Caenorhabditis elegans-Candida albicans infection model identified twelve microbial secondary metabolites capable of conferring an increase in survival to infected nematodes. In this screen, the two compound treatments conferring the highest survival rates were members of the epipolythiodioxopiperazine (ETP) family of fungal secondary metabolites, acetylgliotoxin and a derivative of hyalodendrin. The abundance of fungal secondary metabolites indentified in this screen prompted further studies investigating the interaction between opportunistic pathogenic fungi and Aspergillus fumigatus, because of the ability of the fungus to produce a plethora of secondary metabolites, including the well studied ETP gliotoxin. We found that cell-free supernatant of A. fumigatus was able to inhibit the growth of Candida albicans through the production of a secreted product. Comparative studies between a wild-type and an A. fumigatus ΔgliP strain unable to synthesize gliotoxin demonstrate that this secondary metabolite is the major factor responsible for the inhibition. Although toxic to organisms, gliotoxin conferred an increase in survival to C. albicans-infected C. elegans in a dose dependent manner. As A. fumigatus produces gliotoxin in vivo, we propose that in addition to being a virulence factor, gliotoxin may also provide an advantage to A. fumigatus when infecting a host that harbors other opportunistic fungi.Secondary metabolites are well known for their ability to impede other microorganisms. Reanalysis of a screen of natural products using the  Aspergillus are especially capable of producing a diverse array of these compounds Aspergillus fumigatus secretes more than 226 secondary metabolites including commonly studied polyketides, such as cyclic peptides, alkaloids, and sesquiterpenoids A. fumigatus, termed the epipolythiodioxopiperazines (ETPs), are characterized by an internal disulphide bridge across a diketopiperazine ring, where the first and best characterized member being gliotoxin Microbial secondary metabolites have provided numerous pharmaceutical agents ranging from antibiotics to immunosuppressive compounds. Synthesis of these low molecular weight compounds is not required for normal growth of the microbe, however these compounds may provide several benefits to the organism. Fungi have the ability to produce a plethora of secondary metabolites, typically dependent on the stage of development of the fungus and environmental factors ranging from nutrient concentrations to light and temperature A. fumigatus spores are ubiquitous in the environment and are commonly inhaled. Invasive aspergillosis usually only effects immune-compromised patients  or patients with other medical conditions such as cystic fibrosis, chronic obstructive pulmonary disease, or severe asthma, as the primary route to an established infection is through the lungs Aspergillus species, only those associated with aspergillosis, such as A. fumigatus, A. terreus, A. flavus, and A. niger, produce gliotoxin A. nidulans, a saprobe not normally associated with invasive aspergillosis, does not have the secondary metabolite gene cluster necessary to produce gliotoxin or any other ETP A. fumigatus, the gliotoxin secondary metabolite gene cluster is composed of 12 genes approximately 28 kb in length (the gli cluster) GliP) is required in the first step for the biosynthesis of gliotoxin generating the characteristic diketopiperizine ring A. fumigatus to colonize and remain established within the host by competing for limited available nutritional resources are currently unknown; however gliotoxin has potent antifungal activity against Candida albicans, Cryptococcus neoformans, and other fungi C. albicans and C. neoformans, primarily infect or colonize hospitalized patients and particularly the same patient population as A. fumigatus, providing an environment conducive of pathogen-pathogen interactions between these fungi, in particular within the pulmonary system. For example, concurrent co-infection/colonization of Aspergillus spp. and Candida spp. can occur in patients Candida spp. can colonize the respiratory tract of hospitalized patients, and the ability of a fungus such as A. fumigatus to compete against a previously established Candida spp. colonization may be necessary for the second pathogen to develop an infection.Factors which enable in vivo Candida-infected nematode survival assay to identify secondary metabolites capable of prolonging nematode survival. We found that two members of the ETP class of secondary metabolites we able to significantly increase nematode survival after infection with C. albicans. As pathogenic fungi are capable of producing these compounds within a host, the inhibitory action of gliotoxin against C. albicans was further studied. This research investigates the potential antagonistic activities mediated by secondary metabolites that may be occurring among fungi within a host.Here we reanalyzed the results from a recently published C. albicans-C. elegans antifungal discovery assay allows simultaneous assessment of the ability of a compound to promote survival of infected nematodes and indicate if there is any associated potential toxicity www.ac-discovery.com) was conducted that identified several plant produced saponins that confer an increase in Candida-infected nematode survival C. albicans-infected nematode survival rates greater then 40% were chosen for further discussion.The C. elegans assay at higher concentrations  of the ETP family of secondary metabolites provided the highest worm survival of the natural products screened ; Table 1E)-13-methyltetradec-2-enoic acid substituted analog tunicamycin V gave up to 60% protection to the worms and showed no signs of toxicity in the screen. Several tunicamycin homologues have previously been reported to inhibit C. albicans, and their degree of toxicity varied depending on the fatty-acid side chain Among the other secondary metabolites, compound A15 (tunicamycin V) is a member of the tunicamycin family of antimicrobial agents with a modified fatty acid side-chain  [18]. TuFusarium spp. were active in the assay C. albicans growth at half the concentration as enniatin B1 Importantly, distinct structure activity relationships were discernible between several closely related analogs in the antifungal screen. For example, two members of the enniatin family of natural products, enniatin A (A5) and enniatin B1 (A12), produced by C. albicans treatment has not been extensively studied. However, desferrioxamine increases severity of mycoses caused by some fungi, in particular pathogenic members of the order Mucorales, as the fungi are able to uptake and utilize the iron chelated by the siderophore, although there appears to be no significant difference with C. albicansAnother notable secondary metabolite, desferrioxamine X belongs to the well-studied class of hydroxamate siderophores Ascochyta viciaei and Qo sites of the cytochrome bc1 complex Cylindrocladium iliciola, also acts by inhibiting the cytochrome bc1 complex, however this molecule binds at the Qn site C. albicans-infected nematode survival rate , also referred to as ilicicolin D, is an prenyl-phenol compound that was originally identified in extracts of the fungus A. fumigatus were chosen for additional investigation. This fungus was chosen for further studies with C. albicans and C. neoformans because of the numerous secondary metabolites known to be synthesized and have been characterized, including gliotoxin, the deacetylated version of A3. Although rare, concurrent fungal infection/colonization between A. fumigatus and C. albicans or C. neoformans have been documented Candida spp. from respiratory specimens is generally not indicative of colonization, but rather is the result of contamination of the bronchoscope from the gastrointestinal tract during the examination procedure. However, it is notable that in a study of postmortem examinations, six (4.8%) revealed concurrent Aspergillus spp. and Candida spp. infection and one with Aspergillus spp. and C. neoformans (<1%) The secondary metabolites produced by A. fumigatus was co-inoculated with C. albicans or C. neoformans on plates containing Spider medium at 37°C, yeast colonies proximal to A. fumigatus were unable to grow (data not shown), suggesting that A. fumigatus produces a secreted toxic agent to both C. albicans and C. neoformans at the stationary phase. In order to identify if any secondary metabolites of A. fumigatus were responsible for the growth inhibition to C. albicans and C. neoformans, A. fumigatus supernatant (AFS) was collected. As little as 2 mg of AFS was able to form a zone of inhibition around C. albicans strains DAY185, 95–120, or 98–145 on plates containing Spider medium and grown at 37°C .When  at 37°C ; a zone  at 30°C . AFS wasC. albicans and C. neoformans, the supernatant was heated at 60°C for 2 hours to inactivate potential enzymatic activity. The inhibitory activity of the heat inactivated supernatant (HI-AFS) was assayed on C. albicans DAY185 and was able to produce a zone of inhibition on plates containing Spider medium and grown at 37°C  in the supernatant that are responsible for the inhibition of  at 37°C , similar at 37°C . Taken tA. fumigatus ΔgliP3 mutant strain that does not produce gliotoxin A. fumigatus is responsible for the inhibitory activity of the supernatant to C. albicans and C. neoformans. Unlike the supernatant from the wild type AF293 strain, the supernatant of the ΔgliP isolate failed to produce a zone of inhibition for either C. albicans strains DAY185, 95–120, 98–145, or C. neoformans KN99α, demonstrating that gliotoxin was responsible for the zone of inhibition observed with all the C. albicans and C. neoformans strains. Further studies on the inhibitory activity of commercially available gliotoxin supports that this compound was responsible for the zone of inhibition observed with C. albicans and C. neoformans. The area of the zone of inhibition produced by pure gliotoxin was in a dose dependent manner as observed with AFS . These studies demonstrate that gliotoxin is the major component involved in the inhibitory activity of the A. fumigatus supernatant when grown in Spider medium at 37°C.The with AFS . Pure glin vitro of C. albicans strains DAY185, 95–120, 98–145, and C. neoformans strain KN99α. The MIC of gliotoxin was 2.0 µg/ml for C. albicans and 4.0 µg/ml for C. neoformans (Pure gliotoxin and AFS were used to find the minimum inhibitory concentration (MIC) oformans , whereasoformans . In muriC. albicans is not known, as conflicting studies exist suggesting gliotoxin is produced by some strains of C. albicansC. albicans, the genome of the fungus does not contain a secondary metabolite gene cluster predicted to synthesize an ETP C. albicans is highly susceptible to gliotoxin  by a intracellular redox cycle, where the reduced compound oxidizes to reform the disulfide bridge, producing hydrogen peroxide and superoxide in the process C. albicans DAY185 were further evaluated using the BacLight live-dead staining kit (Molecular probes). Using this system, live fungi with intact membranes fluoresce green, while dead fungi with damaged membranes fluoresce red. The live-dead staining reflects the previous observation where DMSO treated cells were alive (green), and AFS (3.2 mg/ml) and gliotoxin (2.0 µg/ml) treated cells were dead (red) in Spider medium at 37°C. At 30°C, C. albicans yeast cells treated with DMSO and AFS (3.2 mg/ml) were alive  treated D medium  and S1. A. fumigatus is resistant to the effects of gliotoxin, as one of the genes in the gli cluster, gliT, encodes a reductase that confers a high level of self-protection to the compound gliA, encodes a major facilitator transporter that has been shown to be involved in efflux of the ETP A. fumigatus that confer tolerance to gliotoxin that are absent in most other fungi, including C. albicans and C. neoformans. In general there are fewer efflux transporters in C. albicans and C. neoformans when compared to A. fumigatusC. albicans were as susceptible to gliotoxin as the wild-type isolate suggesting the efflux pumps conferring resistance to fluconazole do not confer resistance to gliotoxin  of pure gliotoxin and AFS were determined. The EC50 of AFS and gliotoxin were 1.0 mg/ml and 2.0 µg/ml, respectively  to determine the concentration exhibiting in vitro inhibition of C. albicans growth.The determination of the lowest concentration of the compounds with antifungal activity was accomplished using two-fold serial dilutions of the test compounds in RPMI 1640 media at 35°C for 24 hours C. albicans in the presence of fungal supernatants and gliotoxin was assessed by using the BacLight LIVE/DEAD staining system according to the manufacturer's protocol . The cells which retained the green fluorescence color were live whereas the red fluorescent cells were considered dead.The viability of 50 of gliotoxin and fungal supernatants on worms, the C. albicans-C. elegans co-inoculation assay was performed using the C. elegans glp-4;sek-1 double mutant in all assays Escherichia coli strain OP50 as the food source. The screen medium was 20% brain heart infusion medium  in M9 buffer containing antibiotics kanamycin (90 µg/ml), ampicillin (200 µg/ml), and streptomycin (200 µg/ml). M9 buffer was used to wash the worms as needed and for the diluting the screen media.To measure the ECin vitro fungal growth, followed by visual scoring of live and dead worms based on worm shape, as live worms appear sinusoidal and dead worms are rod shaped. The worms were also tested by using a platinum pick to score live or dead. For the determination of EC50, the test compounds were serially diluted two-fold. Half the maximum effective concentration that conferred 50% survival of the worms was determined as the EC50.At the end of the incubation period, the entire wells were imaged, visually analyzed for Figure S1Inhibition of C. albicans by heat inactivated A.fumigatus supernatant.C. albicans strain DAY185 was grown on YPD media at 30°C and Spider medium at 37°C overnight in the presence of discs containing heat inactivated supernatent from isolate AF293 (HI-AFS) at the indicated concentrations.(TIF)Click here for additional data file.Figure S2Absence of inhibition of C. albicans growth by C. neoformans supernatent.C. albicans strain DAY185 was grown on YPD media at 30°C and Spider medium at 37°C overnight in the presence of discs containing the supernatant of C. neoformans strain KN99α (CNS) and the heat inactivated supernatant of C. neoformans (HI-CNS) at the indicated concentrations.(TIF)Click here for additional data file.Table S1Fungal isolates used in this study.(DOC)Click here for additional data file.


The chemokine receptor CXCR4 regulates cell migration during ontogenesis and disease states including cancer and inflammation. Upon stimulation by the endogenous ligand CXCL12, CXCR4 becomes phosphorylated at multiple sites in its C-terminal domain. Mutations in the CXCR4 gene affecting C-terminal phosphorylation sites are a hallmark of WHIM syndrome, a genetic disorder characterized by a gain-of-CXCR4-function. To better understand how multi-site phosphorylation of CXCR4 is organized and how perturbed phosphorylation might affect CXCR4 function, we developed novel phosphosite-specific CXCR4 antibodies and studied the differential regulation and interaction of three C-terminal phosphorylation sites in human embryonic kidney cells (HEK293). CXCL12 promoted a robust phosphorylation at S346/347 which preceded phosphorylation at S324/325 and S338/339. After CXCL12 washout, the phosphosites S338/339 and S324/325 were rapidly dephosphorylated whereas phosphorylation at S346/347 was long-lasting. CXCL12-induced phosphorylation at S346/347 was staurosporine-insensitive and mediated by GRK2/3. WHIM syndrome-associated CXCR4 truncation mutants lacking the S346/347 phosphosite and the recently identified E343K WHIM mutant displayed strongly impaired phosphorylation at S324/325 and S338/339 as well as reduced CXCL12-induced receptor internalization. Relevance of the S346-S348 site was confirmed by a S346-348A mutant showing strongly impaired CXCL12-promoted phosphorylation at S324/325 and S338/339, defective internalization, gain of calcium mobilization, and reduced desensitization. Thus, the triple serine motif S346-S348 contains a major initial CXCR4 phosphorylation site and is required for efficient subsequent multi-site phosphorylation and receptor regulation. Hierarchical organization of CXCR4 phosphorylation explains why small deletions at the extreme CXCR4 C terminus typically associated with WHIM syndrome severely alter CXCR4 function. Stimulus-dependent phosphorylation of G protein-coupled receptors (GPCRs) represents a major mechanism regulating signal transduction and receptor trafficking G protein-coupled CXCR4, the receptor of the chemokine CXCL12, is essential for embryonic development of various organ systems, serves as HIV coreceptor, and plays a major role in diseases including cancer and inflammation warts, hypogammaglobulinemia, infections, and myelokathexis) and gain-of-CXCR4-function knockin mice carrying a heterozygous R334X truncation mutation, which corresponds to the most frequent WHIM syndrome-causing mutation in humans Early reports analyzing CXCR4 C-terminal truncation mutants described that deletions within the 12 C-terminal residues severely impair CXCL12-induced internalization – an effect expected to reduce CXCR4 desensitization Thus, while phosphorylation occurs at multiple dispersed CXCR4 C-terminal sites  125I-CXCL12 , AMD3100 , Lambda Protein Phosphatase , phorbol 12-myristate 13-acetate , forskolin , epidermal growth factor , staurosporine .CXCL12 ,Phosphosite-specific rabbit antibodies for the S338/339-phosphorylated and S346/347-phosphorylated forms of the CXCR4 receptor were generated against the following peptides: RGGH(pS)(pS)VSTE (S338/339) and VSTESE(pS)(pS)SFHSS (S346/347), which contained two phosphorylated serine residues (pS). The peptides were purified by HPLC, coupled to keyhole limpet haemocyanin and mixed 1∶1 with Freund’s adjuvant for immunization. Specificity of the antisera was tested using dot blot analysis. For subsequent analysis, antibodies were affinity purified against their immunizing peptide and against the non-phosphorylated peptide using the SulfoLink kit . The polyclonal phosphosite-specific antibody for the S324/325-phosphorylated form of the CXCR4 receptor (#5199) was a kind gift of Dr. Jeffrey L. Benovic TM3.1(+) vector . The cDNA for a CXCR4-ST/A mutant lacking C-terminal serines and threonines, a S346-348A mutant, an E343K mutant, and for CXCR4 mutants lacking the last 10 and 17 residues  were generated by gene synthesis  and inserted into CXCR4-WT using the endogenous Sph I site (bp 967) and the Not I extension. Sequence identity was verified by double strand sequencing.cDNA for wildtype CXCR4 (CXCR4-WT) was amplified by reverse transcription PCR from mouse spleen cDNA with Nhe I/Not I extensions. The construct received an N-terminal HA epitope tag or an N-terminal T7 epitope tag and was subcloned into pcDNAHuman embryonic kidney cells  were cultivated in DMEM  supplemented with 10% defined fetal bovine serum , 2 mM L-glutamine, and 100 units/ml penicillin/streptomycin (PAA). JetPEI reagent  was used for transient and stable transfections. Stable transfectants were selected in the presence of 400 µg/ml G418.125I-CXCL12. For homologous competitive radioligand binding, cells were incubated for 2 h at 4°C with 125I-CXCL12 containing increasing amounts of unlabeled CXCL12. Cells were washed twice with ice cold phosphate buffered saline (PBS) and then lysed in 10 mM Tris buffer (pH7.4). IC50 was calculated using the “homologous competitive binding curve” option of the GraphPad Prism 4.0a software. For pulse chase analyses of radioligand/receptor internalization, cells were loaded with 125I-CXCL12 for 2 h at 4°C. Cultures were washed with ice cold PBS and harvested either immediately  or transferred to 37°C to permit internalization. Residual surface-bound 125I-CXCL12 was stripped by a double wash with acidic citrate buffer. Then, cells were lysed in 10 mM Tris buffer. Counts of mock-transfected cultures were subtracted.Binding and internalization assays were performed as described Cells were processed as described Immunoblotting procedures were performed as described An established siRNA-mediated knockdown protocol for GRK2 and GRK3 was used i/o-coupled receptors to Ca2+ mobilization 2+]i measured using the FlexStation3 microplate reader as described Cells were co-transfected with CXCR4 constructs and a 16z44 G protein chimera engineered to link GStatistical tests were done by ANOVA including the indicated post-tests or by Student's t-test using GraphPadPrism software.To investigate multi-site phosphorylation properties of CXCR4, we generated two novel polyclonal phospho-selective antibodies for S338/339 and S346/347 and used them along with a polyclonal antibody for phosphorylated S324/325 The four antibodies were then characterized in immunoblots with lysates from HEK293 cells stably expressing CXCR4 with an N-terminal hemagglutinin (HA) epitope tag. The cultures received vehicle or CXCL12 for 15 min before they were harvested. Aliquots of CXCL12-stimulated lysates were dephosphorylated with Lambda-Protein Phosphatase (λ-PP) before loading. Anti-S341-S352 detected a broad band of the expected size at 47 kDa. The signal was strongly reduced after CXCL12-stimulation and was fully recovered after dephosphorylation . Thus, wThe phospho-selective antibodies produced strong CXCR4-specific signals in CXCL12-stimulated samples and weak signals in non-stimulated and dephosphorylated samples . CXCL12 We then used the four antibodies to examine kinetics of CXCL12-induced phosphorylation. Stable CXCR4 transfectants were harvested immediately before as well as 2, 5, and 15 min after CXCL12 stimulation. Aliquots of the lysates were loaded four times, immunoblotted, and detected with anti-S341-S352 and the three phospho-selective antibodies . Blots wDephosphorylation kinetics were studied after 30 min CXCL12 stimulation. The extended stimulation period was used to ensure maximum phosphorylation. The CXCL12 stimulus was ended by an acidic wash followed by 5, 15, 30, and 60 min ligand-free intervals in the presence of the CXCR4 antagonist AMD3100 . Thus, tIt has been reported that extended CXCL12 stimulation (≥180 min) causes CXCR4 degradation in HEK293 cells Stimulus-dependent phosphorylation of GPCRs is mediated by multiple kinases including protein kinase A (PKA), protein kinase C (PKC), and G protein coupled receptor kinases (GRKs) We then used the PKC inhibitor staurosporine to test the contribution of PKC to the CXCL12- and PMA-induced CXCR4 phosphorylation . This sh125I-labeled 12G5 anti-CXCR4 antibody We then studied the influence of the extreme CXCR4 C terminus on phosphorylation at S324/325 and S338/339. To this end, we used two CXCR4 mutants with an N-terminal T7-tag and deletions of the last 10 and 17 C-terminal residues , which correspond to WHIM syndrome-associated mutations in the human CXCR4 gene CXCL12-induced phosphorylation was studied in transient transfectants 15 min after vehicle and CXCL12 stimulation. Since the phospho-selective antibodies produced some constitutive signals under these experimental conditions, we subjected aliquots of the lysates to Lambda-PP dephosphorylation to demonstrate phosphoselectivity of the signal . CXCL12-Having shown that the CXCL12-promoted phosphorylation at S346/347 precedes that at S324/325 and S338/339 and that phosphorylation at S324/325 and S338/339 is severely reduced when the last ten CXCR4 residues are absent, we hypothesized that phosphorylation at residues S346-S348 is obligate for efficient phosphorylation to occur at the other sites. To test this hypothesis, we generated a HA-tagged S346-348A mutant . After pulse labeling of the extracellular HA-tag with anti-HA antibody in live cells at 4°C and several washes, cells were fixed and detected with a peroxidase-conjugated secondary antibody and a peroxidase-driven color reaction. The assay showed similar labeling intensities for wildtype CXCR4, E343K-CXCR4, and S346-8A-CXCR4 . Mock-trmutation . CXCL12-mutation  and quanmutation . After pmutation . Applicamutation  but littmutation . Consistmutation .i-coupled receptors to the calcium pathway G protein coupling of CXCR4 was examined in calcium mobilization experiments using FLIPR technology. The CXCR4 variants were co-transfected with a 16z44 G protein chimera that was engineered to link GSequential stimulation was applied to determine homologous CXCR4 desensitization. When CXCL12 (80 nM) was administered 180 s after a precedent 80 nM stimulus, it evoked only a small calcium response irrespective of the CXCR4 variant examined (not shown). To circumvent major CXCR4 desensitization, we reduced the CXCL12 concentration in the pre-stimulus to 5 nM. Normalization of the traces with the mean of the first maximum of wildtype CXCR4 showed that the calcium responses to both the first 5 nM stimulus and to the second 80 nM stimulus were larger with CXCR4-E343K and CXCR4-S346-8A than with wildtype CXCR4 , which cUsing novel and established phospho-selective antibodies for the CXCR4 C-terminal sites S324/325, S338/339, and S346/347, we provide evidence that S346/347 represents a major C-terminal phosphorylation site. Upon CXCL12 stimulation, S346/347 reached maximal phosphorylation faster than S338/339 and S324/325. Deletion of S346-S348 reduced CXCL12-promoted phosphorylation at S324/325 and S338/339, caused a gain-of-CXCR4-function and reduced receptor internalization and desensitization. Our findings indicate hierarchical organization of the multiple phosphorylation events taking place at the CXCR4 C-terminal domain and help explain why mutations in the CXCR4 gene which cause small alterations at the extreme C terminus can severely affect CXCR4 regulation and signaling.We established that the rabbit monoclonal anti-S341-S352 antibody UMB-2 is highly selective for CXCR4 and that the antibody recognizes only the non-phosphorylated C terminus. Immunoblots with lysates from CXCR4-overexpressing HEK293 cells showed that 15 minutes CXCL12 stimulation were sufficient to trigger an almost complete loss of UMB-2 signal. The finding that dephosphorylation fully restored the UMB-2 signal indicates that one or several of the potential phosphosites in the UMB-2 epitope  become quantitatively phosphorylated upon CXCL12 exposure. Thus, by detecting dephosphorylated and non-dephosphorylated aliquots of a sample with UMB-2 one can visualize total and non-phosphorylated CXCR4. The difference between the two signals reflects C-terminal CXCR4 phosphorylation and can serve as an indicator for CXCR4 activation. UMB-2 readily detects low endogenous CXCR4 levels in HEK293 cells and brain tissue Our dot blot analyses showed that phosphorylation at the S346/347 site is sufficient to prevent UMB-2 binding. Given that UMB-2 fails to recognize lysates from CXCL12-stimulated cells, we reasoned that the S346-S348 motif might contain a major CXCR4 phosphorylation site. To test this assumption, we developed a phospho-selective anti-pS346/347 antibody. Using this tool, we showed that this site is indeed rapidly phosphorylated upon CXCL12 stimulation. CXCL12-promoted phosphorylation at S346/347 was mediated by GRK2/3 and occurred in a PKC-independent manner. This property renders S346/347 distinct from previously identified phosphosites in the CXCR4 C terminus, which include S324/325, S330, and S338/339 Another distinguishing feature of S346/347 phosphorylation is its permanent nature: whereas S324/325 and S338/339 were rapidly dephosphorylated after CXCL12 washout, major dephosphorylation was not observed for S346/347 during the investigated 60 min ligand-free interval. Ligand-induced phosphorylation of GPCRs typically induces ß-arrestin/receptor interaction Most WHIM syndrome patients carry heterozygous mutations in the CXCR4 gene which eliminate the last 10 to 19 residues of the C terminus But how do WHIM patients lacking CXCR4 mutations Our studies in HEK293 cells show that serine residues 346 and 347 constitute a major CXCR4 phosphorylation site. Elimination of the S346-S348 cluster severely impairs phosphorylation at the intact C-terminal sites S324/325 and S338/339. We suggest that phosphorylation events at the CXCR4 C terminus occur hierarchically requiring initial phosphorylation at S346/347. These results identify a novel structural determinant of CXCR4 regulation and help decipher how mutations affecting the CXCR4 C terminus lead to signaling perturbances and disease.Figure S1Dot blot analysis of phospho-sensitive CXCR4 antibodies.A, Schematic representation of CXCR4 C-terminal residues 322–352. Sequences of peptides #1 - #3 used in the dot blot are underlined. Serine residues 324/325, 338/339, and 346/347 used for phosphorylation in the phosphopeptides are highlighted. B, Decreasing amounts (2–0.125 µg) of non-phospho and phosphopeptides #1 - #3 were blotted onto 4 PVDF membranes and detected with anti-S341-S352, anti-pS346/347, anti-pS338/339, and anti-pS324/325 as indicated. Anti-S341-S352 detects the S341-S352 epitope only when serine residues 346 and 347 are not phosphorylated. The phosphoantibodies recognize their phosphorylated epitopes with high selectivity showing little crossreactivity.(TIF)Click here for additional data file.


Two mutations in FGFR3, G380R and G375C are known to cause achondroplasia, the most common form of human dwarfism. The G380R mutation accounts for 98% of the achondroplasia cases, and thus has been studied extensively. Here we study the effect of the G375C mutation on the phosphorylation and the cross-linking propensity of full-length FGFR3 in HEK 293 cells, and we compare the results to previously published results for the G380R mutant. We observe identical behavior of the two achondroplasia mutants in these experiments, a finding which supports a direct link between the severity of dwarfism phenotypes and the level and mechanism of FGFR3 over-activation. The mutations do not increase the cross-linking propensity of FGFR3, contrary to previous expectations that the achondroplasia mutations stabilize the FGFR3 dimers. Instead, the phosphorylation efficiency within un-liganded FGFR3 dimers is increased, and this increase is likely the underlying cause for pathogenesis in achondroplasia. We further investigate the G346E mutation, which has been reported to cause achondroplasia in one case. We find that this mutation does not increase FGFR3 phosphorylation and decreases FGFR3 cross-linking propensity, a finding which raises questions whether this mutation is indeed a genetic cause for human dwarfism. Receptor tyrosine kinase (RTK)-mediated signaling regulates vital cellular processes such as cell growth, differentiation, and motility Disregulation of the processes that control RTK activation leads to pathologies Thus far, the effect of FGFR3 mutations linked to phenotypes of different severities have been compared and rank-ordered, and mutations associated with more severe phenotypes have been shown to have a more profound effect on FGFR3 signaling. For instance, mutations associated with TD1 have been shown to activate FGFR3 to higher extent than mutations causing ACH To start addressing this question, here we investigate the effect of the G375C mutation, linked to ACH, on FGFR3 phosphorylation and cross-linking, and we compare the results to previously published results for the G380R ACH mutant. The comparison is carried out over a wide range of ligand concentrations, in order to gain insight into the molecular mechanism that underlies the pathology. We observe that the G375C mutation increases FGFR3 phosphorylation in the absence of ligands and at low, but not at high, ligand concentrations, similarly to the G380R mutation. The dimerization propensity of FGFR3 is not affected by the G375C mutation, as inferred from cross-linking experiments. Our results demonstrate that the two achondroplasia mutations, G375C and G380, have the same effect on FGFR3 activity, supporting the idea that identical degrees and mechanisms of over-activation lead to identical phenotypes.In addition, here we investigate a third FGFR3 mutation, G346E. While this mutation is often mentioned in the literature as a genetic cause for ACH The FGFR3/WT plasmid was a generous gift from D. J. Donoghue, University of California, San Diego. The G375C and G346E mutations were created using the QuickChange® XL site-directed mutagenesis kit . All the plasmids were in the pcDNA 3.1(+) vector. Successful incorporation of the mutations was confirmed by sequencing provided by Genewiz .ATCC) were transfected with plasmids encoding FGFR3/WT, FGFR3/G375C and FGFR3/G346E using FuGENE HD (Roche Applied Science) according to the manufacturer's protocol. Cells were cultured in normal medium for 24 h following transfection, and were then starved in serum-free medium for 24 h as previously described fgf1 . Different concentrations of fgf1, ranging from 50 to 2500 ng/ml, were added to the serum-free medium. After incubating for 10 min with ligand, cells were lysed as described Human embryonic kidney cells  suberate, Pierce). Twenty four hours after transfection, cells were incubated with 2 mM cross-linker for 30 min at room temperature and then quenched in 20 mM Tris-HCl for 15 min. After a rinse with ice-cold PBS, the cells were lysed, and the receptors were detected using Western blotting. The cross-linked fraction was calculated as D/SS = DS/(MS+DS), where DS is the intensity of the cross-linked band, and MS is the intensity of the monomeric band Dimeric receptors were cross-linked with a membrane-impermeable linker  antibodies or anti-P-FGFR antibodies. The anti-P-FGFR antibodies used were anti-phospho-Tyr-653/Tyr-654 antibodies , generated to recognize phosphorylated Tyr-653 and Tyr-654 in FGFR1. Since the amino acid sequences of FGFR1 and FGFR3 in the vicinity of these two tyrosines are the same, it also specifically recognizes the analogous Tyr-647 and Tyr-648 in FGFR3 Next we characterized the response of the two FGFR3 mutants to different concentrations of Three independent experiments were performed while varying the ligand concentration. One representative film is shown in In fgf1. Cells that were not stimulated with ligand were used as controls. BS3, a membrane impermeable linker, was used to covalently link the FGFR3 dimers on the plasma membrane. Cells were lysed and the lysates were analyzed using Western blotting. A typical Western blot film is shown in D/S = SD/(SM+SD), where SD is the intensity of the cross-linked band, and SM is the intensity of the monomeric band. The results are shown in fgf1, all three receptors show a moderate increase (∼50% to 100%) in cross-linked fraction. In the presence of ligand, the cross-linked fraction for wild type FGFR3 and the G375C and G380R mutants remain similar , and the G346E cross-linked fraction remains lower (p = 0.005). The results indicate that none of the three ACH mutations increases FGFR3 crosslinking, and thus the dimerization propensity of FGFR3. Therefore, the increase in phosphorylation of the G375C or G380R mutants, shown in Next we used chemical cross-linking to gain insight into the effect of the mutations on FGFR3 dimerization. HEK 293 cells were transfected with plasmids encoding FGFR3/WT, FGFR3/G346E or FGFR3/G375C. Cells were starved for 24 hours, and stimulated with high concentration of Achondroplasia is a bone-growth disorder with phenotypic features which include short arms and legs, and a large head with prominent forehead The G380R mutation accounts for about 98% of the ACH cases. The other 2% are attributed to the G375C mutation in FGFR3 A published model of the wild-type dimer structure, created with the program CHI, predicts that Glycine in position 375 does not face the dimer interface and is exposed to lipids Our work is consistent with previous studies which have reported increased phosphorylation of the G375C mutant, as compared to wild-type Here we observe that FGFR3 phosphorylation in the absence of ligand increases 2.5 times due to the G375C and G380R mutations. The effect is greatest in the absence of ligand and the mutation-induced difference in phosphorylation decreases as more ligand is added. Thus, a question arises if an effect of this magnitude can be responsible for a pathological phenotype. It is possible that the effect is amplified due to defects in mutant FGFR downregulation. It has been shown that the downregulation of FGFR3 mutants linked to skeletal dysplasias is compromised, and this effect prolongs the time that they signal The G346E mutation in FGFR3 has been reported to cause achondroplasia It has been suggested that there is a correlation between the level of FGFR3 phosphorylation and the severity of the disturbance in endochondrial ossification in human skeletal dysplasias. Ornitz and colleagues were the first to explore if such a correlation exists A study by Bellus at al fos transcription in RCJ cells than the G375C mutant, consistent with the fact that TD1 is much more severe than ACH.In a third study, Adar and colleagues compared the effects of the G370C and G371C mutations linked to TD1, to the effect of the ACH G375C mutation different FGFR3 mutations cause the same dwarfism phenotype, and we thus compare the phosphorylation of two mutants linked to the same phenotype, ACH. Unlike previous comparisons, here we compare the phosphorylation over a wide range of ligand concentration, and we perform cross-linking experiments within the native cellular membrane in order to probe the underlying cause for the elevated phosphorylation due to the mutations. We observe identical behaviors of the G380R and G375C mutants in terms of their activities and cross-linking propensities. These data are consistent with the idea that the severity of the phenotype is directly linked to the degree of RTK phosphorylation.All these data suggest that a relationship exists between the activity levels of the mutants and the severities of the associated disorders. Here we seek insight into how Yet, the phosphorylation level of a particular mutant is not the only determinant of a pathological phenotype. For instance, the A391E mutation in FGFR3 increases FGFR3 phosphorylation at low ligand concentration, similarly to the G380R and G375C ACH mutations studied here


Background: KCC3 lacking the two known phosphorylation sites is still regulated by cell swelling and WNK3.Results: Dephosphorylation of serine 96 is necessary for full activation of the cotransporter.Conclusion: Serine 96 is a third phospho-site involved in KCC3 regulation.Significance: The finding of new phosphorylation sites sheds light on an increasingly complex regulation of K+:Cl− cotransporters. +:Cl− cotransporter (KCC) activity is modulated by phosphorylation/dephosphorylation processes. In isotonic conditions, KCCs are inactive and phosphorylated, whereas hypotonicity promotes their dephosphorylation and activation. Two phosphorylation sites (Thr-991 and Thr-1048) in KCC3 have been found to be critical for its regulation. However, here we show that the double mutant KCC3-T991A/T1048A could be further activated by hypotonicity, suggesting that additional phosphorylation site(s) are involved. We observed that in vitro activated STE20/SPS1-related proline/alanine-rich kinase (SPAK) complexed to its regulatory MO25 subunit phosphorylated KCC3 at Ser-96 and that in Xenopus laevis oocytes Ser-96 of human KCC3 is phosphorylated in isotonic conditions and becomes dephosphorylated during incubation in hypotonicity, leading to a dramatic increase in KCC3 function. Additionally, WNK3, which inhibits the activity of KCC3, promoted phosphorylation of Ser-96 as well as Thr-991 and Thr-1048. These observations were corroborated in HEK293 cells stably transfected with WNK3. Mutation of Ser-96 alone (KCC3-S96A) had no effect on the activity of the cotransporter when compared with wild type KCC3. However, when compared with the double mutant KCC3-T991A/T1048A, the triple mutant KCC3-S96A/T991A/T1048A activity in isotonic conditions was significantly higher, and it was not further increased by hypotonicity or inhibited by WNK3. We conclude that serine residue 96 of human KCC3 is a third site that has to be dephosphorylated for full activation of the cotransporter during hypotonicity.The K Erythroid function and differentiation, cancer cell growth and invasiveness, arterial blood pressure regulation, and neuronal excitability are some of the important roles of these membrane proteins  218000) (The Kproteins –3. Inact 218000) –6. Moreo 218000) .+-coupled chloride cotransporters, generally called N(K)CCs, that following the driving force imposed by the Na+:K+:ATPase translocate ions from the outside to the inside of the cell, thus having similar important roles as the KCCs in many physiological aspects, but in the opposite direction  and custom-made primers (Sigma). All mutations were confirmed by sequencing and subcloned back into the appropriate expression constructs.Xenopus laevis oocytes as described previously . All experimental data are based on a minimum of three different experiments. Our institutional committee on animal research approved the use of X. laevis frogs.We assessed the activity of wild type or mutant KCC3 or KCC4 using the heterologous expression system of eviously –15. In bE. coli cells, and 1-liter cultures were grown at 37 °C in LB medium (100 μg/ml ampicillin) until the absorbance at 600 nm was 0.8. Isopropyl β-d-thiogalactopyranoside (30 μm) was then added, and the cultures were grown for further 16 h at 26 °C. Cells were isolated by centrifugation, resuspended in 40 ml of ice-cold lysis buffer, and sonicated  to fragment DNA. Lysates were centrifuged at 4 °C for 15 min at 26,000 × g. The GST fusion proteins were affinity-purified on 0.5 ml of glutathione-Sepharose and eluted in buffer A containing 0.27 m sucrose and 20 mm glutathione.All pGEX-6P-1 constructs were transformed into BL21 E. coli were incubated with active SPAK (Carna Biosciences STLK3 (STK39), product number: 07-130) (1 μg) or kinase-inactive SPAK GST-SPAK(D212A) (1 μg) also purified from E. coli at 30 °C for 60 min in buffer A containing 10 mm MgCl2, 0.1 mm [γ-32P]ATP  in a total reaction volume of 25 μl. The reaction was terminated by the addition of LDS sample buffer. Dithiothreitol (DTT) was added to a final concentration of 10 mm, and the samples were heated at 95 °C for 4 min and cooled for 20 min at room temperature. Iodoacetamide was then added to a final concentration of 50 mm, and the samples were left in the dark for 30 min at room temperature to alkylate cysteine residues. The samples were subjected to electrophoresis on a Bis-Tris 10% polyacrylamide gel, which was stained with colloidal blue and then autoradiographed. The phosphorylated GST-KCC3a(1–175) and GST-NKCC2(1–174) bands were excised, cut into smaller pieces, and washed sequentially for 10 min on a vibrating platform with 1 ml of the following: water; 1:1 (v/v) mixture of water and acetonitrile; 0.1 m ammonium bicarbonate; 1:1 mixture of 0.1 m ammonium bicarbonate and acetonitrile; and finally acetonitrile. The gel pieces were dried and incubated for 16 h at 30 °C in 25 mm triethylammonium bicarbonate containing 5 μg/ml trypsin as described previously  equilibrated in 0.1% trifluoroacetic acid in water. The column was developed with a linear acetonitrile gradient  at a flow rate of 0.2 ml/min, and fractions of 0.1 ml were collected.GST-KCC3a(1–175) and GST-NKCC2(1–174) 10 μg) purified from  μg purif32P-labeled peptides was determined by solid-phase Edman degradation on an Applied Biosystems 494C sequencer of the peptide coupled to Sequelon-AA membrane (Applied Biosystems) as described previously , KCC3 phospho-Ser-96 (residues 80–94 or 89–103 of human KCC3a), KCC3 phospho-Thr-982 (residues 975–989 or 984–998 of human KCC3a), and KCC3 phospho-Thr-1039 (residues 1032–1046 or 1041–1055 of human KCC3a). Secondary antibodies coupled to horseradish peroxidase used for immunoblotting were obtained from Pierce. Specificity of phospho-antibodies was assessed using protein extracts from HEK293 transfected with KCC3 cDNA.with no lysine kinase 3 (WNK3) was used to transiently transfect full-length human KCC3a. To generate the transient cell lines, pre-cultures of HEK293-WNK3 underwent passages every 2–3 days to keep the cells in their exponential growth phase. 2 h before transfection, the cell suspension was centrifuged (300 × g for 5 min), resuspended in fresh medium at 5 × 106 cell/ml, and plated on 60-mm diameter dishes (60% confluence). Then, cells were transfected at 37 °C with the plasmid DNA or empty vector using the calcium phosphate method. Briefly, 5 μg of the full-length KCC3a was suspended in CaCl2 solution at a final concentration of 0.3 m and mixed with 150 μl of 2× HEPES-buffered saline  dropwise while mixing gently. The mixture was left at room temperature for 30 min and then incubated with the cells for 6 h in 37 °C. Cells were then washed two times with 1× phosphate-buffered saline (PBS) and placed in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS, 50 units/ml penicillin, 50 μg/ml streptomycin, and 1 mg/ml G418 in a humidified atmosphere containing 5% CO2 and 95% air at 37 °C. 48 h after transfection, cells were exposed to isotonic  or hypotonic 30% solutions (obtained by reduction of NaCl concentration) for 30 min. After treatment, the cells were harvested in ice-cold lysis buffer , incubated for 10 min and then scraped. Cells homogenates were sonicated and centrifuged , and finally the supernatants were collected.A stably transfected HEK293 cell line developed for a previous study  that hadm 10 Tris-HCl, 150 NaCl, 1 EDTA, 1% Triton (10 μl/oocyte)) supplemented with protease and phosphatase inhibitor mixture (Roche Diagnostics). After clearing the lysate by centrifugation at 10,000 × g at 4 °C for 30 min, total protein was collected from the supernatant. Western blotting was carried out using previously characterized anti-KCC3 rabbit polyclonal antibodies , membranes were incubated with horseradish peroxidase-conjugated secondary antibody in blocking solution  and visualized by chemiluminescence .Groups of 10–20 oocytes were injected with cRNA from various constructs, transferred to Eppendorf tubes, and homogenized at 4 °C in lysis buffer (in mtibodies  and the g for 5 min), and protein concentration was determined by the Bradford method. Then, 25 μg of protein was separated by SDS-PAGE (7.5% acrylamide gel) and transferred onto PVDF membranes (Bio-Rad). Membranes were blocked with TBST containing 5% (w/v) nonfat dried milk and incubated overnight at 4 °C, with rabbit primary antibodies anti-KCC3 (1:1000)  , anti-KCp < 0.05, and the results are presented as mean ± S.E. The significance of the differences between two groups was tested by Student's t test, and the significance of the differences for three or more groups was tested by one-way analysis of variance with multiple comparisons using Bonferroni's correction.Statistical significance is defined as two-tailed A and B) were compared with those of the double mutant KCC3-T991A/T1048A (A and C), in both isotonic and hypotonic conditions. As shown previously (p > 0.001). As reported by Rinehart et al. . However, when oocytes were incubated in hypotonicity, a further increase in activity was observed . Additionally, as depicted in B and C, and reported previously for wild type KCC3 (p < 0.05 versus the absence of WNK3). These observations suggested that there might be an additional phosphorylation site(s) that has to be dephosphorylated to achieve full activation of the cotransporter. This proposal differs from that of Rinehart et al. (D). Thus, total time of exposition to hypotonicity was 20 min in the shorter groups and 90 min in the longer groups. As shown in Fig. 1D, when using a shorter period of hypotonic incubation, the Cl−-dependent 86Rb+ uptake was similar between wild type and double mutant KCC3-T991A/T1048A injected oocytes , whereas when using a longer period of exposition to hypotonic medium, the Cl−-dependent, 86Rb+ uptake induced by wild type was significantly higher than the double mutant.The functional properties of wild type KCC3 , A and BA/T1048A , A and Ceviously , wild tyt et al. , we obseype KCC3 , 18, 19,t et al.  where det et al. –22. Thust et al. D. Thus, A).The evidence shows that phosphorylation and activation of the NKCCs in the N-terminal domain by SPAK and OSR1 mediate WNK kinase signaling –26. TherFig. 232P-labeled KCC3(1–175) was digested with trypsin and analyzed by chromatography on a C18 column for phospho-site mapping. One major 32P-labeled phospho-peptide was observed (B). A combination of solid-phase Edman sequencing and mass spectrometry (C) revealed that this encompassed a peptide phosphorylated at Ser-96. Mutating Ser-96 to Ala prevented phosphorylation of KCC3 by SPAK, thereby confirming that this residue represents the major site of phosphorylation (data not shown).SPAK phosphorylated KCC3(1–175) in a time-dependent manner, showing an ∼18-fold increase in phosphorylation when compared with phosphorylation by active SPAK in the absence of MO25 (data not shown). observed B. A combtrometry C reveale+:Cl− cotransporters shows that the potential phosphorylation site Ser-96 is unique to KCC3 because it is only present in KCC3a and KCC3b isoforms (A), but not in the other KCCs. The double mutant KCC4-T926A/T980A, which is homologous to KCC3-T991A/T1048A, but lacks the Ser-96 residue, is also active in isotonic conditions, but cannot be further increased in hypotonicity (B). Thus, in contrast to KCC3, in the KCC4 isoform that lacks the Ser-96 site, elimination of the two known sites at the C-terminal domain results in full activation of the cotransporter in isotonicity, suggesting that the presence of Ser-96 in KCC3 is responsible for the different functional behavior between KCC3-T991A/T1048A and KCC4-T926A/T980A toward hypotonicity.The sequence alignment of the four Kisoforms A, but notonicity B. Thus, A, similar to wild type KCC3, the KCC3-S96A was inactive in isotonic conditions and was dramatically stimulated by incubation in hypotonicity. The activation was fully prevented by co-expression with WNK3 . Thus, mutation of Ser-96 by itself has no effect on the behavior of KCC3 toward isotonic or hypotonic conditions, as well as to the known inhibitory action of WNK3 on the cotransporter . As shown in Fig. 5B, we were able to demonstrate that wild type KCC3 overexpressed in X. laevis oocytes is indeed phosphorylated at these three residues in isotonic conditions, when the cotransporter is inactive, and is considerably dephosphorylated following incubation in hypotonic conditions, when KCC3 becomes active. We also observed the expected effect of WNK3 kinase co expression; in the presence of WNK3, dephosphorylation of the three sites was partially precluded. This correlates with the uncompleted activation of KCC3 in hypotonic conditions that occurs in the presence of this kinase . However, the level of activity was significantly higher in the triple mutant than in the double mutant , suggesting that the absence of phosphorylation in serine 96 contributed to higher activity of the triple mutant KCC3 in isotonic medium. This is supported by the findings observed during incubation of oocytes in hypotonic conditions. Further activation of the double mutant was noted , whereas no additional effect of hypotonicity was detected in the triple mutant . The Cl−-dependent, 86Rb+ uptake of the triple mutant in isotonicity and hypotonicity was 4.97 ± 0.57 and 5.23 ± 0.57 nmol/oocyte/h, respectively (p = not significant). In addition, co-injection of oocytes with WNK3 cRNA was able to significantly reduce the activity of wild type and double mutant KCC3 in hypotonic conditions, but no inhibitory effect was observed upon the triple mutant .Because Ser-96 residue in the double mutant KCC3-T991A/T1048A was phosphorylated and dephosphorylated similarly as in the wild type KCC3a, but elimination of this site alone had no effect on the cotransporter behavior , to findFig. 6+:Cl− cotransporter isoform KCC3 is only achieved after dephosphorylation of three distinct residues, two of which are located at the C-terminal domain (threonine residues 991 and 1048) and were reported previously by Rinehart et al. . The observed increase was sensitive to WNK3 (C). Because WNK3 cannot phosphorylate neither Thr-991 nor Thr-1048 in the double mutant KCC3-T991A/T1048A, this observation suggested that it was probably reducing the cotransporter activity by phosphorylating Ser-96. In addition, we showed that wild type KCC3 activity in hypotonic medium was significantly higher than that of the double mutant KCC3-T991A/T1048A in the same conditions (A). The reason for the discrepancy appears to be the time of exposure to the hypotonic medium. Rinehart et al.  resulted in full activation in isotonic conditions, suggesting that in this isoform no other sites are involved. Similarly, for NKCCs, between three and five different phosphorylation sites involved in their regulation have been located at the N-terminal domain, from which the master one is conserved (Thr-60 in human NCC), whereas some of the others are unique to one isoform 26A/T980A.The presence of a third site suggests that KCC3 in some cells, tissues, or circumstances requires fine-tuning for its regulation. In this regard, it is worth noticing that we have shown before that an alternatively spliced isoform of KCC3a lacking exon 2 (KCC3a-x2M) is present in several human tissues . Exon 2 Phosphorylation/dephosphorylation processes modulate the activity of the SLC12 cotransporters. Several studies support that promoting phosphorylation by cell shrinkage, activating kinases, or inhibiting protein phosphatases increases the activity of the NKCCs and inhibits the KCCs, thus increasing intracellular chloride concentration, cell volume, and transcellular ion transport capacity. In contrast, promoting dephosphorylation by cell swelling, inhibiting kinases, or activating protein phosphatases reduces the NKCCs and activates the KCCs, thus decreasing intracellular chloride concentration, cell volume, and transcellular ion transport. Because the mode of operation of these secondary transporters is still far from being understood, it is not clear at the moment how the phosphorylation or dephosphorylation of one site can affect the transport activity of the protein, particularly because it is believed that this occurs with proteins that are already in the plasma membrane , 11, 32.In summary, our data support that serine 96 in KCC3 is a third phosphorylation site implicated in the regulation of the cotransporter activity.


BRAF(V600E) has improved drastically after the discovery of the BRAF inhibitor, vemurafenib. However, drug resistance is a recurring problem, and prognoses are still very bad for patients harboring BRAF wild-type. Better markers for targeted therapy are therefore urgently needed.Treatment of metastatic malignant melanoma patients harboring ex-vivo inhibitory effects of vemurafenib and sunitinib on kinase activity status.In this study, we assessed the individual kinase activity profiles in 26 tumor samples obtained from patients with metastatic malignant melanoma using peptide arrays with 144 kinase substrates. In addition, we studied the overall ex-vivo incubation with both vemurafenib and sunitinib caused significant decrease in phosphorylation of kinase substrates, i.e kinase activity. While basal phosphorylation profiles were similar in BRAF wild-type and BRAF(V600E) tumors, analysis with ex-vivo vemurafenib treatment identified a subset of 40 kinase substrates showing stronger inhibition in BRAF(V600E) tumor lysates, distinguishing the BRAF wild-type and BRAF(V600E) tumors. Interestingly, a few BRAF wild-type tumors showed inhibition profiles similar to BRAF(V600E) tumors. The kinase inhibitory effect of vemurafenib was subsequently analyzed in cell lines harboring different BRAF mutational status with various vemurafenib sensitivity in-vitro.Overall kinase activity was significantly higher in lysates from melanoma tumors compared to normal skin tissue. Furthermore, Our findings suggest that multiplex kinase substrate array analysis give valuable information about overall tumor kinase activity. Furthermore, intra-assay exposure to kinase inhibiting drugs may provide a useful tool to study mechanisms of resistance, as well as to identify predictive markers. Metastatic malignant melanoma is associated with a poor prognosis. For decades, patients have been treated with palliative chemotherapy like dacarbazine (DTIC) monotherapy. However, only 10–15% of patients respond to this type of treatment, and for the majority, responses are of short duration only BRAF  is frequently mutated in melanoma (40–50% of cases) and has resulted in the development of BRAF-targeting kinase inhibitors, like vemurafenib (PLX4032) and dabrafenib BRAF(V600E/K) mutations. Initial clinical studies with vemurafenib showed remarkably positive results, with response rates approaching 80% Recent advances in melanoma research have unraveled some of the complexity of the molecular mechanisms of this disease. The oncogene BRAF-signaling pathway, but also more indirectly participating within this particular signaling pathway, might be implicated in cancer progression and drug resistance, and could act as functional biomarkers. In this study, we assayed the kinase activity in protein lysates from tumor samples provided by metastatic melanoma patients using a multiplex kinase substrate array technology. Our primary aim was to identify specific kinase activity profiles of metastatic melanoma and normal skin tissue. Secondly, we aimed to study the ex-vivo inhibitory effects of vemurafenib in order to describe kinases and signaling pathways involved in vemurafenib response, and to compare the findings to the inhibitory effects of in-vitro vemurafenib treatment in metastatic melanoma cell lines. The experiments were repeated with sunitinib, a multi-targeted kinase inhibitor, for comparison of results obtained with vemurafenib, using the same methodological approach.Kinases have become attractive targets for novel anticancer drugs The Regional Committee for Medical and Health Research Ethics approved the study, and each patient provided written informed consent.BRAF, NRAS  oncogene homolog), CDKN2A (cyclin-dependent kinase inhibitor 2A), and TP53 (Tumor protein p53) In total, 26 fresh-frozen tumor samples from patients suffering from stage IV melanoma were collected prior to DTIC treatment at Haukeland University Hospital . The pat3. The number of coupes needed was calculated based on the surface area of the tissue specimen. The tissue samples were kept frozen at all times during the procedure, and stored at −80°C until further use. To avoid contamination, the tumor and normal tissue specimens were prepared separately. The sectioned tissue was lysed with the mammalian protein extraction reagent (M-PER) buffer , supplemented with phosphatase and protease inhibitors , for the determination of kinase activity profiles in the presence and absence of two different inhibitors; vemurafenib  and sunitinib . The protein concentration of lysates was determined using the BCA assay . For each experiment, 15 µg of protein lysate from melanoma tissue or 20 µg of protein lysate from normal skin tissue was added to the reaction mixture, in addition to 400 µM ATP and 12.5 mg/mL of monoclonal fluorescein isothiocyanate-conjugated anti- phosphotyrosine antibody .The tissue specimens were sectioned with a microtome into 10 µm thick coupes, to a total volume of ∼3 mmhttp://www.ebi.ac.uk/arrayexpress/); accession number E-MTAB-1245.Kinase activity profiling was performed using the Tyrosine Kinase PamChip® Array for Pamstation®12  at Akershus University Hospital. Each array consists of 144 peptide substrates, primarily with known tyrosine residues, representing ∼100 different proteins. Three chips can be run simultaneously, and each chip consists of four arrays. The lysates are repeatedly pumped up and down through the porous array, allowing repeat substrate phosphorylation. Based on pilot experiments of increasing concentrations of the individual kinase inhibitors added to melanoma tissue lysates that were incubated on the arrays, concentrations that resulted in ∼50% inhibition of most kinase substrates were chosen for the main experiments. Hence, concentrations of 40 µM vemurafenib and 7.5 µM sunitinib were spiked into the assay mixtures prior to incubation, whereas 1.5% dimethyl sulfoxide was added to mixtures not containing the inhibitors. The samples were run in three technical replicates in the presence or absence of vemurafenib, as paired measurements with and without inhibitor on the same chip. The experimental procedure was repeated with sunitinib. Incubations were commenced for 60 cycles, followed by washing and fluorescence measurement of all peptide spots every fifth cycle. The experiments were run blinded, and the tumor and normal skin tissue lysates were run separately. The microarray data are submitted to ArrayExpress . Signals obtained after subtraction of local array background were used for further analysis. Negative numbers were set to 0.01 and log2-transformed signal values with inhibitor from the corresponding values without inhibitor added. The pairing of measurements with and without inhibitor was taken into account by first calculating the LFC of each chip, and subsequently averaging the LFCs of each chip to obtain the value used in further analysis.For analysis of the ‘basal kinase activity profiles’ (measurements obtained without inhibitor), the technical replicates were averaged. The overall difference between the measurements in the first series, relative to that in the second series was corrected by subtracting the mean of each peptide in the corresponding experimental series (centering), and by averaging the centered results of both experimental series. For analysis of ‘inhibition profiles’ (measurement obtained with inhibitor), values were obtained by calculating the log-fold change (LFC) of each peptide without any further normalization of the data. LFC was calculated by subtracting the logt-tests, and unsupervised multivariate clustering of samples was evaluated with principal component analysis (PCA), both using BioNavigator interfaced to R (The R-project). Supervised multivariate analysis of conditions was performed by applying partial least squares discriminant analysis (PLS-DA), using BioNavigator interfaced to a custom PLS-DA implementation written in Matlab . PLS-DA was performed without any pre-selection of kinase substrates. Prediction performance was evaluated using leave-one-out cross-validation (LOOCV), making sure that the model was optimized completely independent of the test sample Per-peptide differences between conditions were evaluated using two-tailed BRAF wild-type, whereas the patient-3-post and MM200 cell lines harbor the BRAF(V600E) mutation. In this manuscript we entitle the patient-3-post cell line as “vemurafenib-resistant” and the MM200 cell line as “vemurafenib-sensitive”, due to their difference in sensitivity to vemurafenib treatment, as also shown previously 2 flasks at 37°C in 95% air/5% CO2, and subcultured twice a week to maintain exponential growth. The cell lines were confirmed to be mycoplasma-free prior to the experiments.The MelJD, patient-3-post and MM200 metastatic melanoma cell lines were obtained from Professor P. Hersey, University of Sydney, Sydney, NSW, Australia in-vitro treatment with vemurafenib (5 µM) or dimethyl sulfoxide (vehicle) for 1 hour. The cells were harvested by washing the cells twice with 10 ml ice-cold PBS, before adding 4 ml ice-cold PBS and loosening the cells by scraping. To obtain the pellet, the samples were centrifuged  and supernatant was removed. Lysis buffer was added and the samples were vortexed and lysed for 15 minutes on ice. After centrifugation , supernatants were aliqouted and immediately frozen at −80°C. Protein concentrations were measured using a BCA protein assay kit .Cells were exposed to 2-transformed by identical procedures as the data from the patient specimens, before per-peptide differences between conditions  were evaluated using the two-tailed t-tests.Kinase activity profiling was assessed by using 10 µg of total protein from all samples. Lysates from each cell line were run in triplicates. The raw data was logBRAF-, NRAS-, CDKN2A-, or TP53 mutational status) or clinical parameters , including response to DTIC (The majority of the array kinase substrates (80–90%) showed higher phosphorylation levels upon incubation with metastatic melanoma lysates compared with normal skin tissue lysates. The difference ranged up to 5-fold between the two tissue types . Supervi to DTIC .ex-vivo exposure of melanoma tumor lysates to vemurafenib showed reduced kinase substrate phosphorylation levels. Whilst phosphorylation levels of the majority of the kinase substrates were decreased by approximately 50%  mutation was present in 10 out of 26 tumors (38.5%), whereas NRAS(Q61) mutation was present in 6 out of 26 tumors (23%). Unsupervised PCA showed a tendency of separation between BRAF(V600E) and BRAF wild-type tumors (BRAF wild-type and BRAF(V600E) samples based on the inhibition profiles, with an accuracy of 20/26 samples (77%) (BRAF(V600E) was correct for 90% of the samples, whereas for BRAF wild-type tumors, only 75% of samples were correctly classified with PLS-DA, reflecting the interesting observation that a few BRAF wild-type tumors consistently grouped with BRAF(V600E) tumors.e tumors . Predictes (77%) . Classift-tests identified 40 kinase substrates that were significantly affected by ex-vivo vemurafenib (P<0.05), and distinguished between BRAF(V600E) and BRAF wild-type tumors (BRAF(V600E) tumors than in BRAF wild-type tumors (BRAF wild-type tumors invariably grouped together with BRAF(V600E) tumors, exhibiting stronger inhibition in response to vemurafenib than the other BRAF wild-type tumors. No statistically significant differences in phosphorylation profiles were observed between BRAF(V600E) and BRAF wild-type tumors in the absence of ex-vivo vemurafenib incubation (BRAF wild-type and BRAF(V600E) tumors represented kinases mainly involved in the phosphatidylinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signaling network, including processes such as angiogenesis, proliferation, cell cycle progression and apoptosis , patient-3-post (BRAF(V600E)/“vemurafenib-resistant”) and MM200 (BRAF(V600E)/“vemurafenib-sensitive”).To further examine the role of in-vitro vemurafenib treatment. Reduced phosphorylation levels were seen in all cell lysates, with the largest effect seen in lysates from the BRAF wild-type MelJD cells (P<0.05) reduced by vemurafenib in all three cell lines (P<0.05) in patient-3-post and MelJD cells, with 50% (20/40) of them being identical to the kinase substrates identified as differentially affected between BRAF wild-type and BRAF(V600E) in patient specimens  have been identified in half of all melanoma cases BRAFFollowing several decades of nearly complete stagnation in the clinical treatment of patients with metastatic malignant melanoma, we are currently witnessing dramatic improvements regarding therapy. Activating mutations in BRAF- and NRAS mutational status, or response to DTIC therapy. The increased kinase activity observed in our melanoma samples is in agreement with previous studies which shows that kinases are hyperactive in many cancers, acting as a driving force towards tumor proliferation and other growth processes We show that phosphorylation levels of kinase substrates were generally increased in lysates from metastatic melanoma compared to normal skin tissue, indicating high kinase activity. The phosphorylation patterns in melanoma did however not correlate to any clinical or molecular parameters, like ex-vivo inhibitory effects of the BRAF inhibitor, vemurafenib, and the multi-targeted tyrosine kinase inhibitor, sunitinib, in the same set of melanoma samples. Inhibition experiments with sunitinib were performed as an indication that the method worked as expected i.e. multiple kinase substrates were inhibited. Hence, no correlation in inhibition pattern was observed with regard to various clinical and biological parameters.We further analyzed the ex-vivo inhibition profiles upon exposure to vemurafenib showed that a wide range of kinase substrates were affected, indicated by decreased levels of phosphorylation. This was also observed in cell lines treated with vemurafenib in-vitro, regardless of BRAF mutational status. Possible explanations for this comprehensive inhibitory pattern might be due to off-target effects of vemurafenib, as has been suggested by others BRAF-encoded pathway.The ex-vivo vemurafenib revealed a panel of 40 kinase substrates distinguishing the BRAF wild-type and BRAF(V600E) melanoma tumors. Kinases involved in the PI3K and MAPK pathway were among the kinase substrates discriminating the two groups. However, bearing in mind that the 40 discriminating substrates in this analysis appeared from a total number of 144 peptides constituting the kinase activity profiles, the false discovery rate among peptides with a statistical significance level of P<0.05 can be estimated to be about 16%. The 40 kinase substrates were more strongly inhibited when incubated with lysates from tumors harboring BRAF(V600E) compared to BRAF wild-type tumors, which is consistent with previous studies showing that BRAF(V600E) tumors are more responsive to vemurafenib than BRAF wild-type tumors BRAF wild-type tumors is thought to occur through a complex interplay between RAS and RAF dimers, leading to compensatory activation of the MAPK pathway BRAF and NRAS mutations are mutually exclusive in melanoma BRAF(V600E) tumors, RAS activity is low, and the drug is able to bind to the BRAF monomer, blocking its activity completely.Interestingly, inhibition profiles obtained with BRAF samples also exhibited decreased levels of kinase substrate phosphorylation upon exposure to vemurafenib. These samples were classified together with the BRAF(V600E) tumors based on inhibition profiles. In the absence of clinical vemurafenib response data, we speculate whether these patients might benefit from vemurafenib treatment despite the lack of the V600E mutation. When profiling the kinase substrates in BRAF wild-type melanoma cell line (MelJD), we observed that kinase inhibition upon in-vitro vemurafenib treatment occurred to a similar degree as in the vemurafenib-sensitive cell line (MM200) harboring BRAF(V600E). This supports our findings from the patient specimens; that patients with wild-type BRAF may respond to vemurafenib treatment. Increased proliferation in BRAF wild-type cells in response to vemurafenib has, however, been reported as a possibly hazardous event BRAF gene (e.g. BRAF(L597)) In our study, both supervised and unsupervised analyses revealed that some wild-type BRAF(V600E) group showed a lower degree of inhibition in ex-vivo response to vemurafenib. Lower degree of inhibition upon vemurafenib treatment in-vitro was also observed in our vemurafenib-resistant cell line (patient-3-post). This variability in sensitivity towards vemurafenib has previously been observed in several melanoma cell lines, where the presence of BRAF mutations did not guarantee a response BRAF(V600E) mutation.Furthermore, some samples within the BRAF wild-type and BRAF(V600E) melanoma tumor samples after ex-vivo treatment with vemurafenib was similar to the signature obtained with in-vitro vemurafenib treatment between BRAF wild-type and vemurafenib-resistant BRAF(V600E) cells (The 40 kinase substrate signature obtained between E) cells . These rBRAF splicing variants (p61BRAF(V600E)) lacking the RAS-binding domain PTEN) loss leading to elevated PI3K/AKT signaling NRAS(Q61K) mutations leading to activated MAPK pathway signaling BRAF(V600E) colorectal tumors has been shown to correlate with vemurafenib resistance BRAF and affect both the MAPK and PI3K pathway (ex-vivo vemurafenib in melanoma tumors harboring BRAF(V600E) and BRAF wild-type. This was also observed in-vitro, specifically in BRAF wild-type and vemurafenib-sensitive BRAF(V600E) cells, whereas no significant inhibition of EGFR and PDGFRβ was observed in the vemurafenib-resistant cell line   pathway . In our ell line . Additioex-vivo exposure to drugs may identify kinase substrate signatures that correlate to clinical response.In conclusion, our findings show that metastatic malignant melanoma is characterized by high activity of a range of kinases. The multiplex kinase substrate array technology used in the present study proved to be robust and reliable, and provided valuable information. This method may therefore become an important tool for screening of disease-specific functional biomarkers, and thereby pave the way for individualized cancer treatment. Furthermore, Table S1Individual patient characteristics of all metastatic malignant melanoma cases.(XLS)Click here for additional data file.Table S2Mean phosphorylation intensity values of all kinase substrates across twenty-six metastatic malignant melanoma samples and four normal skin tissue samples in the basal data set.(XLS)Click here for additional data file.Table S3ex-vivo vemurafenib and sunitinib.Mean inhibition of 144 kinase substrates in metastatic malignant melanoma samples in response to (XLS)Click here for additional data file.Table S4P<0.05) by ex-vivo vemurafenib incubation in BRAF wild-type and BRAF(V600E) melanoma samples.Kinase substrates identified as significantly differentially affected ((XLS)Click here for additional data file.Table S5BRAF(V600E) and BRAF wild-type tumors.Results from KEGG pathway analysis of kinase substrates involved in the PI3K and MAPK signalling pathways including all 144 kinase substrates, and 40 kinase substrates differentiating between (XLS)Click here for additional data file.Table S6BRAF(V600E) mutations (MM200 and patient-3-post ) and BRAF wild type (MelJD).Kinase inhibitory effects of vemurafenib in melanoma cell lines harboring (XLSX)Click here for additional data file.


Shc acts as a sensor/inducer of oxidative stress and may promote vascular dysfunction. The objective of this study was to investigate the role of p66Shc in tumor necrosis factor TNFα-induced E-Selectin expression and function in human umbilical vein endothelial cells (HUVEC). Exposure of HUVEC to 50 ng/ml TNFα resulted in increased leukocyte transmigration through the endothelial monolayer and E-Selectin expression, in association with augmented phosphorylation of both p66Shc on Ser36 and the stress kinase c-Jun NH2-terminal protein kinase (JNK)-1/2, and higher intracellular reactive oxygen species (ROS) levels. Overexpression of p66Shc in HUVEC resulted in enhanced p66Shc phosphorylation on Ser36, increased ROS and E-Selectin levels, and amplified endothelial cell permeability and leukocyte transmigration through the HUVEC monolayer. Conversely, overexpression of a phosphorylation-defective p66Shc protein, in which Ser36 was replaced by Ala, did not augment ROS and E-Selectin levels, nor modify cell permeability or leukocyte transmigration beyond those found in wild-type cells. Moreover, siRNA-mediated silencing of p66Shc resulted in marked reduction of E-Selectin expression and leukocyte transmigration. In conclusion, p66Shc acts as a novel intermediate in the TNFα pathway mediating endothelial dysfunction, and its action requires JNK-dependent phosphorylation of p66Shc on Ser36.Endothelial cells participate in inflammatory events leading to atherogenesis by regulating endothelial cell permeability via the expression of VE-Cadherin and β-catenin and leukocyte recruitment via the expression of E-Selectins and other adhesion molecules. The protein p66 Endothelial dysfunction plays a major role in the pathogenesis of vascular damage . Typical2+-dependent cell surface glycoprotein that recruits leukocytes under proinflammatory conditions [E-Selectin is a Canditions , and is nditions . Elevatenditions -15. Tight intercellular junctions connecting endothelial cells are required to prevent the vascular contents from leaking into surrounding tissue space . The intShc locus encodes for three different ShcA adaptor proteins with respective Mr of 46, 52, and 66 kDa. Phosphorylation of the 66-kDa isoform, p66Shc, on Ser36 has been associated with negative regulation of the p46/52Shc complex, activation of oxidative stress, and increased cellular apoptosis [Shc in the mouse results in reduced systemic and cellular stress and increased lifespan [Shc to vascular damage and cardiovascular diseases. p66Shc knockout mice are protected from high fat diet-induced atherosclerosis due to decreased oxidative stress and formation of foam cells [Shc protein levels appear to be increased in the aorta and renal cortex of experimental models of diabetes and in circulating leucocytes from diabetic patients [Shc may promote atherogenesis are still largely unknown and the role of p66Shc in cytokine-induced endothelial dysfunction has not been addressed. The mammalian poptosis -25. Genelifespan . In ligham cells ,27, as wpatients . HoweverShc, through its Ser36 phosphorylation, mediates TNFα-induced endothelial cell permeability by disrupting the cadherin–catenin complex and increases leukocyte transmigration through the HUVEC monolayer by increasing E-Selectin expression levels.In this study, we provide evidence that p662, in F12 Kaighn’s medium , supplemented with 10% Foetal Bovine Serum , 100 IU/ml penicillin, 100 µg/ml streptomycin , non-essential amino acids , 25 mg/ml Endothelial Cell Growth Supplement , and 0.1 mg/ml heparin . HUVEC were treated with the JNK inhibitors SP600125 (30 µM for 2 h)  or JNKi peptide [5(6)-FAM-GRKKRRQRRRPPRPKRPTTLNLFPQVPRSQDT-COOH, was synthetized by Primm  and linked to a FITC fluorochrome to visualize its cell entry and accumulation. The promyelocytic cell line HL-60 was cultured in Iscove’s Modified Dulbecco’s Medium  with 100 IU/ml penicillin, 100 μg/ml streptomycin , and 10% of FBS . Cell viability was assessed by trypan blue dye  exclusion. Cell number was calculated using ScepterTM Handheld Automated Cell Counter .HUVEC and HL-60 cells were purchased from ATCC . HUVEC were grown on six-well plates to confluence at 37°C in a humidified incubator gassed with 5% CO peptide  . To obtain HUVEC overexpressing a dominant-negative p66Shc mutant, cells were transfected with an adenoviral construct carrying a Ser36 to Ala36 mutation. An empty adenovirus was used as control for the infection (mock).HUVEC expressing increased levels of the p66eviously . BrieflyShc mRNA and protein expression: siRNA#1  [5’-UGAGUCUCUGUCAUCGCUGTT-3’; 5’-CAGCGAUGACAGAGACUCATT-3’. Both siRNA were designed using the siRNA Target Finder software and synthesized by Qiagen . Two independent siRNAs were used to obtain a selective reduction of E-Selectin expression: siRNA#1 [Shc gene and protein expression was verified by real-time RT-PCR (qPCR) two and three days following transfection, respectively.Two independent siRNAs were used to obtain a selective reduction of p66AUTC-3’) , and siR siRNA#1  was synt siRNA#1  was purc2, 1 mM CaCl2, 4 mM EDTA, 1% Triton X-100, 10% glycerol, 10 mM NaF, and 10 mM NaPP, supplemented with 4% protease inhibitor cocktail , and cleared by centrifugation. Protein concentration was determined by the Bradford assay . Equal protein samples (30-80 µg) were separated on SDS-PAGE gels and electro-transferred onto Hybond-P polyvinylidene difluoride filters . The filters were then probed with the specific primary antibodies, and the immune-reactive bands visualized with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG, goat anti-rabbit IgG, or donkey anti-sheep IgG (H+L) , as appropriate, using an ECL Plus Western Blotting Detection System , and quantified by densitometric analysis using the Versadoc imaging system .Cells were lysed in 50 mM HEPES, pH 7.5, containing 150 mM NaCl, 1 mM MgClShc-phospho-Ser36 was from Calbiochem ; antibodies against MAP kinases (ERK-1/2) were from Zymed Laboratories , polyclonal antibodies against phospho-p42/p44 MAP kinases (Thr202/Tyr204), phospho-SAPK/JNK kinases (Thr183/Tyr185), SAPK/JNK kinases, c-Jun-phospho-Ser63, c-Jun and phospho-p38 kinases (Thr180/Tyr182) were purchased from Cell Signaling Technology ; polyclonal antibody against phospho-IκBα (Ser32), GAPDH and β-actin were from Santa Cruz Biotechnology Inc. ; polyclonal antibody against E-Selectin was from R&D Systems , polyclonal antibody against β-catenin was from Abcam , and polyclonal antibody against VE-cadherin was from Millipore .Polyclonal antibody against Shc was purchased from Transduction Laboratories ; antibody against p66Shc, and β-actin were designed using Primer Express 3.0 . Primer sequences were designed as follows: E-Selectin_For: 5’-GAAGGATGGACGCTCAATGG-3’ and E-Selectin_Rev: 5’-TGGACTCAGTGGGAGCTTCAC-3’; p66Shc_For: 5’-CCCCCAAGCCCAAGTACAA-3’; p66Shc_Rev: 5’-GACCCAGAAGCCCCTTCCT-3’; β-actin_For: 5’-TGGATCAGCAAGCAGGAGTATG-3’ and β-actin_Rev: 5’-GCATTTGCGGTGGACGAT-3’. The PCR reactions were carried out in an ABI PRISM 7500 System . The PCR reactions were carried out under the following conditions: 50°C for 2 min, 95°C for 10 min, 40 cycles at 95°C for 15 sec, and 60°C for 1 min. Relative gene expression levels were determined by analyzing the changes in SYBR green fluorescence during qRT-PCR using the ΔΔCt method. To confirm amplification of specific transcripts, melting curve profiles were produced at the end of each reaction. The mRNA level of each gene was normalized using β-actin as internal control.Total RNA was isolated from HUVEC using the RNeasy Mini Kit . Genomic DNA contamination was eliminated by DNase digestion , and cDNA was obtained using the High Capacity cDNA Reverse Transcription Kit . Primers for E-Selectin, p66546 Fluor anti-mouse or anti-rabbit antibody for 1 h at room temperature  in PBS containing 3% BSA, respectively. Coverslips were mounted on glass slides with Vectashield . Images were acquired on a Leica DM IRE2 & DM IRB confocal microscope . Data were quantified using the Cell Profiler Software (www.cellprofiler.org).Wild-type HUVEC and HUVEC infected with the various adenoviral constructs were grown on coverslips in complete medium under basal conditions or after stimulation with TNFα, and then treated for confocal analysis, as follows. HUVEC were fixed with 3.7% formaldehyde, permeabilized, and incubated with the NF-κB , β-catenin , or VE-cadherin antibody  for 90 min at room temperature followed by the secondary Alexa2), using an aliquot for protein determination. The fluorescence increase (480 nm excitation and 567 nm emission wavelength) caused by the ROS-dependent oxidation of DHE was expressed as arbitrary units normalized by cell protein content.ROS production was detected through the evaluation of dihydroethidium (DHE) oxidation using a Jasco FP6200 spectrofluorimeter . Cells were incubated with 20 mM DHE for 0.5 h at 37°C in a serum-free medium in the dark, then washed with PBS, collected by trypsinization and resuspended in assay buffer  was used to investigate the migration of HL60 through a HUVEC monolayer. HUVEC (105 cells) were seeded onto fibronectin pre-coated cell culture inserts, incubated for 48 h with E-Selectin siRNA#1 (50 nM), E-Selectin siRNA#2 (100 nM), p66Shc siRNA#1 (100 nM), p66Shc siRNA#2 (50 nM), Ad/p66Shc, Ad/p66ShcAla36, or Ad/ mock, and grown to confluency for 72 h. HL-60 (2x105 cells) were added on the endothelial cell layer and left to migrate for 18 h at 37°C. Studies were carried out in the absence or presence of 50 ng/ml TNFα as positive control. Migrated cells were stained and measured by fluorimetry at OD 450 nm after 4 h of incubation at 37°C. The measurements were repeated three times every 15 min.Colorimetric QCMShc, were incubated with 50 ng/ml TNFα for 4 h or left untreated. FITC-dextran was added to the upper chamber for 20 min. The amount of FITC-dextran diffused into the bottom chamber was determined by measuring the fluorescence at 485 nm and 535 nm of excitation and emission, respectively, and expressed as arbitrary units.HUVEC were grown on transwell permeable supports . Control cells and adenovirus-infected cells, overexpressing wild-type or mutant p66P>0.05). Statistical analysis was performed by Student’s t test or one-way ANOVA with Tukey’s multiple comparison test, as appropriate, using Minitab® 15.1. Significance was assumed at a P value of less than 0.05.Data are presented as mean ±SE of at least three independent experiments, unless differently specified in the Figure legend, and are expressed as percentage of control values, as appropriate. Normal distribution of data was assessed by the Kolmogorov–Smirnov test , and then gradually declined . Altogether, these findings indicate that TNFα stimulates the increase of E-Selectin gene expression via JNK but not ERK signaling in HUVEC.Exposure to TNFα induced the phosphorylation of the stress kinases JNK-1 and JNK-2 in HUVEC, with a significant activation peak at 0.5 h , and p66Shc phosphorylation on Ser36 was evaluated. Exposure to TNFα induced a dose-dependent increase in p66Shc Ser-phosphorylation, which was augmented 2-fold with 50 ng/ml of TNFα  were obtained. HUVEC/p66Shc showed normal cellular morphology, similarly to control cells (Shc expression and phosphorylation (A-C), the pattern of JNK and ERK activation was similar in HUVEC/p66Shc and control cells, since JNK-1/2 phosphorylation peaked at 0.5 h , and similar results were obtained in HUVEC/p66Shc . However, after treatment with 50 ng/ml TNFα for 1 h, both leukocyte transmigration and E-Selectin mRNA were induced several-fold , and significantly reduced TNFα-stimulated E-Selectin mRNA . Importantly, though, p66Shc overexpression on Vascular Endothelial (VE)-cadherin and β-catenin expression were investigated next, to explore the potential involvement of p66Shc in regulating vascular permeability. Cells with increased p66Shc expression showed downregulation of VE-cadherin (A and C) and increased expression of β-catenin , and greater FITC-dextran leakage in control HUVEC and even more so in HUVEC/p66Shc  levels were measured in the experimental cells. In HUVEC/p66Shc, ROS levels were increased 8-fold compared to controls . The adenovirus-mediated p66Shc gene transfer augmented the protein levels of mutated p66Shc by approximately 2-3 fold , which was almost completely blocked by pretreatment of cells with the specific JNK inhibitor . Changes in VE-cadherin levels may significantly affect vascular permeability [Shc  [36 phosphorylation of p66Shc for induction of the apoptosis cascade in cells exposed to oxidative stress [Shc pathway  . Our rese stress . Interes pathway , and prepression . The mitroblasts . It was roblasts . A decreroblasts  and the roblasts ,54. In croblasts . In thisal cells . Hence, al cells , in linemulation , and thimulation . This “cnhibitor , even thnhibitor . This phShc, potentially involved in the regulation of E-Selectin gene expression in HUVEC, are still poorly characterized. p66Shc has been shown to phosphorylate and inactivate FOXO3a, which is involved in the modulation of antioxidant defenses in cardiomyocytes [Shc has also been shown to regulate the transcriptional regulator Kruppel-like factor (KLF2), since knockdown of p66Shc in endothelial cells induced mRNA expression of KLF2 and of its target gene thrombomodulin [Shc on E-Selectin. However, we found that selective overexpression or knockdown of p66Shc did not modify KLF2 mRNA levels in HUVEC (data not shown).The downstream targets of p66myocytes . Howeveromodulin . In addiomodulin , suggestShc protein has been shown to be involved in the cellular events leading to atherosclerosis. Overexpression of the p66Shc gene has been shown to reduce nitric oxide generation in endothelial cells, whereas downregulation or deletion of the p66Shc gene improved the impaired endothelial cell-dependent vasodilation [Shc has been evaluated in circulating monocytes, which are key contributors to the vascular damage. The levels of p66Shc in monocytes were significantly increased in patients with type 2 diabetes compared with controls, and were correlated to total plasma 8-isoprostane, a marker of oxidative stress [Shc was found to be increased in monocytes from patients with coronary artery disease, with a direct correlation between p66Shc levels and the number of diseased vessels [Shc, through its regulatory Ser36 phosphorylation, lies downstream of JNK in the TNFα/JNK signaling pathway that controls E-Selectin gene expression in human endothelial cells . Cells were lysed after 48 h, and E-Selectin mRNA levels were measured by qPCR. siRNA Neg, cells transfected with a negative control siRNA. B. Time-course of TNFα-induced JNK-phosphorylation in HUVEC. Representative immunoblots of JNK phosphorylation (top left) and protein content (bottom left), and ratio of phosphorylated to total JNK proteins . C. Time-course of TNFα-induced ERK phosphorylation. Representative immunoblots of ERK-1/2 phosphorylation (top left) and protein content (bottom left), and ratio of phosphorylated to total ERK-1/2 isoforms .(TIF)Click here for additional data file.Figure S2Effects of JNK and ERK inhibitors on TNFα-induced E-Selectin protein levels. HUVEC were pre-treated with 30 mM of the JNK inhibitor SP600125 (Panel A) or the ERK inhibitor PD98059 (Panel B), respectively, for 2 h, and then challenged with 50 ng/ml TNFα for the indicated times . E-Selectin protein levels were evaluated by immunoblotting, using p66Shc protein content as internal control. C. Representative images of HUVEC treated with the JNK inhibitor JNKi. Cells were treated with 10 mg/ml JNKi peptide linked to a FITC fluorochrome for 0.5 h, 1 h or 2 h, and analyzed by fluorescence microscopy. The green staining identifies peptide accumulation inside the cells (left). Cellular morphology was evaluated by optical microscopy (right). Representative images after 2 h of exposure to the JNKi are shown. D. Effects of JNKi on TNFα-induced c-Jun Ser63 phosphorylation and E-Selectin protein expression. HUVEC were preincubated with 10 mg/ml JNKi peptide for the indicated times, and then exposed to 50 ng/ml TNFα for 1 h. E-Selectin protein levels were evaluated by immunoblotting, using Shc protein expression as loading control. c-Jun Ser63 phosphorylation was evaluated as a readout for the activity of the inhibitor, using c-Jun protein content as loading control.(TIF)Click here for additional data file.Figure S336 phosphorylation of p66Shc in HUVEC.Effects of p38 MAPK and PKC-β inhibition on SerA. Effects of the p38 MAPK inhibitor SB203580 on TNFα-induced phosphorylation of p66Shc on Ser36. Cells were pre-treated with 15 or 30 µM SB203580 for 1 h and then exposed to 50 ng/ml TNFα for 0.5 h. Representative immunoblots of p66Shc phosphorylation on Ser36 (top left) and Shc protein content (middle left), and ratio of phosphorylated to total p66Shc protein . B. Effects of the PKC-β inhibitor LY333531 on TNFα-induced phosphorylation of p66Shc on Ser36. Cells were pre-treated with 200 nM LY333531 for 1 h or 24 h and then exposed to 50 ng/ml TNFα for 0.5 h. Representative immunoblots of p66Shc phosphorylation on Ser36 (top left) and Shc protein content (middle left), and ratio of phosphorylated to total p66Shc protein . β-actin protein content was used as loading control. *P<0.05 vs. basal; #P<0.05 vs. TNFα. SB, SB203580; LY, LY333531.(TIF)Click here for additional data file.Figure S4Shc in HUVEC.Overexpression of p66A. Representative immunoblots of p66Shc phosphorylation on Ser36 and of Shc protein content in wild-type HUVEC (HUVEC/wt), HUVEC overexpressing p66Shc (HUVEC/p66Shc), and HUVEC overexpressing p66Shc with Ser36 to Ala mutation (HUVEC/p66ShcAla36). HUVEC were infected with different PFU/ml of adenovirus, as indicated, and cell lysates were subjected to immunoblotting with specific antibodies. B. Quantification of p66Shc protein content in HUVEC/p66Shc (black line) and HUVEC/p66ShcAla36 (grey line) under basal conditions. Protein content of p66Shc is normalized to p52Shc protein content; the p66 Shc/p52Shc ratio in wild-type cells was considered the reference value (PFU=0 in the graph). C. Ratio of basal p66Shc Ser36 phosphorylation to total p66Shc protein content in HUVEC/p66Shc (black line) and HUVEC/p66ShcAla36 (grey line), using the p66Shc phosphorylation/content ratio in HUVEC/wt as reference (PFU=0 in the graph). *P<0.05 vs. controls (wild-type and mock). D. Representative images of HUVEC/wt, HUVEC/p66Shc, HUVEC/p66ShcAla36, and HUVEC/mock. Cells were infected with the different adenoviral constructs and analyzed by fluorescent microscopy. The green staining identifies cells expressing the green fluorescent protein (GFP) encoded by the recombinant adenovirus. Cellular morphology was evaluated by optical microscopy.(TIF)Click here for additional data file.Figure S5Shc.Phosphorylation of JNK and ERK and E-Selectin protein levels in HUVEC overexpressing p66A. Basal and TNFα-stimulated phosphorylation of JNK1/2 in HUVEC overexpressing p66Shc. HUVEC/w.t., HUVEC/p66Shc and HUVEC/mock were incubated with 50 ng/ml TNFα for the indicated times or left untreated. Cell lysates were subjected to immunoblotting with specific antibodies. Representative immunoblots of JNK1/2 phosphorylation and JNK1/2 protein content (top), and ratio of phosphorylated to total JNK proteins (bottom) . B. Basal and TNFα-stimulated phosphorylation of ERK1/2 in HUVEC overexpressing p66Shc. HUVEC/w.t., HUVEC/p66Shc and HUVEC/mock were incubated with 50 ng/ml TNFα for the indicated times or left untreated. Cell lysates were subjected to immunoblotting with specific antibodies. Representative immunoblots of ERK-1/2 phosphorylation and protein content (top), and ratio of phosphorylated to total ERK-1/2 isoforms (bottom) . *P<0.05 vs. basal. C. Basal and TNFα-stimulated E-Selectin protein levels in HUVEC overexpressing p66Shc. HUVEC/p66Shc and HUVEC/mock were incubated with 50 ng/ml TNFα for the indicated times or left untreated. Cell lysates were subjected to immunoblotting with specific antibodies. Representative immunoblots of E-Selectin protein levels (left), and the ratio of E-Selectin to GAPDH (used as loading control) protein levels in multiple experiments (right) are shown . *P<0.05 vs. basal; #P<0.05 vs. HUVEC/mock.(TIF)Click here for additional data file.Figure S6Shc in TNFα regulated NF-κB pathway.Role of p66 HUVEC/wt (A), HUVEC/mock (B), HUVEC/p66Shc (C), and HUVEC/p66ShcAla36 (D) were left untreated or incubated with TNFα for 15 min, and then analyzed by immunofluorescence. Adenovirus-infected cells are shown in green, NF-κB antibody staining is in red, and TOPRO-stained nuclei are in blue. The fuchsia staining results from the merging of red and blue, indicating NF-κB localization in the cell nucleus. E. Cell lysates were subjected to immunoblotting with ph-Ser32IκBα antibody using β-actin as loading control. Representative immunoblots (left) and the ratio of ph-Ser32IκBα to β-actin protein levels from multiple experiments (right) are shown . *P<0.05 vs. basal.(TIF)Click here for additional data file.Figure S7Shc.siRNA-mediated knockdown of p66A. p66Shc mRNA levels in siRNA-transfected HUVEC. HUVEC were transfected with 50 nM or 100 nM of two different siRNAs specific for p66Shc (siRNA#1 and siRNA#2). Cells were lysed 48 h or 72 h later, and p66Shc mRNA levels were measured by qPCR. wt, wild-type cells; Lipo, cells treated with Lipofectamine™ only; Negative siRNA, cells transfected with a negative control siRNA. B and C. Effects of siRNA-mediated knockdown of p66Shc on E-Selectin protein expression under basal conditions and following TNFα stimulation in HUVEC pretreated with SP600125 (B) and PD98059 (C). HUVEC were transfected for 72 h with 100 nM siRNA#1 and or negative control siRNA, and preincubated with SP600125 or PD98059 , using DMSO as control, before treatment with 50 ng/ml TNFα for 1 h. Lysates were analyzed by immnoblotting, using p52Shc and p46Shc as controls. Blackbars, cells transfected with a negative control siRNA; whitebars, cells transfected with 100 nM siRNA#1.(TIF)Click here for additional data file.