While allosteric activation of AMPK is triggered only by AMP, binding of both ADP and AMP has been reported to promote phosphorylation and inhibit dephosphorylation at Thr172. Because cellular concentrations of ADP and ATP are higher than AMP, it has been proposed that ADP is the physiological signal that promotes phosphorylation and that allosteric activation is not significant in vivo. However, we report that: AMP is 10-fold more potent than ADP in inhibiting Thr172 dephosphorylation; only AMP enhances LKB1-induced Thr172 phosphorylation; and AMP can cause >10-fold allosteric activation even at concentrations 1–2 orders of magnitude lower than ATP. We also provide evidence that allosteric activation by AMP can cause increased phosphorylation of acetyl-CoA carboxylase in intact cells under conditions in which there is no change in Thr172 phosphorylation. Thus, AMP is a true physiological regulator of AMPK, and allosteric regulation is an important component of the overall activation mechanism. •AMP and ADP both inhibit AMPK dephosphorylation and inactivation, but AMP is more potent•AMP, but not ADP, promotes Thr172 phosphorylation and AMPK activation, and only by LKB1•Allosteric activation by AMP is significant even at cellular ATP concentrations•Allosteric activation of AMPK by AMP occurs in intact cells Once activated by metabolic stresses that inhibit ATP production or accelerate ATP consumption, it triggers metabolic changes that act to restore energy homeostasis, switching on catabolic pathways that generate ATP while inhibiting anabolic pathways and other ATP-requiring processes . AMPK exy active . Howevery active  causes cThe regulatory adenine nucleotide-binding sites on the γ subunits are formed by four tandem CBS repeats . CrystalEven before the identity of the upstream kinases had been determined, AMP binding had been reported to both promote phosphorylation  and inhi50) was at 2.6 ± 0.3 μM, whereas the half-maximal effect of ADP was at 23 ± 3 μM, almost 10-fold higher. We repeated the assays in the presence of 5 mM ATP , the EC50 values for AMP and ADP were 25-fold and 3- to 4-fold lower, respectively, than the ATP concentration. Parallel analysis of Thr172 phosphorylation  family phosphatase. Inhibition by AMP of Thr172 dephosphorylation by a PPM phosphatase, which would be insensitive to the added okadaic acid, would lead to an apparent increase in net phosphorylation -based assay that allowed simultaneous measurement of peptide substrate and product and adenine nucleotides, it was reported that commercial preparations of ATP were contaminated with low levels of AMP, which could also be generated during assays, most likely by nonenzymic breakdown of ADP . To mini50 for activation by AMP = 5.3 ± 0.4, 22 ± 1.1, and 137 ± 14 μM; IC50 for inhibition by AMP = 1.9 ± 0.092, 7.1 ± 0.33, and 22 ± 3 mM; degree of activation by AMP = 5.4 ± 0.2, 10 ± 0.5, and 13 ± 1.3-fold. Thus, even when the ATP concentration was 5 mM  distinct from those used by adenine nucleotides, causing both allosteric activation and inhibition of Thr172 dephosphorylation . The effTo confirm that ACC phosphorylation in G361 cells was catalyzed by AMPK, we generated cells stably expressing a FLAG-tagged, kinase-inactive mutant (D157A) of AMPK-α2. This mutant was expected to act in a dominant-negative manner by competing with the endogenous α subunits (primarily α1) for binding to β and γ. −/− and AMPK-α2−/− mouse embryo fibroblasts (AMPK KO MEFs)   and coexKO MEFs) . We coulKO MEFs) C. As exp2+ ionophore A23187 in G361 cells and A23187 and berberine in human embryonic kidney 293 (HEK293) cells and estimated, using two different methods, the proportion of the α subunits that became phosphorylated.Although bacterially expressed AMPK complexes can be activated >100-fold in cell-free assays by stoichiometric phosphorylation of Thr172 , it remaFirst, we quantified the signal obtained by western blotting using anti-pT172 antibody in lysates of cells treated with and without A23187 and/or berberine. The signal was calibrated using equivalent amounts of bacterially expressed human α1β2γ1 complex (inactive α1-D157A mutant) phosphorylated by increasing amounts of LKB1 until phosphorylation reached a maximum. By making the assumption that this corresponded to stoichiometric phosphorylation, and quantifying the blots by densitometry, we were able to estimate the stoichiometry of phosphorylation in the intact cells. As a second approach, we immunoprecipitated AMPK from the cells and incubated the precipitates with recombinant CaMKKβ and MgATP to fully activate them. The treatment with A23187 in the G361 cells caused a 5.3-fold increase in AMPK activity, but subsequent incubation of the immunoprecipitates with CaMKKβ caused further large activations to reach similar final values , increasing after A23187 treatment to 13%–18%. In HEK293 cells, which express LKB1, the basal phosphorylation was initially much higher, i.e., 23%–27%. This increased to 36%–37% in response to A23187 and 50%–60% in response to berberine. The increases in phosphorylation in G361 cells due to A23187 were therefore 4-fold, and the increases in HEK293 cells due to A23187 and berberine were 1.5- and 2-fold, respectively. Thus, although 50%–60% of the α subunit became phosphorylated in berberine-treated HEK293 cells, the actual increases in Thr172 phosphorylation were quite modest.(1)Dephosphorylation of Thr172 by PP2Cα is inhibited by ADP and AMP, with the effects of both being antagonized by ATP. However, AMP is about 10-fold more potent than ADP.(2)2+ when ATP was omitted; (ii) the effect on LKB1-mediated phosphorylation and activation was specific to AMP; if it had been due to inhibition of dephosphorylation, both AMP and ADP should have been effective.AMP promotes Thr172 phosphorylation in cell-free assays by LKB1, but not CaMKKβ; ADP had no effect with either upstream kinase. This effect of AMP was not due to contamination with a PPM family protein phosphatase  because:(3)2+ and activates CaMKKβ). These experiments revealed that the allosteric effect of AMP in intact cells could be at least as important, if not more important, than the effect of increased Thr172 phosphorylation.In cell-free assays, a large (13-fold) allosteric activation of AMPK by AMP occurred even when the concentration of ATP was 5 mM, within the normal physiological range . We alsoThree major conclusions, summarized in the model shown in D for AMP binding at the “tight” (2.5 μM), rather than the “weak” (80 μM), site. Our very similar estimates of EC50 for the effects of AMP on dephosphorylation (3 μM) and allosteric activation  also throw doubt on the proposal by 50 for the effect of AMP to promote phosphorylation by LKB1  is closer to their estimate of the affinity for AMP at the weak site.Our results confirm previous results showing that the effect of AMP on Thr172 dephosphorylation is mimicked by ADP . By carrUnlike Our results reinforce the view that AMP, not ADP, is the critical physiological activator of AMPK, and that the name AMP-activated protein kinase remains appropriate. We have confirmed that AMP acts via three mechanisms C, two ofSome recent reviews  have casOur results in the AMPK knockout MEFs expressing the T172D mutant of AMPK-α1  led to increased ACC phosphorylation , and sufficient recombinant PP2Cα to yield about 70% inactivation in the absence of added nucleotide. AMP, ADP, or ATP were added at concentrations indicated in the figure legends, and aliquots were removed for western blotting or kinase assays. Kinase assays were performed immediately with a further dilution of 100-fold, which was sufficient to prevent dephosphorylation during the assay. Where added, the 5′-ectonucleotidase CD73 was at 3.9 μg/ml ; it was diluted 150-fold into final kinase assays, where it did not cause a significant effect.Rat liver AMPK  was incubated at 30°C in Na HEPES , NaCl (150 mM), Brij 35 (0.02% v/v), dithiothreitol (1 mM) with MgClC to give ≈95% inactivation. Okadaic acid was added (5 μM) to inhibit PP2AC. Dephosphorylated AMPK (2 μg/ml) was then incubated with recombinant LKB1-STRAD-MO25 complex (4 μg/ml) or CaMKKβ (1.2 μg/ml) together with MgCl2 (5 mM) and ATP (200 μM) in the same buffer, and incubation continued for 10 min, with AMP or ADP at concentrations given in the figures or legends. Aliquots were removed at the times stated for kinase assays or western blotting. When added, 5′-nucleotidase was at 1.8 μg/ml; it was diluted a further 150-fold into final kinase assays and had no effect on these. To confirm that AMPK was not contaminated with a PPM phosphatase, we incubated AMPK that had not been treated with PP2AC in buffer A with or without LKB1, MgCl2 (5 mM), or PP2Cα exactly as for the rephosphorylation experiments, but omitting ATP. After 15 min at 30°C, aliquots were subject to kinase assay or western blotting.Rat liver AMPK  was incubated in Na HEPES , NaCl (50 mM), Brij 35 (0.02% v/v), and dithiothreitol (1 mM) for 15 min at 30°C with sufficient PP2A2 concentrations also increased  to maintain a constant excess of [Mg2+] over [ATP]. Where allosteric activation by AMP was being studied, we used the SAMS peptide as substrate. For immunoprecipitate assays, AMPK was precipitated from lysates of G361 cells (50 μg protein) by incubation at 4°C for 2 hr on a roller mixer, with equal amounts of AMPK-α1 and -α2 antibodies noncovalently coupled to protein G Sepharose . After washing, immunoprecipitates were assayed for AMPK activity using the AMARA peptide  and washed into medium containing zeocin (100 μg/ml) after 48 hr. The medium was replaced every 3–4 days until foci of cells could be identified and expanded. The incorporation of the FRT site was confirmed by monitoring β-galactosidase activity. A construct encoding human AMPK-α2 (D157A mutant) was amplified using primers designed to encode a 5′-Kpn1 site and a C-terminal FLAG tag followed by a 3′-Xho1 site. The resulting PCR product was inserted into pcDNA5/FRT. The G361 cells containing an FRT site were transfected with Effectene (QIAGEN) in the absence of zeocin, using the plasmids pOG44 (encoding Flp recombinase) and pcDNA5/FRT/α2D157A at a ratio of 9:1. After 48 hr, hygromycin B (100 μg/ml) was added to the medium, and the medium was changed every 3–4 days until foci of cells could be identified and expanded.G361 cells stably expressing an integrated Flp recombinase target (FRT) site were generated using the Flp-In System (Invitrogen) following manufacturer’s instructions. Briefly, G361 cells were transfected with the pFRT2 (5 mM) for 15 min at 30°C in 50 mM Na HEPES (pH 7.4), 1 mM dithiothreitol, 0.02% (v/v) Brij 35. The reaction was stopped by the addition of SDS, with further incubation at 70°C for 10 min. Samples (corresponding to 20–40 ng of AMPK) were loaded onto the same Bis-Tris 4%–12% polyacrylamide gels as lysates (20–40 μg protein) from cells that had been treated with DMSO, A23187, or berberine. Membranes were probed with the indicated antibodies, and band intensities were quantified using LI-COR Odyssey software.A polycistronic plasmid expressing human α1β2γ1 was generated by the Division of Signal Transduction Therapy, University of Dundee, using previously described methodology . This waAMARA peptide as above.In a second approach, cell lysates were immunoprecipitated with AMPK-α1 and AMPK-α2 antibodies covalently bound to protein G Sepharose. The Sepharose beads were incubated with CaMKKβ and MgATP for 30 min at 30°C and washed several times in Na HEPES buffer (pH 7.4) to remove CaMKKβ. AMPK activity was then determined using the eq.(ADP:ATP)2 2 , where Kq = 1.05 .Total AT∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; ns, not significant).Unless stated otherwise, data presented are mean ± SEM (n = 3), with all replicates in intact cell experiments from separate dishes of cells. Unless stated otherwise, statistical analysis (GraphPad Prism 5 for Mac OSX) was by ANOVA, using Bonferroni’s multiple comparison test of selected data sets (Additional experimental procedures are provided in the


The transcription factor E2F4 controls proliferation of normal and cancerous intestinal epithelial cells. E2F4 localization in normal human intestinal epithelial cells (HIEC) is cell cycle-dependent, being cytoplasmic in quiescent differentiated cells but nuclear in proliferative cells. However, the intracellular signaling mechanisms regulating such E2F4 localization remain unknown.APC and KRAS or BRAF genes, known to deregulate GSK3/β-catenin and MEK/ERK signaling, respectively.Treatment of quiescent HIEC with serum induced ERK1/2 activation, E2F4 phosphorylation, E2F4 nuclear translocation and G1/S phase transition while inhibition of MEK/ERK signaling by U0126 prevented these events. Stimulation of HIEC with epidermal growth factor (EGF) also led to the activation of ERK1/2 but, in contrast to serum or lysophosphatidic acid (LPA), EGF failed to induce E2F4 phosphorylation, E2F4 nuclear translocation and G1/S phase transition. Furthermore, Akt and GSK3β phosphorylation levels were markedly enhanced in serum- or LPA-stimulated HIEC but not by EGF. Importantly, E2F4 phosphorylation, E2F4 nuclear translocation and G1/S phase transition were all observed in response to EGF when GSK3 activity was concomitantly inhibited by SB216763. Finally, E2F4 was found to be overexpressed, phosphorylated and nuclear localized in epithelial cells from human colorectal adenomas exhibiting mutations in The present results indicate that MEK/ERK activation and GSK3 inhibition are both required for E2F4 phosphorylation as well as its nuclear translocation and S phase entry in HIEC. This finding suggests that dysregulated E2F4 nuclear localization may be an instigating event leading to hyperproliferation and hence, of tumor initiation and promotion in the colon and rectum. E2F4 gene causes a reduction in the number of erythrocytes due to impaired proliferation of progenitors in bone marrow ATP was obtained from PerkinElmer . PP1 phosphatase was obtained from New England Biolabs Ltd. . All other materials were from Sigma-Aldrich  unless stated otherwise.MEK inhibitors U0126 and PD184352 were purchased from LC Laboratories  and GSK3 inhibitor SB216763 was purchased from Sigma-Aldrich . Antibodies against E2F4 (C-20), cyclin D1 (C-17), cyclin A (H-432), p27 (C-19), ERK1 (C-16), ERK2 (C-14) and HA-probe (F-7) were all purchased from Santa Cruz Biotechnology Inc. . Phospho-ERK1/2 (Thr202/Tyr204) #9101, phospho-GSK3β (Ser9) #9336, phospho-glycogen synthase (GS) (Ser641) #3891, phospho-ERK5 (Thr218/Tyr220) #3371 and phospho-Akt (Ser473) #9271 were from Cell Signalling Technology . β-actin monoclonal antibody (clone C-4) and recombinant active ERK1 were obtained from Millipore . pRb and Ki67 monoclonal antibodies (clone 35) were purchased from BD BioSciences  and phospho-serine antibody (clone 16B4) from BIOMOL International (#SA-337). Alexa Fluor® 488 goat anti-rabbit and Alexa Fluor® 568 goat anti-mouse antibodies were from Invitrogen . ATP (3 μCi/assay) was added and the reaction initiated by the addition of recombinant active ERK1 (5 ng) and incubation at 30°C. After 5 min, reactions were stopped with Laemmli’s buffer. Samples were boiled and radiolabeled E2F4 was separated from antibodies on SDS-PAGE gels. Results were visualized by autoradiography. After radiography, gels were transferred onto PVDF membranes and immunoblotted with HA antibody.293T cells were transfected with pCDNA3.1 empty vector or pCDNA3.1 HA-tagged-wild-type E2F4 or mutants using Lipofectamine 2000 (Invitrogen) according to the recommended manufacturer protocol. Cells were lysed in Triton lysis buffer 48 h after transfection. To immunoprecipitate E2F4, cleared lysates (1 mg) were pre-incubated with HA antibody  to which protein A Sepharose CL-4B beads were added for an additional hour. Immunocomplexes were washed four times with Triton lysis buffer and twice with kinase assay buffer . Two days after transfection, luciferase activity was measured as previously described [293T cells were seeded in 6-well plates and co-transfected by lipofection  with 0.1 μg of thymidine kinase-luciferase reporter, 0.25 μg of the relevant expression vector (pcDNAneo 3) containing wild-type E2F4 or mutants and 0.25 μg of the relevant expression vector containing DP-2. The pRL-SV40 APC (exon 15), KRAS (exons 1 and 2) and BRAF (exon15) were amplified by PCR and the presence of mutations was detected by direct sequencing . Paired tissues were lysed in Triton lysis buffer and immunoblotted as described above.Samples of colorectal adenomas and paired normal tissues (at least 10 cm from the adenoma) were obtained from patients undergoing surgical resection. Patients did not receive neoadjuvant therapy. Tissues were obtained after patient’s written informed consent, according to the protocol approved by the Institutional Human Subject Review Board of the Centre Hospitalier Universitaire de Sherbrooke. All tissues were frozen in liquid nitrogen within 30 min from resection. Tissues were embedded, cryosectioned and immunostained, as described previously [in situ indirect immunofluorescence from three independent experiments are shown.Assays were performed in triplicate. Typical Western blots shown are representative of three independent experiments. Representative results of The authors declare that they have no competing interests.MCP performed the molecular and cellular studies, participated in the study design and drafted the manuscript. SC and CL participated to the molecular studies and to the critical reading of the manuscript. JCC participated in the study design and is the leader of the colorectal cancer tissues biobank. NR conceived the study, and participated in its design and coordination and helped draft the manuscript. All authors read and approved the final manuscript.Subconfluent HIEC were serum-deprived for 36 h, treated or not (DMSO) during 10 min with the MEK inhibitors U0126 (20 μM) or PD184352 (5 μM) and then stimulated with 100 ng/ml EGF for 30 min. Thereafter, cells were lysed and proteins were analyzed by SDS-PAGE for Western blot analysis for the expression of phosphorylated ERK5, phosphorylated AKT, total AKT and β-actin.Click here for fileSubconfluent HIEC were serum-deprived for 36 h, treated or not (DMSO) during 10 min with the MEK inhibitors U0126 (20 μM) or PD184352 (5 μM) and then stimulated with 10% serum for 30 min. Thereafter, cells were lysed and proteins were analyzed by SDS-PAGE for Western blot analysis for the expression of E2F4, ERK2, phosphorylated ERK2 and β-actin. Representative of three experiments.Click here for file


The development of adipocytes from their progenitor cells requires the action of growth factors signaling to transcription factors to induce the expression of adipogenic proteins leading to the accumulation of lipid droplets, induction of glucose transport, and secretion of adipokines signaling metabolic events throughout the body. Murine 3T3-L1 pre-adipocytes sequentially express all the proteins necessary to become mature adipocytes throughout an 8–10 day process initiated by a cocktail of hormones. We examined the role of Clk/STY or Clk1, a cdc2-like kinase, in adipogenesis since it is known to be regulated by Akt, a pivotal kinase in development. Inhibition of Clk1 by a specific inhibitor, TG003, blocked alternative splicing of PKCβII and expression of PPARγ1 and PPARγ2. SiRNA depletion of Clk1 resulted in early expression of PKCβII and sustained PKCβI expression. Since Clk1 is a preferred Akt substrate, required for phosphorylation of splicing factors, mutation of Clk1 Akt phosphorylation sites was undertaken. Akt sites on Clk1 are in the serine/arginine-rich domain and not the kinase domain. Mutation of single and multiple sites resulted in dysregulation of PKCβII, PKCβI, and PPARγ1&2 expression. Additionally, adipogenesis was blocked as assessed by Oil Red O staining, adiponectin, and Glut1 and 4 expression. Immunofluorescence microscopy revealed that Clk1 triple mutant cDNA, transfected into pre-adipocytes, resulted in excluding SRp40 (SFSR6) from co-localizing to the nucleus with PFS, a perispeckle specific protein. This study demonstrates the role of Akt and Clk1 kinases in the early differentiation of 3T3-L1 cells to adipocytes. Messenger RNA splicing is a synonymous or post-transcriptional modification of pre-mRNA of eukaryotic cells, whereby noncoding introns are removed and exons are joined to provide new protein templates. Alternative splicing (AS) is a variation where exons of pre-mRNAs are linked by AS by inclusion or exclusion to produce transcripts with different protein coding sequences. It is predicted that >92–94% of genes undergo AS with variation between tissue types and developmental stages in vitro. Between days 4–6 of the differentiation process, PKCβI splicing is suppressed while PKCβII splicing increases. We hypothesized that the PI3Kinase/Akt/Clk1 signaling pathway was important for adipoctye differentiation for regulating PKCβII splicing since pAkt(S473) is activated very early in adipogenesis. The role of Clk1 in this process was unknown. To examine this question, a chemical inhibitor of Clk1 and 4 isoforms, TG003, siRNA for Clk1, and mutants of Clk1 with Akt phosphorylation sites mutated to alanine were used to determine their effect on PKCβII splicing and the progression of adipogenesis by markers PPARγ adiponectin, GLUT4, Glut1, and Oil Red O staining of accumulating lipid droplets.We demonstrated that PKC βII was spliced in 3T3-L1 adipocytes in a developmentally regulated manner 2. Confluent cells were differentiated (day 0) in DMEM high glucose with 10% fetal bovine serum , 10 µg/mL bovine insulin (Sigma), 1 mM dexamethasone (Sigma), and 0.5 mM isobutyl-1-methylxanthine (Sigma). On day 2, media was replaced with DMEM high glucose, 10% FBS, and bovine insulin. Day 4 and afterwards, cells were cultured in DMEM high glucose plus 10% FBS.Mouse 3T3-L1 pre-adipocytes obtained from American Type Tissue Culture repository, ATCC  were maintained and passaged as pre-confluent cultures in DMEM high glucose  with 10% newborn calf serum  at 37°C and 10% COGeneration of Clk1 mutant cDNA was generated using QuickChange Site-Directed Mutagenesis Kit (Stratagene). The Clk1 template was denatured at 95°C. The mutagenic primers containing the desired mutation(s) were annealed at 55°C and primers extended using PfuUltra DNA polymerase at 68°C. The parental DNA was digested with Dpn I enzyme. The pure mutated DNA was transformed into competent cells and harvested. The mutated fragment was confirmed by sequencing.6 cells/100 ul, then combined with 2 ug DNA, 100 ul cell line nucleofector solution L, selected nucleofector program A-033 to transfer, immediately transfer the sample into collagen pre-coated 6-well plate.Clk siRNA oligonucleotides were obtained from Dharmacon (SMARTpool). The DharmaFECT1 transfection reagent was used in siRNA transfections. 3T3-L1 cells were seeded in 6-well plates (80% confluent). SiRNA reagent was mixed with Opti-MEMI media (Invitrogen) and DharmaFECT1 reagent. The transfection mixture was added to each well afterwards. Non-target control siRNA (Dharmacon) was used as negative controls. The knock-down efficiency was measured at 24, 48 and 72 hour time points with RT-PCR. Myc-Clk over-expression was obtained using Amaxa Cell Line Nucleofector Kit L (Lonza). 3T3-L1 cells were trypsinized and suspended at 2×10Fast Protease Inhibitor Tablet, Sigma) and phosphatase inhibitors . Lysates (40 µg) were run on an SDS-PAGE gel and transferred to Hybond-C Extra nitrocellulose membranes . Membranes were blocked and probed in 5% non-fat dried milk. The only exception was when probing for PKCβII, where pig gelatin (Bio-Rad) was used for blocking (3%), primary (1%) and secondary (1%). Detection was performed using SuperSignal West Pico Chemiluminescent substrate . Antibodies used were as follows: PPARγ 81B8, pIRβ Tyr 1150/1151, pAkt Ser473 #4058, and adiponectin #2789 , GLUT-1, GLUT4 C-Terminus 07-1404 , β-actin A5441 (Sigma). PKCβII NH2–(GC) EGFSFVNSEFLKPEVKS-COOH (aa 657–673) was raised by BioSynthesis Inc.  and purified using Nab Protein A Plus Spin Kit (Pierce #89948). Monoclonal antibody 104 was from ATCC. SRp40 (SFSR5), SRp55 (SFSR6) antibodies were described previously 3T3-L1 cells were harvested in lysis buffer containing protease inhibitors .RNA was extracted using RNA Bee , according to manufacturer’s protocol. Reverse Transcriptase was performed using Omniscript RT kit  according to manufacturer’s protocol. PCR reactions were performed to determine Clk 1 mRNA levels using JumpStart Ready-Start amplification mix (Sigma) and previously published primers. The forward Clk 1 primer was 5′ATGAGACATTCAAAGAGAACTTACT 3′ and the reverse Clk 1 primer was 2O (2×) and 2 ml 60% isopropanol for 5 min at RT. After drying, 1 ml of Oil Red O solution (0.35 g/100 ml isopropanol then filtered) was added, and incubated 10 min with gentle shaking. Cells were then viewed using brightfield microscopy and digital photos captured.3T3-L1 cells grown in tissue culture plates were washed in PBS and 10% formalin was added for 10 min at room temperature (RT). After discarding, 2 ml fresh formalin was incubated for at least 1 hr. Cells were washed with 2 ml H3T3 L1 pre-adipocytes were grown to 80% confluence in 6-well plates on glass coverslips and plasmids indicated were transfected into cells with Lipofectamine™. After differentiation for 4 days, coverslips were washed in warm PBS and 2% formaldehyde in PBS was added to fix the cells for 15 min at 37°C. Cells were permeabilized with 10% TritonX-100 and blocked in 1% BSA with anti-goat IgG in PBS. The primary antibody was diluted 1∶100 and incubated for 1 hr in 2% BSA, 10% NP-40. After washing with PBS, the secondary antibody (anti-mouse or anti-rabbit HTC) was added in 1% BSA at room temp for 30 min. For DAPI, 1 g/ml of solution was incubated with the slides for 2 min. Slides were mounted and sealed. Images were acquired by inverted fluorescent microscopy using a Nikon Eclipse TE2000-U using a 20× objective with 0.38 µM/pixal.We hypothesized that Clk1 levels were increased developmentally during adipogenesis since splicing from PKCβI to PKCβII was increased dramatically between days 4–6 of differentiation as well as expression of the two splice variants of PPARγ on day 2. Clk1 mRNA levels were measured by RT-PCR in 3T3-L1 pre-adipocytes during the course of adipogenesis. The two splice variants of Clk1 mRNA were detected. A shift in the ratio of full length to truncated form, lacking the kinase domain, occurred during adipocyte differentiation with the full-length transcript predominating on days 2–6. After terminal differentiation, the truncated form of Clk1 exceeded the full length on day 8 . Clk1 isWe previously demonstrated that the depletion of Clk1 by siRNA or inhibition of the kinase by a general Clk inhibitor, TG003, or an upstream inhibitor of Akt activation, LY294002, blocked insulin-regulated splicing of PKCβII in muscle cells There were also differences in the expression of other alternatively spliced proteins with TG003 treatment. The alternative splicing of PPARγ is due to the use of alternative promoters as depicted in Since TG003 was not a specific inhibitor for Clk1, Clk1 siRNA was used to knock down the isoform that was shown to be upregulated with differentiation. Clk1 was focused on as it was known to be important for SRp40 phosphorylation and PKCβ splicing Clk1 kinases are conserved throughout eukaryote evolution from Saccharomyces cerevisiae (KNS1), Arabidopsis thaliana (AFC1-3), and Drosophila melanogaster (DOA), mouse (Clk/Sty) to human  The role of the n-terminal lobe in substrate specificity is only speculated, however, as a kinase inactive mutant, Clk1 colocalizes with other splicing factors to nuclear speckles causing them to dissolve. Here, we mutated only the Akt sites in Clk1 predicted to be specific for recognizing SR proteins, while leaving the kinase domain active. In this experiment, we looked at the ability of Clk1-AAA  mutants to regulate PKCβ and PPARγ splicing and adiponectin expression as a measure of adipogenesis. When Clk1-AAA was transfected into 3T3-L1 preadipocytes, PKCβII levels were elevated on day 0 and throughout differentiation . PKCβI lSince the triple mutation of Akt sites on Clk1 (Clk1-AAA) had multiple effects on splicing and protein expression, individual site mutations of Akt phosphorylation motifs were made and the single mutant Clk1 plasmids were transfected into 3T3-L1 cells and cell lysates were isolated on days 0–10 during differentiation for Western blots of Clk1 phosphorylation activity using and antibody that recognizes the phospho-Clk substrate motif, mAb104 We previously demonstrated the role of Akt2 and Clk/STY in regulation PKCβ alternative splicing via SRp40 Expression of major adipogenic proteins was probed by western blot in cell lysates obtained at days 0–10 during differentiation following transfection of the single Clk1 mutants. The adipogenesis program was blocked in cells transfected with the single Clk1 mutants prior to differentiation. As with Clk1-AAA, when all three Clk sites were mutated in a single construct, and adiponectin expression was blocked  and day 4 after 2 days in induction media. We visualized SRp40, as in our lab it is known to regulate PKCβII alternative splicing. As a control, cells were stained with a nuclear stain, DAPI, FITC conjugated SRp40 (SFRS5), and TRITC conjugated PSF, a PTB-associated splicing factor, and a mouse and human paraspeckle-associated protein The functional role of Clk kinase in alternative splicing is mediated by its substrates, serine-arginine rich “SR” splicing factors. Clk1  is regulated by Akt phosphorylation in response to insulin Clk1 kinase phosphorylates and colocalizes with nuclear splicing factors cis-elements within exons Clk kinase activity is essential for phosphorylation of the splicing factors that recognize First, siRNA was used to deplete Clk1 in pre-adipocytes. This depleted cells of both Akt substrate motifs and the kinase activity of one Clk isoform. It resulted in the early expression of PKCβII and continued expression of PKCβI beginning on day 0 of differentiation. Hence, a dysregulation of splicing was observed in contrast to the tight regulation governing the timed switch from one splice variant to the other between days 4–6. There was also depletion of PPARγ1 and a reduction in PPARγ2 expression, and this was also different from the inhibition observed with TG003, an inhibitor of both Clk1 and 4 kinases. Thus, without Clk1, cells failed to produce a transcription factor known to induce adipogenesis, PPARγ. It was also noted that mAb104, a phospho-Clk substrate antibody, no longer recognized a heavily phosphorylated SRp40 band despite no changes in its protein expression. However, in the absence of Clk1, SRp40 became a better substrate for Akt phosphorylation and this suggests the inability of SR proteins to co-localize to the nucleus for further Clk1 phosphorylation once they have been phosphorylated by Akt The mutation of Clk1 on all three Akt phosphorylation sites leaving its kinase domain intact, produced similar results to siRNA. PKCβII was heavily expressed early on and PPARγ1 expression was depleted. Overexpression of the triple Clk1 mutant had a similar effect to the depletion of Clk1 using siRNA for Clk1. Without available Akt sites in Clk1, SR proteins were not properly phosphorylated resulting in a disruption of splicing. It was also noted here that SRp40 did not colocalize to nuclear speckles with PSF. The dysregulation of PKCβII is likely due to improper SRp40 phosphorylation by Clk1 and the inability of the SR protein to interact with RNA PolII during transcription. During adipogenesis, the splicing of PKCβII is regulated developmentally via co-transcription To further determine the role of each Akt site in Clk1 recognition of SR proteins, single Akt sites were mutated and constructs transfected into 3T3-L1 pre-adipocytes. Single site mutations resulted in different developmental patterns of SR protein phosphorylation. In control cells, identification of five predominant phosphorylated SR proteins is possible given their molecular weights and mobilities on SDS-PAGE. When Serine36 was mutated to alanine, SRp30a and b protein phosphorylation was reduced. This protein doublet represents ASF/SF2 (SFSR1) and SC35 (SFSR2) proteins, SR proteins known to function in constitutive splicing and alternative splicing. When Threonine 122 was mutated, the phosphorylation of SRp40 (SFSR5) was disrupted. The Clk phosphorylation states of all five proteins (including SRp55 (SFSR6) and SRp75 (SFSR4)) were significantly blocked when Serine 139 was mutated. Each mutation increased PKCβII expression and increased the phosphorylation of SRp40 by Akt as was also demonstrated with Clk siRNA. None of the mutations, however, interfered with the affinity of Akt for Clk1 or altered SRp40 levels or any other SR protein levels. The block of adipogenesis by single mutations in Clk1 was documented by lack of Glut4, Glut 1 and adiponectin expression as well as failure of the cells to develop Oil Red O staining lipid droplets.Since phosphorylation of SR proteins by Clk is thought necessary for their nuclear roles, Clk1-AAA, was used to demonstrate this. Immunfluorescence microscopy showed that another nuclear paraspeckle protein, PSF, no longer co-localized with SRp40 when mutant Clk1-AAA was expressed during adipogenesis. The SRp40 remained perinuclear unlike the SRp40 in control cells which was detected in nuclear as well as in distinct perinuclear areas.The finding that Clk1 is involved in adipogenesis is not surprising as the kinase was discovered while searching for tyrosine kinases involved in differentiation programs The orchestration of Akt and Clk to regulate splicing emerges here when the Akt sites on Clk1 are mutated and the expression of key adipogenic proteins are disrupted including PPARγ, Glut4, Glut1, and adiponectin. This pathway is likely to be important not only is adipogenesis, but in other lineage specific commitment pathways. Mutation of similar Akt sites in Clk2 resulted in cells that were no longer resistant to survival following ionizing radiation


V600E mutation leading to constitutive signaling of MEK-ERK pathways causes papillary thyroid cancer (PTC). Ras association domain family 1A (RASSF1A), which is an important regulator of MST1 tumor suppressor pathways, is inactivated by hypermethylation of its promoter region in 20 to 32% of PTC. However, in PTC without RASSF1A methylation, the regulatory mechanisms of RASSF1A-MST1 pathways remain to be elucidated, and the functional cooperation or cross regulation between BRAFV600E and MST1,which activates Foxo3,has not been investigated.The BRAFV600E positive thyroid cancer cells. The FoxO3 transactivation is augmented by RASSF1A and the MST1 signaling pathway. Interestingly, introduction of BRAFV600Emarkedly abolished FoxO3 transactivation and resulted in the suppression of p21 and p27 expression. The suppression of FoxO3 transactivation by BRAFV600Eis strongly increased by coexpression of MST1 but it is not observed in the cells in which MST1, but not MST2,is silenced. Mechanistically, BRAFV600Ewas able to bind to the C-terminal region of MST1 and resulted in the suppression of MST1 kinase activities. The induction of the G1-checkpoint CDK inhibitors, p21 and p27,by the RASSF1A-MST1-FoxO3 pathway facilitates cellular apoptosis, whereasaddition of BRAFV600E inhibits the apoptotic processes through the inactivation of MST1. Transgenic induction of BRAFV600Ein the thyroid gland results in cancers resembling human papillary thyroid cancers. The development of BRAFV600Etransgenic mice with the MST1 knockout background showed that these mice had abundant foci of poorly differentiated carcinomas and large areas without follicular architecture or colloid formation.The negative regulators of the cell cycle, p21 and p27, are strongly induced by transcriptional activation of FoxO3 in BRAFV600E is associated with the inhibition of MST1 tumor suppressor pathways, and that the activity of RASSF1A-MST1-FoxO3 pathways determines the phenotypes of BRAFV600E tumors.The results of this study revealed that the oncogenic effect of BRAF V600E is the most common of these activating mutations, especially in papillary thyroid cancer, where it is found at a frequency of 40–70% V600E-positive thyroid cancer cell lines and BRAFV600E transgenic mice, this mutation is responsible for tumor initiation, transformation, growth, proliferation and dedifferentiation V600E-positive tumors has revealed that the missense valine to glutamic acid mutation increases kinase activity, promoting the constitutive activation of MEK-ERK signaling V600E tumors are not fully characterized V600E in thyroid cancer remain to be identified.Activating mutations in the BRAF gene are found at high frequency in various human cancers, and BRAFde novo promoter methylation in the early stages of thyroid tumorigenesis V600E. However, promoter hypermethylation of RASSF1A could only be detected in a relatively small percentage of PTC (20 to 32%) V600E might function during the development of PTC whichdoes nothave RASSF1Apromoter methylation.The tumor suppressor gene RASSF1A (Ras association domain family 1A) is epigenetically inactivated through V600E thyroid tumors. Several molecular mechanisms which are possibly regulated by BRAFV600E may control FoxO3 activity in thyroid cancer. First, as RET/PTC kinase inhibits FoxO3 transactivation through an Akt/PKB dependent pathway, BRAFV600E might also activate Akt/PKB signaling pathway V600E could inhibit FoxO3 activation via the ubiquitin-proteasome pathway V600E could act through crosstalk with the RASSF1A-MST1-FoxO3 pathway.FoxO3 transactivation is effectively inhibited by RET/PTC (rearranged in transformation/papillary thyroid carcinomas) kinase V600E. This resulted in the identification of novel cross-talksignaling between BRAFV600E and MST1, thereby demonstrating the functional activity of the RASSF1A-MST1-FoxO3 tumor suppressor system. Furthermore, in vivo experiments showed that MST1 knockout mice exhibited more aggressive BRAFV600E tumor phenotypes.Based on these hypotheses, we decided to investigate the regulation of the MST1-FoxO3 pathway by BRAF5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCAAGATGGCGGCGCTGAGCGGT-3′, and antisense, 5′- GGGGACCACTTTGTACAAGAAAGCTGGGTCTCAGTGGACAGGAAACGCCC-3′. To generate vectors for human BRAFV600E and BRAFG469A, the Site-Directed PCR Mutagenesis Kit  was used to introduce the missense mutation into the wild-type BRAF sequence. SiRNAs for MST1 and RAF-1 were purchased from Invitrogen .The pCMV5-Myc-FoxO3 plasmid was purchased from Addgene  and pcDNA3.1/CT-GFP-FoxO3 was constructed using the CT-GFP Fusion TOPO Expression Kit according to the manufacturer's protocol . Expression vectors (pME18) for Flag epitope-tagged forms of human MST1, MST1-N (residues 1–326), MST1-C (residues 327–487), HA-RASSF1A and 3X-IRS reporters have been described previously 5/35 mm dish) were transfected with siRNA at a concentration of 50 nM using the HiPerfect Transfection Reagent  and/or with the indicated vectors using Fugen6 Transfection Reagent . The MEK inhibitor (U0126) and PI3K inhibitors  were purchased from Cell Signaling Technology .FRO cells and 293T cells were cultured in RPMI and DMEM supplemented with 10% fetal bovine serum Primary antibodies against p21, p27, phospho-ERK, total ERK, phospho-Akt, total Akt, MST1, Actin, FoxO3, Myc , BRAF , RASSF1A  and Flag  were purchased from the indicated companies.Renilla luciferase  using Lipofectamine in OptiMEM (Invitrogen). Luciferase levels were determined using the Dual Luciferase Assay Kit (Promega). Firefly and Renilla luciferase activities were measured sequentially from the same sample using a Fusion Alpha-Microplate Analyzer . For each sample, firefly luciferase activity was normalized to Renilla luciferase activity.To assay the activity of FoxO3, 293T cells were transfected with 3X-IRS-Luc, or p27-Luc and pRL-SV40, which encodes All immunoprecipitation and immunoblotting procedures were performed at 4°C. Cells were washed twice with PBS and lysed in lysis buffer containing protease inhibitors. Lysates were centrifuged at 14,000 g for 15 min at 4°C. For the IP analysis, lysates were precleared with protein A/G beads (Santa Cruz Biotechnology) for 30 min, and supernatants were incubated with the indicated primary antibody for 3 h with agitation. Protein A/G beads were then added to the mixture and incubated for at least 2 h at 4°C. Beads were washed with lysis buffer and the immunoprecipitates were subjected to immunoblotting. Cell lysates were separated by SDS-PAGE, and transferred to nitrocellulose membranes. Membranes were blocked with TBS containing 5% milk and 0.1% Tween20 for 1 h and then incubated overnight with primary antibody at 4°C. Membranes were washed in TBS/T and incubated with secondary antibodies conjugated to horseradish peroxidase  for 1 h at RT. Blots were developed using the LumiGLOChemiluminescent substrate .V600E and Flag-MST1, respectively . Cells were observed with a CellomicsArrayScanV HCS reader .293T cells were grown on 6-well plates and transfected with the indicated plasmids using LipofectAmine (Invitrogen). Twenty-four hours after transfection, the cells were washed three times with PBS and fixed in 3.7% formaldehyde for 20 min. Cells were permeabilized with PBS containing 0.1% Triton X-100 and 0.1 M glycine at RT, incubated for 15 min, washed three times with PBS, and blocked with 3% BSA in PBS for 10 min at RT. Cells were incubated with primary anti-Myc and anti-Flag for 1 h at 37°C, washed three times with PBS, and incubated for 1 h at 37°C with Cy5-conjugated anti-Rabbit and TRITC-conjugated anti-Mouse for Myc-BRAF2, 1 mM DTT, phosphatase inhibitor mixture, 10 µM unlabeled ATP, and 1 µCi [γ-32P]ATP. The reaction was terminated by the addition of Laemmli sample buffer and phosphorylated proteins were detected by SDS-PAGE followed by autoradiography.MST1 was immunoprecipitated from cell lysates and the washed precipitates were incubated for 30 minutes at 30°C with 1 µg histone H2B (Roche) in 25 µL kinase assay buffer comprised of 40 mM HEPES-NaOH (pH 7.4), 20 mM MgCl600E, as indicated, and then stained with DAPI and terminal deoxynucleotidyltransferase–mediated dUTP nick end labeling (TUNEL) reagents.The proportion of apoptotic cells was determined asthe percentage of TUNEL-positive cells among all DAPI-stainedcells.Image based apoptosis assays were carried out using the APO-BrdU TUNEL Assay Kit (Invitrogen). Cells was transfected with FoxO3, MST1, and/or BRAFVV600E FVB/N mice with a bovine thyroglobulin promoter have been described previously V600E mice were crossed with MST1-null mice to obtain Tg-BRAFV600E/MST1-null mice and Tg-BRAFV600E littermates. These mice were sacrificed at 16 weeks and their thyroids were removed after genotyping. Thyroid tissues were fixed in 10% formalin and embedded in paraffin. Five µM-thick sections were prepared and stained with H&E. Immunohistochemical analysis of paraffin embedded thyroid tissue was also performed using anti-cyclin D1 rabbit monoclonal antibody . ImageJ (http://rsbweb.nih.gov/ij/) for microscopy was used to estimate areas without follicular architecture or colloid formation in thyroid tissues. Tg-BRAFV600E/MST1 null mice (n = 10) and Tg-BRAFV600E littermates (n = 10) were collected and three sections of each mouse were used for immunohistochemical and imageJ analysis. Differences in the number of undifferentiated foci and areas without follicular architecture or colloid formation between both groups were assessed by the Mann-Whitney U-test. All animal experiments were approved by the Institutional Animal Care and Use Committee of the Chungnam National University School of Medicine .The strategy and methods used for the generation of thyroid specific transgenic BRAFP<0.05.All statistical analyses were performed using SPSS Version 16.0 for Windows .Results were expressed as the means ± standard deviations. Statistical differences between two groups were analyzed by the Mann-Whitney U test. Differences were considered to be significant at V600E, the effect of BRAFV600E on RASSF1A-mediated p21 and p27 induction was evaluated. Interestingly, BRAFWT did not affect the induction of p21 and p27 by RASSF1A, whereas BRAFV600E effectively decreased p21 and p27  and PI3K inhibitors  were used to block these pathways and observe the effect of BRAFV600E on RASSF1A-induced FoxO3 transactivation. As shown in V600E-mediated suppression of p21 and p27 induction by RASSF1A was not reversed by the MEK inhibitor U0126 or the PI3K inhibitors Wortmannin and LY294002, showing that BRAFV600E inhibits the RASSF1A-induced p21 and p27 upregulation independently from the ERK and PI3K-Akt/PKB signaling pathways. Recently, RASSF1A was shown to interact with MST1 via its SARAH domain and increase MST1 kinase activity To confirm that RASSF1A induces FoxO3 transactivation, RASSF1A was transiently transfected into FRO cells harboring the BRAF mutated alleles and then p21 and p27, the known transcriptional targets of FoxO3, were assessed by immunoblotting  and p27 , suggestThe transcriptional activity of FoxO3 is regulated by posttranslational modifications such as phosphorylation, acetylation, nitrosylation and sumoylation V600E inhibited RASSF1A-induced p21 and p27 upregulation, the effect of BRAFV600E on FoxO3 transactivation by RAASF1A was assessed. As shown in V600E abrogated the RASSF1A-induced FoxO3 transactivation. Furthermore, MST1-induced FoxO3 transactivation was also repressed by BRAFV600E, indicating that BRAFV600E inhibits the RASSF1A-MST1-FoxO3 pathway , MST1 showed dominant cytoplasmic localization (red), and merged images revealed a combined color (lilac) that suggests the co-localization of BRAFV600E and MST1. To quantify FoxO3-fluorescence, the nuclear GFP-fluorescence signal intensity was analyzed using the BioApplication of CellomicsArrayScan, following the manufacturer's protocol. In agreement with the immunofluorescence images, MST1 increased intensity of the nuclear GFP-fluorescence signal, and BRAFV600E inhibited the nuclear detection of GFP-fluorescence  assays were carried out to define the direct interaction of BRAFV600E and MST1. As shown in V600E was co-precipitated with Flag-tagged MST1 in lysates from 293T cells transfected with BRAFV600E and Flag-tagged MST1 and vice versa. To verify the region of MST1 important for the interaction with BRAFV600E, IP assays using MST1 deletion constructs, such as MST1 N-terminal (residues 1–326) and C-terminal (327–487) constructs  and MST1 null mice were designed. Tg-BRAFV600E mice were crossed with MST1 null mice and the phenotypic differences between Tg-BRAFV600E/MST1 null mice and their Tg-BRAFV600E littermates were analyzed at 16 weeks V600E/MST1 null mice showed aggressive PTC tumor phenotypes compared with their Tg-BRAFV600E littermates. Although the presence of lymph node metastasis was not observed in either of the two groups, Tg-BRAFV600E/MST1 null mice showed abundant foci of undifferentiated carcinomas and large areas without follicular architecture or colloid formation . Similarly, male Tg-BRAFV600E/MST1 null mice showed a higher number of undifferentiated foci than their Tg-BRAFV600E littermates . The total area composed of sheets of spindle cells without follicular architecture or colloid formation was estimated and ImageJ (http://rsbweb.nih.gov/ij/) was used to quantify these areas and analyze the differences between Tg-BRAFV600E/MST1 null mice and their Tg-BRAFV600E littermates . In male mice, Tg-BRAFV600E/MST1 null mice also showed a larger area without follicular architecture or colloid formation compared to their Tg-BRAFV600E littermates . This in vivo study suggests that the direct interaction between BRAFV600E and MST1 could affect the behavior of papillary thyroid cancer. Another piece of evidence demonstrating the aggressive tumor phenotype in these Tg-BRAFV600E/MST1 null mice was the frequent presence of muscle invasion -mediated degradation V600E did not alter HA-FoxO3 protein levels, as shown by Western blot analysis, and, interestingly, treatment with a MEK inhibitor did not abolish the BRAFV600E-mediated inactivation of FoxO3. In the present study, BRAFV600E did not induce phosphorylation of Akt/PKB and, furthermore, the addition of PI3 kinase inhibitors did not affect the inhibition of FoxO3 by BRAFV600E. Therefore, BRAFV600E did not affect FoxO3 transactivation by a PI3-Akt/PKB-dependent signaling pathway, whereas RET/PTC inactivates FoxO3 by Akt/PKB activation V600E.Taken together,these data suggest that FoxO3 inactivation might be a common process in the carcinogenesis of PTC and that BRAFV600E inhibits FoxO3 transactivation through a MEK-ERK–, Akt/PKB–, or RAF-1–independent pathway. The results of the present study suggest the existence of a novel FoxO3 inhibitory mechanism that might operate in BRAFV600E-induced PTC.BRAFV600E-induced PTC, which led to the evaluation of BRAFV600E regulation of MST1-FoxO3 signaling pathways. A reporter assay revealed that MST1-induced FoxO3 transactivation was completely inhibited by co-transfection with BRAFV600E. In addition, over-expression of MST1 prevented BRAFV600E-induced FoxO3 inactivation. These data suggested that cross-regulation between MST1 and BRAFV600E might play a critical role in regulating FoxO3 transactivation. Supporting this idea, immunofluorescence studies revealed not only the BRAFV600E-mediated inhibition of MST1-induced FoxO3 nuclear translocation, but also a direct interaction between MST1 and BRAFV600E, which was confirmed through IP assays that demonstrated that BRAFV600E binds to the C-terminal dimerization domain of MST1.MST1 kinase had a counter-regulatory effect on FoxO3 activation and localization compared with Akt/PKB V600E might affect MST1 kinase activity. Actually, our in vitro kinase assay clearly showed that MST1 kinase activity on histone H2B was markedly decreased by BRAFV600E. Another possible mechanism of MST1 regulation may be the inhibitory phosphorylation of MST1 by BRAFV600E. Prior research has shown that Akt/PKB can phosphorylate the Thr387 residue of MST1 and prevent its proteolytic activity, providing a mechanism for the Akt/PKB-mediated inactivation of MST1-FoxO3 pathways V600E-induced MST1 phosphorylation was not assessed, it remains possible that BRAFV600E may phosphorylate and inactivate MST1 through direct interaction with the C-terminal domain of MST1.In fact, the dimerization domain of MST1/2 mediates MST1 kinase activity by interacting with binding partners such as RASSF1A and hWW45 in vivo model was generated to investigate the cross-regulation between BRAFV600E and MST1, and the results obtained with transgenic mice were consistent with the in vitro generated data. Although MST1 knockout mice did not show any thyroid gland pathological changes compared to wild-type mice, Tg-BRAFV600E/MST1 null mice showed an aggressive phenotype, supporting the hypothesis that MST1 might have a modifying effect on BRAFV600E-induced carcinogenesis. Although the lack of an appropriate antibody for the specific immunohistochemical detection of serine 207 residue in FOXO3 phosphorylated by MST1 kinaseprevented the detection of FoxO3 activity modified by MST1, these results, coupled with the results of in vitro assays, suggest that BRAFV600E may alleviate the tumor suppressor function of RASSF1A-MST1 and vice versa.Few mouse models exist for the study of tumor suppressor systems in PTC. In the present study, a new V600E and MST1 kinase plays a crucial role in determining tumor behavior in PTC. This novel interaction might provide advanced insight into the pathogenesis of the disease, and form the basis for novel drug development for the treatment of BRAFV600E-induced PTC. In light of this, it will be important to delineate the precise regulatory mechanism of the RASSF1A-MST1 pathway and verify the action of BRAFV600E on this signal pathway in human PTC.In conclusion, the present study suggests that a direct interaction between oncogenic BRAFFigure S1Renilla luciferase activity and expressed as relative-fold change compared to basal luciferase activity. All data are presented as mean±SD: (*) P<0.01 between two groups.MST1 significantly increases 3XIRS reporter activities. (A) 293T cells were cultured in 12 well dishes until they reached 80% confluence, and co-transfected with 3XIRS Luc (100 ng/well), FoxO3 (0.5 µg/well), and MST1 (0.5 µg/well) as indicated for 24 hours. To verify transfection efficacy, total lysates were immunoblotted with anti-HA, anti-Flag, and anti-Actin antibodies. (B) Cells were co-transfected with 3XIRS Luc (100 ng/well), FoxO3 (0.5 µg/well), and MST1 (indicated amount/well). Total lysates were immunoblotted with anti-HA, anti-Flag, and anti-Actin antibodies. For each sample, firefly luciferase activity was normalized to (TIF)Click here for additional data file.Figure S2V600E inhibits MST1-induced nuclear translocation of FoxO1. 293T cells were cultured in a six-well dish until they reached 80% confluence and co-transfected with FoxO3-GFP (0.5 µg/well), Flag-MST1 (0.5 µg/well), and Myc-BRAFV600E (0.5 µg/well) as indicated. Twenty-four hours after transfection, the cells were prepared for subcellular fractionation using the Nuclear/Cytosol Fractionation kit . The markers, origin recognition complex subunit 1 (ORC1) and β-tubulin, were used to verify the identity and purity of the nuclear and cytosolic fractions, respectively. Based on these markers, a good overall yield was obtained without mixing of the fractions.BRAF(TIF)Click here for additional data file.Figure S3V600E mediated FoxO3 inhibition was not altered by RAF-1. 293T cells were cultured in 12 well dishes until they reached 80% confluence and co-transfected with 3XIRS Luc (100 ng/well), FoxO3 (0.5 µg/well), BRAFV600E (0.5 µg/well), and SiRAF-1  for 24 h as indicated. Total lysates were immunoblotted with anti-HA, anti-BRAF, anti-RAF-1, and anti-Actin antibodies. For each sample, firefly luciferase activity was normalized to Renilla luciferase activity and expressed as relative-fold change compared to basal luciferase activity. All data are presented as mean±SD: (*) P<0.01 between two groups.BRAF(TIF)Click here for additional data file.Figure S4V600E suppresses FoxO3 transactivation via a MEK/ERK-, PI3 kinase-independent pathway. 293T cells were cultured in 12 well dishes until they reached 80% confluence, and co-transfected with 3XIRS Luc (100 ng/well), FoxO3 (0.5 µg/well), and BRAFV600E (0.5 µg/well) as indicated for 24 h. MEK inhibitor  and PI3 kinase inhibitors  were added. Total lysates were immunoblotted with anti-HA, anti-BRAF, anti-pERK, anti-ERK, anti-pAkt/PKB, anti-Akt/PKB, and anti-Actin antibodies. For each sample, firefly luciferase activity was normalized to Renilla luciferase activity and expressed as relative fold change compared to basal luciferase activity. All data are presented as mean±SD. Abbreviations: U0, U0126; WT, Wortmannin; LY, LY294002; and Con, control.BRAF(TIF)Click here for additional data file.


Left ventricular noncompaction (LVNC) is a cardiomyopathy with important prognostic implications. Identification of patients with LVNC using current criteria is challenging. Fractal analysis is a novel approach which summarises global LV trabecular complexity as a continuous variable, the fractal dimension, enabling the establishment of ethnically and disease-appropriate reference ranges for trabeculation.We aimed to determine the reproducibility and accuracy of the fractal method compared to other LVNC criteria. We used these results to estimate sample size requirements for population studies of trabeculation. Two independent observers analyzed CMR data from 20 non-selected LVNC cases  and 40 non-selected healthy volunteers . Datasets were evaluated using the fractal method . This reproducibility was greater than that calculated for the other LVNC analysis techniques Figure . LikelihFractal analysis of LV trabeculae demonstrates good reproducibility and is more accurate in diagnosing LVNC than other techniques. The fractal dimension, if used in clinical trials would more than halve the number of patients required to detect clinically relevant differences in trabecular complexity.J.C.M is supported by the Higher Education Funding Council for England.G.C. is supported by the University College London through a Graduate Research Scholarship and by the European Union through a Science and Technology Research Grant.This work was undertaken at the University College London Hospital and University College London, which receive a proportion of funding from the Department of Health's National Institute for Health Research Biomedical Research Centres funding scheme.


Oxidative stressors such as hydrogen peroxide control the activation of many interconnected signaling systems and are implicated in neurodegenerative disease etiology. Application of hydrogen peroxide to PC12 cells activated multiple tyrosine kinases , and Pyk2) and the serine-threonine kinase ERK1/2. Peroxide-induced ERK1/2 activation was sensitive to intracellular calcium chelation and EGFR and c-Src kinase inhibition. Acute application and removal of peroxide allowed ERK1/2 activity levels to rapidly subside to basal serum-deprived levels. Using this protocol, we demonstrated that ERK1/2 activation tachyphylaxis developed upon repeated peroxide exposures. This tachyphylaxis was independent of c-Src/Pyk2 tyrosine phosphorylation but was associated with a progressive reduction of peroxide-induced EGFR tyrosine phosphorylation, EGFR interaction with growth factor receptor binding protein 2, and a redistribution of EGFR from the plasma membrane to the cytoplasm. Our data indicates that components of peroxide-induced ERK1/2 cascades are differentially affected by repeated exposures, indicating that oxidative signaling may be contextually variable. ROS were originally recognized as active products produced from phagocytic neutrophils [κB, activated protein-1, phospholipase A2, and mitogen-activated protein kinases (MAPKs) such as the extracellular signal-regulated kinase (ERK), Akt, and Jun kinase [Our current knowledge of the complexity of cellular signaling systems is only now allowing us to appreciate the true depth of connectivity in what was previously thought to be collections of linear transduction cascades. It is clear from many lines of study that the structure of signaling systems is far more complex than first thought –4. Cellutrophils , which wtrophils . With ren kinase .2O2 in response to a variety of physiological stimuli such as cytokines, peptidergic growth factors and neurotransmitters [2O2, even at subtoxic concentrations, affects the function of various proteins including transcription factors, phospholipases, protein kinases and phosphatases, ion channels, G proteins, G protein-coupled receptors, and receptor tyrosine kinases [2O2 is the control of cellular tyrosine phosphorylation. H2O2 plays a key role in reversible protein phosphorylation through modulation of protein-tyrosine phosphatases, the lipid phosphatase, and tumor suppressor PTEN as well as multiple receptor tyrosine kinases, potentially through direct oxidation of their catalytic cysteine residues [2O2 therefore has been recognized as an important signaling mediator in growth factor functionality and more specifically, cell signaling-regulated phosphotyrosine posttranslational modification [in vitro experiments, such as those mentioned previously, the signaling effects of H2O2 are usually explored in the context of persistent and chronic exposure to H2O2. As the generation and functioning of H2O2 may be controlled by humoral signaling systems in physiological settings, it is highly likely that, in vivo, the presence of H2O2 may vary from time to time according to physiological neurotransmission or hormonal release patterns. To investigate the effects of intermittent H2O2 exposure, we studied the effects of repetitive H2O2 exposure to neural PC12 cells with regards to cellular tyrosine kinase and ERK-activation properties. Both singular and repeated exposure of PC12 cells to H2O2 resulted in rapid increases in whole-cell protein tyrosine phosphorylation, specific phosphotyrosine content increases in receptor and nonreceptor tyrosine kinases and the potent activation of ERK. Repeated exposures to H2O2, however, resulted in the differential tachyphylaxis of specific components of the peroxide-dependent ERK-activation mechanisms. Such evidence may suggest that, in vivo, a complex interplay between multiple H2O2-controlled signaling processes may underlie its multiple physiological activities.Hydrogen peroxide is the major physiologically relevant form of ROS due to its relatively high stability. Many nonphagocytic cells are now known to produce Hsmitters –16. Libe kinases . One of residues –24. H2O2fication . In manyβ-cyclodextrin (MeβC), sucrose, filipin, and the protein kinase A inhibitor H-89 were obtained from Sigma-Aldrich . PP2 (non-receptor Src-family tyrosine kinase inhibitor), wortmannin (phosphoinositide 3-kinase inhibitor), BAPTA-AM , PD98059 (MEK1/2 inhibitor) and AG1478  were all obtained from EMD Bioscience . Qproteome subcellular compartmentalization purification kits were obtained from Qiagen . The generic anti-phosphotyrosine antisera, PY99, anti-epidermal growth factor (EGF), anti-actin, anti-catalase, non-phosphorylated ERK1/2, anti-Grb2, anti-epidermal growth factor receptor, anti-lamin A, anti-Tim23 and anti-annexin V antisera were all obtained from Santa Cruz Biotechnology . An agarose-preconjugated PY20 (anti-phosphotyrosine) slurry was also obtained from Santa Cruz Biotechnology. Anti-phosphosite-specific anti-Pyk2 (anti-phospho-Tyr402/Tyr881) and anti-c-Src (anti-phospho-Tyr418) were obtained from Invitrogen  and BD Bioscience respectively. Anti-active, (phosphor-)ERK1/2 antisera were obtained from Cell Signaling Technology . Non-phosphorylated anti-Pyk2 antisera were obtained from BD Bioscience.Cell culture grade hydrogen peroxide, monodansylcadaverine (MDC), methyl-http://www.atcc.org/), were maintained as previously described [2 atmosphere. Prior to any cellular challenges with hydrogen peroxide, the cells were serum deprived for 16 h by removing the horse and bovine serum content of the RPMI growth medium and addition of 10 mM HEPES to compensate for the reduced buffering capacity caused by serum withdrawal. All PC12 cells were maintained in 100-mm plates coated with poly-d-lysine . To experimentally degrade the exogenously-applied hydrogen peroxide, bovine liver catalase  was added to the experimental media at a concentration of 40 U/mL. Bovine liver catalase was inactivated with a 95°C incubation for 10 minutes.Rat pheochromacytoma cells (PC12), obtained from the American Type Tissue Culture Collection  of generic phosphotyrosine proteins was achieved using 20 μL of a 50% slurry of anti-PY20 affinity agarose (Santa Cruz Biotechnology) with agitation for 16 h at 4°C. Immunoprecipitation of Pyk2 or EGFR was achieved using the addition of 5 μg of the specific anti-protein sera plus 30 μL of a protein-A/G-conjugated agarose slurry (EMD Bioscience) followed by agitation for 16 h at 4°C. Immune complexes were washed three times with ice-cold NP40-based lysis buffer and transferred to a clean microcentrifuge tube before addition of 20 μL of 2× Laemmli sample buffer. Immunoprecipitates were resolved by SDS-PAGE and electrotransferred to polyvinylidene difluoride  membrane for protein immunoblotting (IB). Polyvinylidene difluoride membranes were blocked in a bovine serum albumin-based solution  solution for 1 h at 37°C before immunoblotting. Immunoblotting of whole-cell lysates for unphosphorylated/phosphorylated ERK1/2 or inactive/active c-Src (Tyr-418 autophosphorylated) was performed using specific primary antisera  at a 1 : 1000 dilution with subsequent addition of a 1 : 7000 dilution of alkaline phosphatase-conjugated anti-mouse or rabbit IgG as a secondary antibody (Sigma Aldrich). Immunoblotting of anti-phosphotyrosine immunoprecipitates, resolved with SDS-PAGE, for phosphotyrosine content was performed using a 1 : 1000 dilution of PY99 antisera with a subsequent addition of a 1 : 7000 dilution of alkaline phosphatase-conjugated anti-mouse IgG as a secondary antibody (Sigma). Immunoblotting of anti-Pyk2 or EGFR immunoprecipitates was performed using either anti-PY20 , anti-phosphosite-specific (Tyr-402 or Tyr-881) anti-Pyk2 antisera  or with anti-Grb2 antisera . Specifically isolated Qproteome cell compartments  were identified using 1 : 1000 dilutions of specific primary antisera, CE1-annexin-V, CE2-Tim23, or CE3-lamin-A followed by 1 : 7000 dilutions of species-specific alkaline phosphatase-conjugated secondary antisera . Visualization of alkaline phosphatase-labeled proteins was performed using enzyme-linked chemifluorescence  and quantified using a Typhoon 9410 PhosphorImager . Image density measurements were performed from phosphorimager-captured images using L-Process version 2.2 and Image-Gauge version 4.2 (Fuji Image Systems). Western blot image density was measured per square visual pixel (px2) as arbitrary units (AUs) from which background (B) signals were subtracted. Therefore western image density is represented throughout as (AU-B)/px2. Statistical analysis of relative protein expression values was performed using an unpaired Student's t-test in GraphPad Prism version 5.0.After challenges with hydrogen peroxide, PC12 cell monolayers were placed on ice, washed twice in ice-cold Dulbecco's phosphate-buffered saline, and lysed in a Nonidet P-40 (NP40-)based solubilization buffer, as described previously . SolubilμM a lack of further phosphotyrosine increase was seen (μM), it was demonstrated that this effect was achieved and reached its maximum after 10 minutes of acute stimulation (Short term application (10 minutes) of hydrogen peroxide to serum-deprived PC12 cells resulted in the dose-dependent increase in the generic phosphotyrosine content of multiple proteins identified using poylacrylamide gel resolution of anti-phosphotyrosine (PY20) immunoprecipitates derived from cell lysates. The dose response relationship, however, was not perfectly sigmoidal, as at peroxide concentrations greater than 200 was seen . Employimulation . This tiμM) of hydrogen peroxide resulted in the significant increase of the levels of tyrosine-418 phosphorylation of the non-receptor tyrosine kinase c-Src  application of multiple doses  did not greatly elevate global calcium levels (data not shown). Direct inhibition of the upstream ERK1/2-phosphorylating kinase, MEK1/2, with PD98059  resulted in near complete inhibition of the peroxide-induced ERK1/2 signal. The peroxide-induced ERK1/2 activation signal was, however, largely resistant to inhibition of phosphoinositide-3-kinase (PI-3K) or protein kinase A (PKA) as neither H-89 nor wortmannin preincubation affected the peroxide-induced ERK1/2 activation . To create protein samples for R1, the simple “acute-recovery” process was performed. To create protein samples for secondary (R2), tertiary (R3), and quaternary (R4) responses, individual “acute-recovery” peroxide applications were made and were separated by 30 minutes of recovery time , we noted that along with the expected reduction in ERK1/2 activation, at the R4 response point, the ability of the hydrogen peroxide to induce Pyk2 auto-tyrosine phosphorylation (indicative of Pyk2 tyrosine kinase activation—measured with anti-phospho-Tyr-402-Pyk2 sera) and increase generic Pyk2 tyrosine phosphorylation was not significantly affected Figures . As withAs we had noted that repeated exposures to peroxide reduced both the ERK1/2 activation and the degree of EGFR tyrosine phosphorylation, but not that of Pyk2 or c-Src, we decided to investigate whether additional signaling components of ERK1/2-activating complexes were affected. The growth factor receptor binding protein 2 (Grb2) is a bifunctional adapter protein involved in multiple ERK-activating multiprotein complexes. Grb2 is typically associated with Ras-guanine nucleotide exchange factors such as son of sevenless (Sos). Upon activation of scaffolding molecules employed in ERK1/2 activation cascades, for example, EGFR, Pyk2, or the focal adhesion kinase (FAK), the auto-tyrosine phosphorylation of these proteins attracts Grb2 to the plasma membrane via interaction of its SH2 domains with phosphotyrosine residues on the scaffolds. Grb2 interacts with Sos via its SH3 domains associating with proline-rich regions of Sos. At the plasma membrane, Sos can then facilitate exchange of guanosine triphosphate for guanosine diphosphate at juxtamembrane monomeric G proteins such as Ras. As Grb2 association with scaffolds, such as EGFR or Pyk2, is vital for their capacity to activate ERK1/2 cascades, we investigated the relative effects of R1- versus R4-level peroxide exposures upon Grb2 interaction with EGFR or Pyk2. As we have seen previously, successive peroxide exposures reduced the degree of peroxide-induced EGFR tyrosine phosphorylation Figures . When thμM MDC or hypertonic 0.45 M sucrose) significantly attenuated the peroxide-induced EGFR redistribution (βC (10 μM) or filipin (5 μg/mL)) failed to alter R4 peroxide-induced EGFR redistribution (n = 3)) using a repeated exposure paradigm analogous to the peroxide stimulations. As both repeated peroxide and EGF mediated an effective redistribution of EGFR from the plasma membrane fraction, we also found that exogenous EGF application  was able to partially blunt the subsequent ERK1/2 response to peroxide stimulation ). Therefore, it seems that the peroxide-mediated effects on the EGFR are similar in several ways to the normal functioning of the EGF/EGFR system.It has been demonstrated by many researchers that the molecular and biochemical nature of a specific ERK1/2-activating scaffold has profound effects upon the physiological mechanisms of ERK1/2 signaling , 25. To ribution . Inhibitribution . When thribution . These dIn this study, we have shown that in response to acute exposure of PC12 cells to hydrogen peroxide, multiple ERK1/2-activating mechanisms can be stimulated. We demonstrated that hydrogen peroxide exposure resulted in the elevation of the phosphotyrosine status of the non-receptor tyrosine kinase c-Src as well as the EGFR and Pyk2. Multiple studies have demonstrated that alterations in cellular oxidative state are a potent modulator of multiple diverse and interconnected intermediary cell metabolism signaling mechanisms , 30, 31.


Mast cells (MCs) are tissue resident cells of hemopoietic origin and are critically involved in allergic diseases. MCs bind IgE by means of their high-affinity receptor for IgE (FcεRI). The FcεRI belongs to a family of multi-chain immune recognition receptors and is activated by cross-linking in response to multivalent antigens (Ags)/allergens. Activation of the FcεRI results in immediate release of preformed granular substances , generation of arachidonic acid metabolites, and production of pro-inflammatory cytokines. The FcεRI shows a remarkable, bell-shaped dose-response behavior with weak induction of effector responses at both low and high  Ag concentrations. This is significantly different from many other receptors, which reach a plateau phase in response to high ligand concentrations. To explain this unusual dose-response behavior of the FcεRI, scientists in the past have drawn parallels to so-called precipitin curves resulting from titration of Ag against a fixed concentration of antibody (Ab) in solution (a.k.a. Heidelberger curves). Thus, for high, supra-optimal Ag concentrations one could assume that every IgE-bound FcεRI formed a monovalent complex with “its own Ag”, thus resulting in marginal induction of effector functions due to absence of receptor cross-linking. However, this was never proven to be the case. More recently, careful studies of FcεRI activation and signaling events in MCs in response to supra-optimal Ag concentrations have suggested a molecular explanation for the descending part of this bell-shaped curve. It is obvious now that extensive FcεRI/IgE/Ag clusters are formed and inhibitory molecules and signalosomes are engaged in response to supra-optimal cross-linking (amongst them the Src family kinase Lyn and the inositol-5′-phosphatase SHIP1) and they actively down-regulate MC effector responses. Thus, the analysis of MC signaling triggered by supra-optimal crosslinking holds great potential for identifying novel targets for pharmacologic therapeutic intervention to benefit patients with acute and chronic allergic diseases. Mast cells are central players in allergic diseases like hay fever and asthma. When allergens bind to specific receptors  on the surface of mast cells, various intracellular events are triggered, resulting in the secretion of inflammatory mediators, such as histamine and proteases. These mediators are responsible for the well known symptoms of allergies . In principle, receptors for different ligands are expressed by every cell, enabling each cell to sense its environment and react to it in an appropriate fashion. Usually, the concentration of the ligand correlates with the extent of the ligand-induced cellular response. Intriguingly, this is not the case for the FcεRI. High, so-called supra-optimal allergen concentrations result in only weak activation of mast cells, whereas lower concentrations of allergen can cause strong activation. The reason is that high allergen concentrations engage inhibitory mechanisms, which do not allow for the induction of mediator secretion. In recent years, several intracellular proteins involved in such inhibitory mechanisms have been identified and their functional interactions are currently being elucidated. Thus, further investigation of this cell-intrinsic shut-off mechanism and identification of additional proteins involved should enable researchers to define novel molecular targets for pharmacologic intervention. The aim would be to exploit this knowledge to actively silence the mast cell during pollen season.Mast cells (MCs) are hemopoietically derived tissue resident cells that are concentrated in tissues close to the external environment, i.e., the skin and mucosal membranes of the intestine and airways . One of 2) [The FcεRI on murine MCs and basophils consists of an α-subunit, a β-subunit, and two disulfide-bridged γ-subunits (αβγ2)   . The β-s2) , which, 2) . Lyn has2) ,14. Of n2) . Though 2) . Moreove2) . Thus, a2) -19. For 2) , the Fcε2) . The β-c2) ,21. More2) ,23. The 2) . In huma2) ,25.30-40-HSA [Many receptors, like receptor tyrosine kinases and cytokine receptors, require ligand-mediated dimerization for their activation . The dos0-40-HSA . AlternaIn 1973, Becker et al. investigated IgE/FcεRI redistribution and histamine release in human basophils in response to increasing concentrations of anti-IgE Abs . They foHow would secretion of histamine be suppressed when FcεRIs are supra-optimally cross-linked? Baird and coworkers reported that stimulation of IgE-bound FcεRI by polyclonal Abs against IgE induced a detergent-resistant association of these complexes with the cellular cytoskeleton . In thorThus, suppressed degranulation in response to supra-optimal Ag appeared not to be due to reduced FcεRI crosslinking, as depicted in Figure 2+ ions from the ER with subsequent influx of extracellular Ca2+ via store-operated Ca2+ (SOC) channels [2+ mobilization in response to sub- as well as supra-optimal Ag concentrations and strong responses after optimal cross-linking [2+ mobilization at supra-optimal was not simply weaker than at optimal Ag concentrations, but the sustained pattern  changed to a transient one , suggesting active negative regulation. Consistent with this, early overall tyrosine phosphorylation events were slightly enhanced in supra-optimally vs. optimally stimulated MCs [3), the product of the PI3K-catalyzed reaction, to yield PI-3,4-P2[2+ mobilization [All these studies indicated that the bell-shaped dose-response curve of MC degranulation might not reflect the level of signaling taking place inside the cells. Thus, it was important to elucidate the signaling pathways controlling MC activation in response to differing concentrations of Ag. A critical step in regulating IgE-induced degranulation is the release of intracellular Cachannels ,38,39. T-linking . Interesated MCs ,40. Partated MCs ,41,42. SPI-3,4-P2,44. Thislization .Related to this, the SFK Lyn has been shown to phosphorylate and activate SHIP1 in MCs and degranulation studies with Lyn−/− BMMCs have revealed that these cells do not display the descending part of the bell-shaped degranulation curve . InteresHow could one envision the functional coupling between supra-optimally cross-linked FcεRIs and such an inhibitory signalosome? Intriguingly, the β-subunit of the FcεRI, which has been carefully described by Kinet and colleagues as the amplifier of FcεRI-mediated activation signals ,49, has Interestingly, analyzing GPVI-mediated dense granule secretion from platelets, Kunapuli and coworkers found that functional interactions between Lyn, PKC-δ, and SHIP1 negatively regulate this process as well . GPVI is3 phosphatases are expressed that participate in regulation of the PI3K pathway, namely the 5′-phosphatase SHIP2 and the 3′-phosphatase PTEN. SHIP2 has been knocked-down by shRNA in BMMCs and shown to result in stronger FcεRI-induced degranulation at every Ag concentration tested, compared to cells treated with control shRNA [2+ mobilization [2+ mobilization [2+ mobilization as well as degranulation in response to FcεRI activation [3 levels in unstimulated MCs [In MCs, two additional prominent PIPol shRNA . Howeverlization . On the lization . These rtivation . Unfortuated MCs .3, SHIP1 would suppress various PIP3-dependent molecules/pathways, one or more of them critically involved in Ca2+ mobilization and degranulation (e.g. phospholipase C-γ1 (PLC-γ1), PLC-γ2, and the tyrosine kinase Btk [3, PI-3,4-P2 is generated, which is known to specifically interact with PH-domains of certain signaling proteins, e.g. the adaptor proteins Bam32/DAPP1, TAPP1, and TAPP2 [2+ mobilization and degranulation in response to supra-optimal Ag concentrations, suggesting that Bam32 represents an effector of SHIP1’s negative activity [How SHIP1 controls degranulation in response to supra-optimal Ag concentrations is not entirely clear at the moment. Because of its structure, SHIP1 incorporates catalytic as well as adaptor functions . Even onnase Btk ,59). Secnd TAPP2 . Relatedactivity . Intriguactivity .3[With respect to SHIP1’s adaptor or scaffolding function, increasing numbers of interaction partners of SHIP1 have been and continue to be identified. SHIP1 contains an N-terminal SH2-domain, a centrally located 5′-phosphatase domain, and a C-terminus containing several proline-rich sequences as well as two NPxY motifs . The SH23.Finally, proteins containing so-called phosphotyrosine-binding (PTB)-domains, such as the adaptor proteins Shc and p62Dok1, have been demonstrated to bind to SHIP1’s C-terminal NPxY motifs upon their phosphorylation -73. Sincmotheaten (me) mice [viable motheaten (mev) mice [me mice and measured decreased Ca2+ mobilization and degranulation in me BMMCs in response to Ag [mev mice, which were found to degranulate in an augmented fashion compared to wild-type BMMCs [As far as other possible negative regulators of supra-optimal FcεRI-induced MC degranulation are concerned, Xiao et al. showed that both SHIP1 and SHP-1 interacted with the FcεRI β-chain and that their phosphorylation was controlled by Lyn . Howeverme) mice  and viabev) mice ) have nose to Ag . On the pe BMMCs . The obspe BMMCs . Unfortu2+ mobilization was shown in Cbl-b-deficient BMMCs. This resulted in augmented degranulation at every Ag concentration studied with the most prominent effect at high, supra-optimal concentrations [2+ mobilization, and degranulation [The Cbl family of E3 ubiquitin-protein ligases comprises three mammalian members, c-cbl, Cbl-b, and Cbl-c. These proteins poly-ubiquitinate various cellular proteins, thereby targeting them for proteasomal degradation . Applyintrations , suggesttrations ,84 & unptrations . Overexpnulation . Unexpecnulation , and Jnknulation . Again, Immune complex-induced coaggregation of the FcεRI with the inhibitory, low-affinity receptor for IgG, FcγRIIB (CD32), results in suppression of FcεRI-mediated MC activation . In contWithout a doubt the field has come a long way since the precipitin-type model of supra-optimal FcεRI activation. As stated in this review, several proteins can be assigned inhibitory functions in response to supra-optimal Ag concentrations Figure . HoweverThe bell-shaped dose-response behavior represents a remarkable feature of the FcεRI. Nature provides us with an efficient shut-off mechanism at high, supra-optimal Ag concentrations, which could be employed to alleviate allergic symptoms. By deciphering the molecular language of inhibitory signaling in response to supra-optimal Ag concentrations one might identify novel targets for pharmacologic therapeutic interventions to benefit patients with acute and chronic allergic diseases. A deeper understanding of this mechanism(s) should also help to elucidate the molecular events that occur during hyposensitization of allergy patients. Finally, the bell-shaped dose response pattern is not unique to the FcεRI, but can also be found with other receptors that signal in an ITAM-dependent manner, such as the FcγRIII (CD16) and the B cell antigen receptor ,98. ThusThe author declares that he has no competing interests.