F are not homologous with those of other family members, indicating that cystatin F might bind distinct protease targets  because of carbohydrate addition on a cryptic noncanonical (Asn-X-Cys) N-glycosylation site. Either this site, or a neighboring canonical site, sufficed to specify sorting of cystatin F to lysosomal compartments. We further show that cystatin F made and secreted by one cell can be internalized and activated within a different cell through mannose 6-phosphate (M6P)-mediated targeting. Thus, immune cells expressing cystatin F may regulate in transthe protease activity of bystander cells.Here, we investigate how cystatin F is retained within cells by analysis of cystatin F mutants that either cannot dimerize or lack specific oligosaccharide chains. We show that cystatin F molecules that cannot dimerize are still efficiently targeted to the endosomal system. Moreover, a mutant of cystatin F lacking both canonical N-glycosylation accounted for the resolution of distinct forms of cystatin F on SDS–PAGE To investigate the trafficking of cystatin F, we generated various mutant forms and analyzed these initially in the human kidney epithelial cell line 293T, which does not express cystatin F. The cells were transfected, and after 48 h, the relative amount of cystatin F in cell lysates (L) and in the culture medium (M) was assessed. Expression of wild-type cystatin F leads to significant secretion of the protein into the culture medium as we and others have previously reported ,13,16. NCystatin F dimerizes through two disulfide bridges involving Cys26 on one subunit and Cys63 on the other To confirm targeting to the endocytic pathway, transfected cells were fixed and stained with antibodies against cystatin F and a marker of the late endosomal/lysosomal compartment (CD63). As shown in Although covalent dimer formation was circumvented by substitution of the two cysteines involved in disulfide bond formation with serine, noncovalent dimer formation was still plausible. In other words, dimerization of C26,63S through noncovalent interaction may account for its lysosomal localization and secretion. To address this, wild-type and C26,63S cystatin F were subject to size exclusion chromatography on a column calibrated with standard proteins of known molecular weight and stokes’ radii. Elution of cystatin F was initially followed by absorption (280nm) of the eluted fractions and then confirmed by western blot . As expeN-linked sugar at a site homologous to Asn62 in humans. We generated mutant forms of human cystatin F where one or both Asn residues were altered to Ser  and transfected these into 293T cells. Culture medium and cell lysates were then analyzed for the presence of cystatin F and digested with PNGase to assess its glycosylation status. Similar to wild-type cystatin F, all mutant forms of cystatin F migrated as a single band following treatment with PNGase. Puzzling, however, the gel mobility of the N62S mutant in the absence of PNGase digestion was similar to that of wild type , the N61,62S mutant and the triple mutant were largely absent and found mostly in the medium . Consistecretion . These rN-linked glycans that become modified with M6P and use the cation-dependent or -independent M6P receptors  for targeting the endocytic pathway 32P] orthophosphate in 293T cells, and this label was removed by treatment with endo-β-N-acetylglucosaminindase H (EndoH), suggesting phosphorylation occurred on high mannose glycans. In contrast, [35S]-labeled cystatin F migrated faster following treatment with EndoH, but the label was not removed. Next, we infected mouse fibroblasts, including those lacking the CI-MPR, with a retrovirus expressing wild-type cystatin F. Whereas L929 cells, which express wild-type levels of both MPR, retained a proportion of cystatin F and converted it to the monomeric form, no intracellular cystatin F could be detected in L cells lacking the CI-MPRs . As expected, the cell lysates also contained cystatin F, much of which was converted to the active monomeric form  analysis to distinguish wild type (CD45.1+) from cystatin F null (CD45.2+) cells  are secreted from cells, suggesting that their primary function is to inactivate potentially dangerous proteases that have been secreted or lost from cells into the extracellular space thereby protecting from inflammatory reactions associated with aberrant release of these proteases –25. WhilThe mechanism by which cystatin F is retained within cells and matured in the endocytic compartment is addressed here. We demonstrate that dimerization is not necessary for intracellular cystatin F to be directed to the lysosomal pathway but rather a carbohydrate-dependent sorting mechanism operates. Our findings provide an explanation for why cystatin F is glycosylated when almost all other family members are not. Cystatin M/E is also glycosylated and is found in the lamellar granules of the cells of the stratum granulosum of the skin as well as in extracellular locations To our knowledge, cystatin F is the first lysosomal protease inhibitor shown to be M6P modified. This appears to be integral for proper targeting to the lysosomal compartment as judged by the failure of cells lacking MPRs to retain cystatin F. An earlier report identified cystatin F as one of several secreted proteins recovered from an MPR column, although it was not clear if the association was direct or indirect, for example through a binding partner modified with M6P N-linked oligosaccharides at Asn62 and 115, whereas murine cystatin F lacks the second sugar. Consistent with conservation of glycosylation at position 62 in mammalian cystatins  in the Asn-X-Cys motif is involved in the dimerizing disulfide bond, presumably recognition of the motif and addition of core oligosaccharide to Asn61 must occur before dimerization and loss of the free thiol. However, this has not been formally demonstrated.Human cystatin F carries two ystatins , we showthus far . Other pthus far ,21,33.Whin vivo.High concentrations of reducing agents are required to reduce the cystatin F dimer making a proteolytic mechanism for monomerization more likely and linking cystatin F activation to endosomal proteolytic activity rather than reducing potential ,16. BaseSequestration of cystatin F from the secretory pathway by an M6P/MPR mechanism raised the possibility that secreted dimeric cystatin F might be taken up into cells using the same system, as first described by Neufeld and colleagues for lysosomal enzymes + T cells often show heterogeneous levels of expression. Therefore, secretion and internalization of cystatin F in the closely confined in vivomilieu may even out these differences in expression. Cystatin F may be transferred efficiently from one cell to another not only because of its M6P tag but also because, unlike other cystatins found in the external milieu, it is unable to bind protease targets and therefore will not be sequestered by extracellular cysteine proteases. Several precedents exist for in vivoacquisition of lysosomal enzymes by one cell type following secretion by another. For example, cathepsin L-deficient thymocytes acquired this enzyme from surrounding epithelial cells rescuing a defect in antigen presentation and development of natural killer T cells in vivofrom the glomerular filtrate into kidney proximal tubule cells and reconstituted a cathepsin B deficiency in vivo, our results clearly indicate that this may take place. Conceivably, given that cathepsin C is one of the targets of cystatin F, this may be a way of attenuating granule protease-driven inflammatory reactions.These results raise the possibility that immune cells secreting cystatin F may be able to regulate protease activity in neighboring cells that either do not express cystatin F or express insufficient levels. Cystatin F expressing primary cell populations such as CD8N-linked M6P-modified sugar can be added to Asn61. Although the dimeric form is not crucial for intracellular targeting, it appears to be necessary for endocytic uptake. Moreover, dimerization ensures the latency of the inhibitor, which only becomes active following activation by proteases. The possibility that cystatin F is activated by the same protease systems that it inhibits is currently under investigation.In summary, our study sheds light on the unusual features of the cystatin F molecule. Glycosylation and more specifically M6P modification serve to target it to the endocytic pathway during biosynthesis, and for a proportion of molecules, by endocytosis from the external medium . In the Cell culture media were purchased from Gibco unless otherwise indicated. H-Gly-Phe-AMC and Z-Phe-Arg-AMC were purchased from Bachem. PNGase and EndoH were purchased from Roche. Rabbit polyclonal antibodies directed against full-length human cystatin F (FL Ab) or against an N-terminal peptide  have been described previously ml- glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin. L cells devoid of the CI-MPR (CI-MPR KO), and those reconstituted with the CI-MPR (KO + CI-MPR) were kindly provided by T. Braulke  and were cultured in the same medium as L929 cells. Wild-type MEF, MPR knockout fibroblasts and those reconstituted with the human CI-MPR were generous gifts from R. Pohlmann  and B. Hoflack  and were cultured as described + T lymphocytes were cultured from the bone marrow and spleen, respectively, of C57BL/6 mice as described previously (+ (CD45.1+) mice were a kind gift from P. Crocker . Cystatin F knockout mice were generated under contract by Artemis Pharmaceuticals GmbH and will be fully described elsewhere.The cell lines 293T and L929 were cultured in DMEM supplemented with 10% fetal calf serum (FCS), 2 meviously ,47. C57BTCCTGCACGAACGACATGTTCTTGTTC and GAACAAGAACATGTCGTTCGTGCAGGAGTTGAACTTTTCAACACTG; N61,62S, CAGTGTTGAAAAGTTCAGCTCCTGCACGAACGACATGTTCTTGTTC and GAACAAGAACATGTCGTTCGTGCAGGAGCTGAACTTTTCAACACTG and N115S, GACTTCCAAACCAGCCACACCTTGAAGCAGACTCTGAGCTGCTAC and GTAGCAGCTCAGAGTCTGCTTCAAGGTGTGGCTGGTTTGGAAGTC. Underline denotes substituted residues. All mutagenesis was performed using the Quik Change Site-Directed Mutagenesis kit. Glycosylation of cystatin F was assessed by treating cell lysates, conditioned medium or immunoprecipitated cystatin F with PNGase to remove the entire glycan or EndoH to selectively remove high mannose forms of the glycans according to manufacturer’s guidelines.Mutagenesis was performed using plasmids encoding full-length human cystatin F cDNA in a pcDNA-DHFR expression vector as described previously mcitrate, pH 5.4, 150 mmNaCl, 2 mmMgCl2 and 1% Triton-X-100. Culture media and cell lysates were analyzed for cystatin F expression by western blot using antibodies directed against the whole protein (FL Ab) unless otherwise noted. Protein concentrations in cell lysates were quantified by Bio-Rad Protein Assay (Bio-Rad). Equivalent concentrations of cystatin F in culture medium were determined by western blot and normalized before internalization assays, as described below.293T cells were transfected with plasmids containing either full-length (wild type) or mutant cystatin F following manufacturer’s instructions . Human cystatin F was transduced into murine L929 and L cells using the retroviral vector pBMN-I-GFP  as described previously mH-Gly-Phe-AMC (cathepsin C) or 40 mmZ-Phe-Arg-AMC (cathepsin L) in assay buffer containing 150 mmNaCl, 2 mmethylenediaminetetraacetic acid, 5 mmDTT and 100 mmcitrate, pH 5.5. All fluorometric assays were performed on a FLUOstar Optima Fluorimeter (BMG) reading excitation at 360 nm and emission at 460 nm. Cathepsin activity in 293T cells (cathepsin C) and L929 cells (cathepsin L) are expressed as the rate of AMC release over time.Cathepsin activities were determined from 293T or L929 cell lysates following transfection or incubation with cystatin F-enriched media, as described below. Ten micrograms of postnuclear cell lysates was assayed using either 50 mmphosphate buffer and 0.15 M NaCl, pH 7.2. Protein was loaded in 0.8 mL of equilibration buffer in detergent-free conditions at a flow rate of 0.5 mL/min at room temperature on the ÄKTA fast protein liquid chromatography system (Amersham Pharmacia) collecting 1 mL fractions. The elution volumes of cystatin F and standard proteins (Ve) were converted into Kav using the following equation; Kav = (Ve − Vo)/(Vc − Vo), where Vo is the void volume as determined by blue dextran and Vc is the calculated geometric column volume (Vc = r2∏L). The Stokes’ radius of cystatin F was determined using a linear calibration plot of stokes’ radii versus (−log Kav)1/2Stokes’ radius determination of wild-type dimeric cystatin F, C26,63S cystatin F and standard proteins were performed by size exclusion chromatography using an Amersham HiLoad 26/60 Superdex 75 column (Amersham Pharmacia). The column was pre-equilibrated in 50 m35S] labeling, transfected 293T cells were preincubated in Met/Cys-free media (Sigma), labeled for 45 min with 0.4 mCi/mL [35S] Met/Cys  and chased for 3 h in complete RPMI-medium containing 100 μg/mL methionine and 500 μg/mL cysteine. For [32P] orthophosphate labeling, transfected 293T cells were incubated for 30 min at 37°C in phosphate-free MEM containing 10% dialyzed FCS and for another 4 h in the same medium supplemented with 0.5 mCi/mL [32P] orthophosphate (Perkin Elmer). Culture medium was removed and cells were washed extensively and then lysed in buffer containing 1% Triton-X-100. Radiolabeled cystatin F from culture medium and lysates were immunoprecipitated using cystatin F antibodies (FL Ab) as described 125I-labeling experiments, 20 μg of purified recombinant cystatin F was labeled with 0.5 mCi [125I]-Na (Amersham) using the IODO-GEN reagent (Pierce) following manufacturer’s instructions.For -labeled cystatin F, L929 cells were incubated with 500 μg/mL [125I] cystatin F in the presence or absence of 10 mmM6P at 4°C. The cells were washed three times with PBS to remove unbound protein and incubated at 37°C for 30 min to allow for internalization. Before lysis, cystatin F remaining on the plasma membrane was removed by acid stripping . Control cells were maintained at 0°C throughout. Percent internalization was determined by dividing the acid stable fraction following acid washing at 30 min from the acid labile fraction at 0 min. Primary murine CD8+ T lymphocytes (CTL) and dendritic cells were isolated from the spleen and bone marrow of C57Bl/6 mice as described  and CD45.2 (APC) were detected by flow cytometry using directly conjugated antibodies. Internalization of cystatin F was determined by surface staining of either CD45.1 (APC) or CD45.2 (APC) followed by intracellular staining for cystatin F (FL Ab) using the Fix & Perm cell permeabilization kit .To follow the internalization of cystatin F, L929 cells were cultured at 5 × 10escribed ,47. Priml-lysine-coated coverslips for 48 h before staining. Cells were then fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton-X-100 and costained with rabbit anti-cystatin F (FL Ab), and either mouse anti-CD63 or sheep anti-TGN46 in the presence of 1% (w/v) BSA as described l-lysine-coated coverslips for 18 h and treated with cystatin F-containing conditioned media or control media as described above. Primary antibodies were detected using Alexa-488 or Alexa-594-conjugated anti-rabbit (Invitrogen), TR-conjugated anti-sheep (pseudo-colored green) (Jackson Laboratory) and Alexa-488 anti-mouse (Invitrogen) secondary antibodies. Stained cells were mounted on glass slides and imaged on a LSM 510 META confocal microscope using a 100× PlanFluar/NA 1.45 objective and lsm510 software (Carl Zeiss Inc.) followed by processing with Photoshop (Adobe).Transfected 293T cells were grown on poly-


Recent advances in oncology have lead to identification of a plethora of alterations in signaling pathways that are critical to oncogenesis and propagation of malignancy. Among the biomarkers identified, dysregulated kinases and associated changes in signaling cascade received the lion's share of scientific attention and have been under extensive investigations with goal of targeting them for anti-cancer therapy. Discovery of new drugs is immensely facilitated by molecular imaging technology which enables non-invasive, real time, dynamic imaging and quantification of kinase activity. Here, we review recent development of novel kinase reporters based on conformation dependent complementation of firefly luciferase to monitor kinase activity. Such reporter system provides unique insights into the pharmacokinetics and pharmacodynamics of drugs that modulate kinase signaling and have a huge potential in drug discovery, validation, and drug-target interactions. Cell signaling is a mode of communication by which the intracellular information is conveyed from the site of instigation to the site of action. Recent advances in molecular profiling technologies such as microarrays and proteomics along with synergistic growth in the field of bio-informatics, have actuated our appreciation of signaling changes in patho-physiological conditions and led to identification of unique disease biomarkers -5. For eMolecular imaging is a recent area of investigation that attempt to develop suitable probes for noninvasive visual representation of biological processes at the cellular and molecular level in the whole organism and the modalities and instrumentation to support the visualization and quantification of these processes. This is an attempt to bridge the gap between discovery of biomarkers and their deployment in clinic. At present molecular imaging is still largely in the animal experimental phase but promises to bring dramatic change in the way in which a disease is diagnosed, staged and treated. In clinical oncology it will allow oncologists to diagnose cancer at an earlier stage based on molecular characterization, predict the risk of precancerous lesion progression, quantify activities of specific molecules related to tumor growth, invasion and metastasis, select a rational molecular therapy and assess the efficacy of chemo and radio therapeutic agents in real time -21. Overin vitro and in vivo. For monitoring signaling cascade or activity of specific biomarker in vivo, firefly luciferase is the reporter of choice as 30% of the light generated by firefly luciferase has an emission spectra above 600 nm, a region where the signal attenuation by the absorbing and scattering properties of live mammalian tissue is minimal [Discovery of reporters that are genetically encoded and generate light such as fluorescent proteins and luciferases in conjunction with the development of instrumentation for real time functional imaging of their activity has offered researchers powerful tools to perform noninvasive studies of dynamic biological process in intact cells and whole organisms. These optical reporter systems have extensively utilized in molecular imaging of signaling pathways mainly because of their efficiency for sequential imaging, operational simplicity, and substantial cost benefits. Bioluminescent firefly luciferase based reporters are widely used for non-invasive, real-time, repetitive imaging both  minimal ,34. A ma minimal -41 Addit minimal -46.in vitro and FRB-FKBP12 interaction in vivo in real time non-invasively. On similar lines Paulmurugan and Gambhir [Protein complementation assay have garnered a lot of lime light for monitoring protein-protein interaction, kinase and protease activities . Here, t Gambhir  develope Gambhir  where th Gambhir  reported Gambhir ,57-59.Protein kinases are one of the principle regulators of signaling cascades influencing majority of cellular decisions -65. Protbioluminescent Akt reporter, (BAR) was constructed by fusion of an Akt consensus substrate peptide and phospho-amino acid binding domain (FHA2) which were flanked by the amino- (N-Luc) and carboxyl- (C-Luc) terminal domains of the firefly luciferase reporter molecule  but decreased thereafter  would be identified. In contrast to other cell based reporter screens, which are fraught with false positives, the kinase reporter described here is a "gain of function assay" wherein the inhibition of kinase activity results in an increase in bioluminescence. For example, compounds that kill cells  or those that inhibit luciferase activity may show up as false positives in typical luciferase/fluorescent/enzyme based assays. However, specific inhibition of reporters like BAR result in an increase in bioluminescence activity and thus non-specific cytotoxic agents are eliminated. Such carefully designed screening methodology will enable us to narrow down the number of positive compounds to a smaller group of "true positives".Optimum activity and specificity of a kinase is dependent upon its subcellular localization. For example, Akt is recruited to the plasma membrane by PI-3 kinase-generated D3-phosphorylated phosphoinositides which bind to the Akt PH domain and induce the translocation ,76. At thttp://www.kinasenet.ca, http://www.phosphosite.org, http://www.kinase.com. Experimentally verified phosphorylation sites are available at http://phospho.elm.eu.org and for prediction of phosphorylation site http://www.cbs.dtu.dk/services/NetPhos/ or http://scansite.mit.edu may be useful.As discussed above, the BAR can be adapted for other protein kinases including receptor or non-receptor Tyr and Ser/Thr kinases by using a suitable substrate and a specific phospho-amino acid binding protein domain. We have successfully adapted this platform for monitoring GSK3β/CK1α kinase activities using a β-catenin substrate sequence . Towardsin vitro and in vivo. This greatly facilitates the real time visualization of the aberrant oncological signaling and will play an important role in monitoring therapeutic outcome, drug-target validation as well as identification of next generation of drugs.In summary, molecular imaging reporters for kinases provide a unique opportunity to monitor cellular pathways both The authors declare that they have no competing interests.SN and MSB wrote the review and made the figures. AR and BDR revised the manuscript.


Drosophila serine/threonine kinase, promotes apoptosis and restricts cell growth and proliferation. Its mammalian homolog MST2 has been shown to play similar role and be regulated by Raf-1 via a kinase-independent mechanism and by RASSF family proteins through forming complex with MST2. However, regulation of MST2 by cell survival signal remains largely unknown.Hippo, a in vitro kinase and in vivo labeling assays, we show that IGF1 inhibits MST2 cleavage and activation induced by DNA damage through the phosphatidylinosotol 3-kinase (PI3K)/Akt pathway. Akt phosphorylates a highly conserved threonine-117 residue of MST2 in vitro and in vivo, which leads to inhibition of MST2 cleavage, nuclear translocation, autophosphorylation-Thr180 and kinase activity. As a result, MST2 proapoptotic and growth arrest function was significantly reduced. Further, inverse correlation between pMST2-T117/pAkt and pMST2-T180 was observed in human breast tumors.Using immunoblotting, Our findings demonstrate for the first time that extracellular cell survival signal IGF1 regulates MST2 and that Akt is a key upstream regulator of MST2. MST2 and its close homologue MST1 are members of the germinal center kinase group II (GCK II) family of mitogen-activated protein kinase (MAPK)–related kinases that includes the more distantly related kinases MST3, MST4, LOK, SOK, and SLK. Unlike other members, MST1 and MST2 contain a Ste20-related kinase catalytic domain in the N-terminal region followed by a noncatalytic tail that contains an autoinhibitory domain, a dimerization domain, and two nuclear export sequences at the COOH terminus Drosophila, Hippo, a homolog of mammalian MST2, restricts cell growth and cell proliferation and promotes cell death by interaction with the tumor suppressors Salvador (Sav)/WW45 and Warts (Wts)/Lats1/Lats2, which result in inhibition of transcription and/or degradation of cyclin E and DIAPs Drosophila ortholog of the mammalian transcription co-activator yes-associated protein (YAP) In Accumulated evidence shows that Akt and its downstream targets constitute a major cell survival pathway. Akt inhibits the programmed cell death in a number of cell types induced by a variety of stimuli through regulation of down stream molecules in vitro and in vivo, which leads to inhibition of MST2 cleavage and kinase activity as well as nuclear translocation. Furthermore, Akt activation is inversely correlated with autophosphorylation of MST2-T180 but paralleled with MST2-T117 phosphorylation in breast tumors. Collectively, our findings suggest that MST2 is a bona fide substrate of Akt and that Akt could play a critical role in regulation of the Hippo/MST2 pathway.A previous study showed that EGF stimulation caused a drop of MST1 kinase activity Stauroporine, LY294002 were obtained from Sigma . DMEM and fetal bovine serum were purchased from Invitrogen Co. . Anti-MST2 (#3952), -pMST2-Thr180 (#3681), -Akt (#9272), -pAkt-Ser473 (#9271), -actin (#4967) and -cleaved PARP (#9541 and #9544) antibodies were from the Cell Signaling Technology . Anti-GFP antibody was purchased from Santa Cruz Biotechnology . COS7 and human embryonic kidney (HEK) 293 cells were purchased from The American Type Culture Collection  and cultured at 37°C and 5% CO2 in DMEM supplemented with 10% fetal bovine serum. Eighty primary human breast cancer specimens were obtained from patients who underwent surgery at H. Lee Moffitt Cancer Center and approved by Institutional Review Board. Each sample contains at least 80% tumor cells, confirmed by microscopic examination.EcoRI-BamHI sites. MST2 specific primers are: ccggaattcatggagcagccgccggcg (5′primer), and cgcggatccaaagttttgctgccttct (3′primer). Flag-MST2-T117A and Flag-MST2-T117D were produced with mutagenesis kit (Stratagene) using wild-type MST2 as template. Wild type MST2 and mutant MST2 were also cloned to pEGFP-C1 (Clontech) at EcoRI-BamHI sites. GST-MST2 and GST-MST2-T117A were created by PCR amplification of 150 nucleotide fragment (a.a. 100–150) which include T117 and T117A sites using Flag-MST2-T117A and Flag-MST2-T117D as templates. The fragments were cloned to pGEX-4.1 vector at BamHI-EcoRI sites. The MST2 plasmids were confirmed by DNA sequencing. Akt expression constructs were previously described Flag-tagged MST2 was created by PCR amplification of human Fetal Marathon-Ready cDNA (Clontech). The PCR products were cloned to p3XFLAG-CMV-10 vector (Sigma) at in vitro kinase assay. Protein kinase assays were performed as previously described 2, 10 mM MnCl2, and 1 mM dithiothreitol using myelin basic protein (MBP) as substrate. After incubation at room temperature for 30 min the reaction was stopped by adding protein loading buffer and proteins were separated on SDS-PAGE gels. Each experiment was repeated three times and the relative amounts of incorporated radioactivity were determined by autoradiography and quantified with a Phosphoimager (Molecular Dynamics).Immunoprecipitation and immunoblotting were performed as previously described 32P] Pi (0.5 mCi/ml) in phosphate- and serum-free DMEM medium for 4 h and then lysed. Cell lysates were subjected to immunoprecipitation with anti-Flag antibody. The immunoprecipitates were separated by 7.5% SDS-PAGE and transferred to membranes. The phosphorylated MST2 band was visualized by autoradiography.COS7 cells were co-transfected with kinase-dead Flag-MST2 and constitutively active Akt. Following 48 h incubation, cells were labeled with [Cells were seeded into 60-mm dishes and grown in DMEM supplemented with 10% FBS for 24h, treated with STS (30 µM). Apoptosis was determined by Tunel assay using an in situ cell death detection kit  and caspase 3/7 assay kit (Promega). These experiments were performed three times in triplicate.Since MST2 is cleaved and activated upon apoptotic stimuli and is required for DNA damage-induced apoptosis in different types of cells, we initially examined if extracellular cell survival signal regulates MST2 cleavage and activation. COS7 cells were treated with staurosporine  together with and without IGF1 for 2 h. Consistent with previous studies, STS alone induces MST2 cleavage and autophosphorylation of Thr180, an indicator of MST2 activation, as well as apoptosis. However, addition of IGF1 largely reduced STS effect towards MST2. PI3K inhibitor LY294002 inhibited the IGF1 action . FurtherIn vitro MST2 kinase activity was also inhibited by ectopic expression of wild-type and constitutively active Akt  considerably induces MST2 activation and cleavage as well as apoptosis in Akt-null MEF but not Akt-reconstituted cells . Moreovetive Akt .112RLRNKT117) in a kinase domain of MST2 LIEDEIA-amide). We first characterized the specificity of this antibody. HEK293 cells were co-transfected with constitutively active HA-Akt and full-length wild-type and T117A mutant Flag-MST2. After immunoprecipitation with anti-Flag antibody, immunoblotting analysis with anti-pMST2-T117 antibody revealed that Akt phosphorylates wild-type but not T117A mutant MST2  is higher than that of MST1 versus Hippo (50%). Previous studies have shown that Hippo/MST is autophosphorylated and cleaved by caspases in response to apoptotic stimuli. The cleaved N-terminal kinase domain  subsequently translocates into the nucleus where it interacts and phosphorylates tumor suppressors Salvador (WW45) and Warts (Lats1/2), which result in inhibition of transcription and/or degradation of DIAPs and cyclin E leading to apoptosis and cell growth arrest t Thr117 .A previous study has demonstrated that Raf-1 kinase binds to MST2 and prevents its dimerization and autophosphorylation of Thr180, which results in inhibition of MST2 activation and proapoptotic activity 112RLRNKT117) within its N-terminal kinase domain  and immunoblotting with indicated antibodies (B).(2.13 MB TIF)Click here for additional data file.