These results also showed that the S693D group is more effective in protecting both non-neuronal and neuronal cells from apoptotic death than the S637D group. Altogether, our data suggest that GSK3beta-mediated phosphorylation at Ser693 of Drp1 may be associated with mitochondrial elongation via down-regulating apoptosis, but not autophagy upon H2O2 insult.Multiple phosphorylation sites of Drp1 have been characterized for their functional importance. However, the functional consequence of GSK3beta-mediated phosphorylation of Drp1 remains unclear. In this report, we pinpointed 11 Serine/Threonine sites spanning from residue 634∼736 of the GED domain and robustly confirmed Drp1 Ser693 as a novel GSK3beta phosphorylation site. Our results suggest that GSK3beta-mediated phosphorylation at Ser693 does cause a dramatic decrease of GTPase activity; in contrast, GSK3beta-mediated phosphorylation at Ser693 appears not to affect Drp1 inter-/intra-molecular interactions. After identifying Ser693 as a GSK3beta phosphorylation site, we also determined that K679 is crucial for GSK3beta-binding, which strongly suggests that Drp1 is a novel substrate for GSK3beta. Thereafter, we found that overexpressed S693D, but not S693A mutant, caused an elongated mitochondrial morphology which is similar to that of K38A, S637D and K679A mutants. Interestedly, using H89 and LiCl to inhibit PKA and GSK3beta signaling, respectively, it appears that a portion of the elongated mitochondria switched to a fragmented phenotype. In investigating the biofunctionality of phosphorylation sites within the GED domain, cells overexpressing Drp1 S693D and S637D, but not S693A, showed an acquired resistance to H Glycogen synthase kinase-3 (GSK-3) is a serine/threonine kinase originally found to inactivate the enzyme glycogen synthase by phosphorylation Drp1 is one of the dynamin related proteins, which is a large protein reported to be comprised of an amino-terminal GTPase domain, middle domain, insert B and a GTPase effector domain (GED) as shown in 2O2-induced apoptosis rather than inducing autophagy.Regarding the functional regulation of Drp1, post-translational modifications such as sumoylation, S-nitroylation, ubiquitination, and phosphorylation are thought to be an important part of its regulation 634–690 as a possible GSK3beta binding domain. Protein sequences of Drp1 orthologies among different species were aligned by Clustal W2  of Drp1 in addition to a dephosphorylated (S693A) mutant. Both of these two Drp1 mutants were confirmed to lose their GSK3beta-mediated phosphorylation in vitro  was used as prey or bait protein in the yeast two hybrid system, it can interact with Drp1 truncate 2 fragment (Drp1502–736). This finding suggests that Drp11–489 is important for intra-molecular interaction. In addition, the entire GED domain is also required for the inter-molecular interactions . Altogether, these data suggest that Drp1634–690 (truncate 5) might be important to both the inter-/intra-interactions of Drp1 assembly (502–736), the results showed no differences between these three Drp1 mutants and wild-type Drp1. Our data indicated that S693D, S637D and K679A are unlikely to impair intra-molecular interaction and Ser693 may not be involved in Drp1 inter-/intra-molecular interactions (The GTPase Effect Domain (GED) has been suggested to regulate the GTPase activity of dynamin related proteins via inter-/intra-molecular interactions assembly . Surprisractions . Neverthractions . In the ractions . In concin vitro GTPase assays, we found that phosphomimetic His-Drp1 S693D exhibited the same decreased GTPase activity as the S693A and K38A mutants  to stimulate GTP hydrolysis after dynamin assembly  mutants . The datTo elucidate whether the Drp1phosphorylation by GSK3beta affects mitochondrial morphology, we transfected HeLa cells with constructs expressing different mimetic phosphorylated Drp1 mutants. Five different mutations  were tested to evaluate the biological functionalities involved in regulating mitochondrial morphology, especially with respect to reported disruptions of Drp1 functions in controlling inter-/intra-molecular interaction (S637D). The GFP-expressing cells were examined for their protein expression see  and were2O2 since the elongated mitochondrial morphology has been linked to a protective effect against both apoptosis and autophagy. After treating HeLa cells with 500 µM H2O2 for 24 hours, the mitochondrial networks with respect to GFP alone, GFP-Drp1 wild-type (wt) and the S693A group did show a phenotype of fragmentation, in which almost 80% of the mitochondria of cells underwent fragmentation  exert a protective effect for HeLa cells to combat Hwt group . In cont93 cells . GSK3betin vitro, decreased mitochondrial fission, and caused elongated mitochondrial morphology. Finally, we concluded that GSK3beta-mediated phosphorylation at Ser693 may be associated with elongated mitochondrial morphology and correlated to acquired resistance against H2O2-induced apoptosis via down-regulating cytochrome c, caspase-3, -7 and PARP activities, but does not induce autophagyIn the present study, we reported the possible functional roles of GSK3beta interaction as well as GSK3beta-mediated phosphorylation of Drp1 at the GED domain. We successfully identified the GSK3beta phosphorylation site at Ser693 of Drp1. We demonstrate that GSK3beta binds to Drp1 via 634–690 residue and phosphorylates Ser693 in the GED domain. Using a mimetic phosphorylated mutant of S693D, we also found that expressed S693D Drp1 mutant resulted in decreased GTPase activity 1–489 and Drp1502–736 or between two Drp1502–736 fragments in a yeast two hybrid system. Moreover, the K679A and S693D Drp1 mutations also do not interfere with inter-/intra-molecular interactions of Drp1 truncated fragments  corresponding to Drp1502–736 and/or the GED-GTPase domain (intra-molecule) refers to Drp11–489 and Drp1502–736. These two kinds of interactions are rationalized as two major molecular mechanisms consequent to the decrease in GTPase activity of Drp1. Therefore, our finding that Ser693 is only responsible for GSK3beta-mediated phosphorylation, but not involved in the inter-/intra-molecular interactions of Drp1 monomers, may be indirectly supported by these data. Obviously, the Drp1 K679 is more likely to be important for GSK3beta binding and Ser693 is a GSK3beta phosphorylation site.In this report, a new Drp1 phosphorylation site, Ser693, was identified and phosphorylation at this site was shown to lead to elongated mitochondrial morphology. Although the role of GED in dynamin assembly is widely accepted to be correlated to its GTPase activity, the mechanism by which mutation in GED leads to conformational change of Drp1 and cooperatively increases GTPase activity remains uncertain. Chang et al. found that the S637D mutant of Drp1 impairs intra-molecular interactions of the GED domain and middle domains, indicating that S637D mutation may be associated with a conformational change in the GED that interferes with the inter-domain interactions of Drp1 monomer ragments . The incGSK3beta . InteresGSK3beta  and 3C. Drp1 exhibits a basal level of GTP hydrolysis that is enhanced by self- assembly/oligomerization. GED in Drp1 is required for activating its GTPase activity, though the underlying mechanisms are still poorly understood. The function of GED in regulating dynamin has been suggested via two distinct ways. First, it may regulate GTPase activity through self-assembly or highly ordered GED-GED interaction. Recently, evidence from protein structural studies has revealed that GED may biophysically serve as a docking site for GTPase binding and exerts stimulatory effects 2+ influx Tremendous progress has been achieved in elucidating the functional roles of phosphorylation on Drp1, providing significant evidence that mitochondria dynamics are regulated by phosphorylation and by various signaling cascades including CaMKIα, PKA, and Cdk1/Cyclin B It has been suggested that mitochondrial fragmentation is associated with apoptotic and non-apoptotic cell death 2O2-induced mitochondrial fragmentation and ensuing apoptosis, but does not provoke protective autophagy. Here we demonstrate that Drp1 is phosphorylated by GSK3beta at Ser693 and by PKA at Ser637 leading mitochondria to undergo rapid elongation through the attenuation of GTPase activity, down-regulating cytochrome c release, capase-3, -7 and PARP activation , neuron-like cells, were cultured in D-MEM/F12 medium (Gibco) supplemented with 10% FBS, 1% nonessential amino acids (Gibco), 100 IU/ml penicillin, and 100 mg/ml streptomycin (Gibco) at 37°C in a humidified 5% CO2 incubator. Cells were passaged when they reached 80–90% confluence at 1: 5–6 with 0.05% trypsin. The Drp1 cDNA expression constructs have been described previously 5 cells were transfected using 2 µg DNA and 3 µL of Lipofectamine 2000. After 24 hours, cells were harvested in radioimmunoprecipitation assay (RIPA) buffer. For immunofluorescence, cells were transfected with 1 µg of DNA and 1.5 µl Lipofectamine 2000 and cultured for 24 hours. Thereafter, 5×104 cells were seeded on coverslips and immunostained with fluorophore-conjugated antibodies. For H2O2 insult, HEK293, HeLa and SH-SY5Y cells were transfected with GFP alone, GFP-tagged Drp-1 wild-type and other mutants for 24 hours. Then cells were treated with 500 µM H2O2 for another 24 hours, then observed by confocal fluorescent microscopy or harvested for western blotting analysis. For inhibition of GSK3beta and PKA signaling, HeLa cells were transfected with GFP-tagged Drp1wt or other mutants for 24 hours. Then cells were treated with10 mM LiCl and 10 µM H89 for another 24 hours. Mitochondrial morphology of HeLa cells was observed by staining with Mitotracker under confocal microscopy. Cell nuclei were counter-stained by using 4′, 6-diamidino-2-phenylindole .HeLa and HEK293 cells (ATCC) were cultured in DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/mL) and streptomycin (100 µg/mL) at 37°C and 5% COTo construct the pACT2-Drp1 plasmid for the yeast two-hybrid working assay, DNA fragments encoding Drp1 were amplified by PCR using high fidelity polymerase (Roche). The truncate 1–6 of Drp1 were amplified by PCR. These amplified fragments were digested by BamHI and XhoI, and they were also introduced into the BamHI and XhoI sites of the pACT2 or pAS2-1 vector . Full-length Drp1 was inserted into the pEGFP-C1 vector using the BamHI and XhoI restriction sites. Full-length or truncated with or without point-mutated Drp1 fragments were also inserted into the pET-32a (+) vector using BamHI sites. Site-directed mutagenesis experiments to create the Drp1 mutants  were carried out using the Quickchange system. All procedures were done according to the manufacturer's protocol (Stratagene) with minor modifications. The nucleotide sequencing was performed using a BigDye terminator v3.1 kit and the extended products were resolved on an ABI PRISM™ 3730 Genetic Analyzer (Applied Biosystems).Standard techniques were used to carry out yeast two-hybrid screening Lysates were prepared using chilled cells on ice for 30 minutes in RIPA buffer, cleared by centrifugation. For detecting cytochrome C, we prepared cell lysates carefully avoiding destroy mitochondria using Mitochondria/Cytosol Fractionation Kit (Millipore) even though we did not prepare the mitochondrial/cytosolic fractions separately. Protein concentration was determined by the Bradford method and 20 µg per lane of lysate was resolved by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred onto nitrocellulose membranes. Nonspecific binding was blocked by 1 hour incubation with blocking buffer before membranes were probed overnight at 4°C with primary antibodies  diluted in blocking buffer ). After extensive washing with TBS-T, specific bands were detected on Hyperfilm™  using horseradish peroxidase (HRP)-conjugated secondary antibodies and the ECL detection system .2, 0.1 mM ATP, 0.5 mM PMSF, 10% glycerol, and 10 Ci of [γ-32P]ATP , 3000 Ci/mM]. The assays were carried out for 15 minutes at 30°C. The reaction was stopped by adding 2× sample buffer. Samples were denatured at 95°C for 5 min and separated by 8% SDS-PAGE. Signals were detected via autoradiography.The kinase reaction was carried out as described previously Escherichia coli BL21 (DE3) using Ni-Q affinity resin (QIAGEN). GTP hydrolysis by recombinant Drp1 was assayed using a simple, continuous, coupled GTP regenerating assay as described elsewhere The cDNA of wild-type or mutant Drp1 was cloned into pET32A for 6His-Drp1 fusion protein expression. The soluble His-tagged wild-type or mutant Drp1 variants were purified from Cells were pre-incubated with 100 nM MitoTracker (Invitrogen) for 30 minutes, fixed in 3.7% paraformaldehyde (PFA) for 15 minutes and permeabilized with 0.1% Triton for 15 minutes at room temperature. Nonspecific binding was blocked for at least 30 minutes using blocking buffer (10% horse serum in PBS). Next, cells were incubated for 2 hours with primary antibodies in blocking buffer, washed 4 times with PBS, 5 minutes each, and incubated with FITC-anti-mouse IgG diluted 1∶300 in PBS. After washing again, the coverslips were mounted using Vectashield® (Vector Labs) containing DAPI (1.5 µg/mL), and wide-field epi-fluorescence images were acquired at room temperature with an Olympus FlowView confocal microscope system. Mitochondrial morphology was categorized by mitotracker staining in GFP-Drp1 cells expressing WT, K38A, S637D, K679A, S693A, or S693D as described in the study. Digital microscopic images of cells were acquired with a confocal microscope as described above.Data from densitometry analysis and confocal fluorescent microscopy were indicated as continuous variables, and then subjected to testing for inter-group difference by paired T-test using PAWS Statistics version 18.0 software . For multi-group comparisons, one-way ANOVA was used with Tukey's post-hoc test. A p-value less than 0.05 was viewed as statistically significant.Figure S1The protein levels of ectopic expression of GFP-tagged Drp1 wt and mutants in HeLa cells.(TIF)Click here for additional data file.Figure S2Mitochondrial morphology of HeLa cells with Drp1 wt and K679A mutant. HeLa cells were transfected with GFP-tagged Drp1 wt or K679A mutant for 24 hours. Mitochondrial morphology was observed by confocal fluorescent microscope with Mitotracker dye. Cell nuclei were counter-stained by DAPI. Insets are magnifications of the Mitotracker signal at the indicated areas. Inset 1 represents the non-transfected cells, and inset 2 indicates the transfected cells.(TIF)Click here for additional data file.Figure S32O2-induced mitochondrial fragmentation and ensuing apoptosis but does not induce autophagy.Overexpressed Drp1 S693D can protect against H HEK293 cells were transfected with GFP alone, GFP-tagged Drp-1 wild-type and other mutants for 24 hours. Then cells were treated with 500 µM H2O2 for another 24 hours, and were lysed and detected by Western blotting using anti-cytochrome c, -caspase 3, -caspase 7, -PARP, -Bcl2, -LC3, -Beclin 1 and -Atg5 antibody, respectively. beta-actin served as a protein loading control. The data are representative of three independent experiments.(TIF)Click here for additional data file.


CHLH mutant, rtl1, indicated that CHLH specifically affects ABA-induced stomatal closure, but also that CHLH did not serve as an ABA receptor in Arabidopsis thaliana. However, the molecular mechanism by which CHLH engages in ABA-mediated signaling in guard cells remains largely unknown. In the present study, we examined CHLH function in guard cells and explored whether CHLH expression might influence stomatal aperture. Incubation of rtl1 guard cell protoplasts with ABA induced expression of the ABA-responsive genes RAB18 and RD29B, as also observed in wild-type (WT) cells, indicating that CHLH did not affect the expression of ABA-responsive genes. Earlier, ABA was reported to inhibit blue light (BL)-mediated stomatal opening, at least in part through dephosphorylating/inhibiting guard cell H+-ATPase (which drives opening). Therefore, we immunohistochemically examined the phosphorylation status of guard cell H+-ATPase. Notably, ABA inhibition of BL-induced phosphorylation of H+-ATPase was impaired in rtl1 cells, suggesting that CHLH influences not only ABA-induced stomatal closure but also inhibition of BL-mediated stomatal opening by ABA. Next, we generated CHLH-GFP-overexpressing plants using CER6 promoter, which induces gene expression in the epidermis including guard cells. CHLH-transgenic plants exhibited a closed stomata phenotype even when brightly illuminated. Moreover, plant growth experiments conducted under water-deficient conditions showed that CHLH transgenic plants were more tolerant of drought than WT plants. In summary, we show that CHLH is involved in the regulation of stomatal aperture in response to ABA, but not in ABA-induced gene expression, and that manipulation of stomatal aperture via overexpression of CHLH in guard cells improves plant drought tolerance.The Mg-chelatase H subunit (CHLH) has been shown to mediate chlorophyll biosynthesis, as well as plastid-to-nucleus and abscisic acid (ABA)-mediated signaling. A recent study using a novel  Furthermore, we found that CHLH reduced the ABA mediated de-phosphorylation of guard-cell H+-ATPase. Finally, we obtained direct evidence that overexpression of CHLH in guard cells confers drought tolerance on Arabidopsis plants by increasing the sensitivity of stomatal guard cells to ABA.In the present study, we further analyzed CHLH function in guard cells and tried to manipulate stomatal aperture using A. thaliana ecotype Columbia gl1 (glabrous1) served as wild-type (WT) plants. The background ecotype of the rtl1 mutation is gl1  is the background ecotype of abi1-1 . Plants were grown in soil under a 16-h fluorescent light (50 μmol m−2 s−1)/8-h dark cycle at 24°C at a relative humidity of 55–75% for 4–6 weeks in a dedicated growth room  supplemented (or not) with 20 μM ABA in the dark for 1 h at 24°C. For protein blot analysis and detection of 14-3-3 protein, GCPs were incubated in the above buffer with or without 20 μM ABA in the dark for 10 min at 24°C.GCPs from 5-week-old rosette leaves (~10 g) were enzymatically prepared as described by Ueno et al. . To analRAB18 (At5g66400) and RD29B (At5g52300) transcripts, the following primer sets were used: for RAB18, 5′-TGTAACGCAGTCGCATTCG-3′ and 5′-CACATCGCAGGACGTACATACAT-3′; and for RD29B, 5′-CGAGCAAGACCCAGAAGTTCAC-3′ and 5′-TTACCCGTTACACCACCTCTCA-3′. Relative quantification of target gene expression levels was achieved using the comparative Ct (threshold cycle) method, and expression levels were normalized to that of TUB2 (At5g62690) transcripts  amplified using the primer pair 5′-AAACTCACTACCCCCAGCTTTG-3′ and 5′-CACCAGACATAGTAGCAGAAATCAAGT-3′. Three replicates were prepared in each experiment and the assays were repeated three times.Real-time quantitative polymerase chain reaction (PCR) was performed as described by Kinoshita et al.  using th2. The 14-3-3 proteins and CHLH were detected using an anti-14-3-3 protein (GF14ø) antibody and an anti-CHLH antibody, respectively  at 10 μmol m−2 s−1 was applied to RL-illuminated epidermal fragments, thus being superimposed on the background RL, for 2.5 min. The phosphorylation status of epidermal guard cell H+-ATPase was immunohistochemically evaluated using the method of Hayashi et al.  with 3% (w/v) bovine serum albumin Fraction V  and incubation proceeded at 37°C overnight. All samples were washed six times for 5 min each time with PBS and incubated with Alexa Fluor 488 goat anti-rabbit IgG  at a dilution of 1:500 in PBS with 3% (w/v) BSA at 37°C for 3 h in the dark. After washing a further six times for 5 min each time with PBS, each specimen was mounted on a glass slide using 50% (v/v) glycerol. Specimens were observed under a fluorescence microscope  using the narrow excitation band-pass filter set BP460–480HQ BA495–540HQ  to detect signal from Alexa Fluor 488. A Hg arc lamp was used as a source of excitation. Fluorescent images were collected using a CCD camera  and processed with the aid of DP2-BSW software (Olympus). To estimate fluorescence intensities, all exposure times were identical (334.79 ms). The fluorescence intensities of stomata (>30 stomata/treatment) were determined via a zone densitometry method using CS Analyzer version 3.0 (Atto).Epidermal fragments were incubated for 20 min under background red light  at 50 μmol mi et al. . EpidermStomatal apertures were measured as described by Inoue et al. . The stoPlants were grown in soil for 3 weeks under normal condition and then subjected to drought stress by withholding water for 18 days  in a manner causing the 3′-end of the CHLH coding sequence to be fused in-frame to the 5′-end of GFP. CHLH-GFP was amplified by PCR with insertion of XbaI sites upstream of the CHLH start codon and downstream of the GFP stop codon using the primer pair 5′-GCCTCTAGAAAAATGGCTTCGCTTGTGTATTCTCCAT-3′ and 5′-GCCTCTAGATTACTTGTACAGCTCGTCCATGC-3′. The PCR product was ligated into the vector pPZP211-CER6 previously constructed by our group , which was next transferred to plants using the floral dip method , which shows ABA-insensitive phenotype in stomatal closure  both before and after BL illumination. The observed changes in the phosphorylation level of H+-ATPase were not attributable to differences in the amounts of H+-ATPase between strains; the treatment used had no effect on the levels of guard cell H+-ATPase in WT or rtl1 plants. These results suggest that CHLH mediates not only the induction of ABA-induced stomatal closure but also the ABA inhibition of BL-induced phosphorylation of guard cell H+-ATPase.Previous work indicated that ABA inhibits BL-mediated stomatal opening at least in part via induction of the dephosphorylation of guard cell H Figures . In addiCHLH is reportedly associated with open-stomata and ABA-insensitive phenotypes of stomatal guard cells  of ABA, although a high level of ABA (20 μM) induced stomatal closure in both types of plants. It should be noted that the differences of stomatal aperture between wild type and CHLH-transgenic plants in Figure +, which facilitates stomatal opening, for 2.5 h under light in Figure CHLH in guard cells confers drought tolerance via promotion of stomatal closure and that this is probably attributable to enhanced sensitivity of guard cells to ABA.Next, we examined drought tolerance in e Figure . The reschlh mutant, rtl1, indicated that CHLH specifically affected ABA-induced stomatal closure , have been shown to become phosphorylated in guard cells in response to ABA, and such phosphorylation is most likely mediated by the PYR/PYL/RCARs-PP2Cs-SRK2/SnRK2s pathway (Takahashi et al., rtl1 mutant exhibited normal ABA-dependent phosphorylation of AKSs (Figure CHLH has no effect on PYR/PYL/RCARs-PP2Cs-SRK2/SnRK2s pathway-mediated ABA-dependent phosphorylation of AKSs in guard cells.A previous study using a novel y Figure . Recentls Figure . The res+-ATPase in the stomatal guard cells of A. thaliana (Hayashi et al., +-ATPase in the epidermis of a single rosette leaf, and thus eliminates the need to prepare GCPs from a large number of plants. Moreover, the method can be used to detect guard cell responses under more natural and stress-free conditions. Protoplast preparation stresses plants. We took advantage of these properties to examine the effect of ABA on BL-induced phosphorylation of guard cell H+-ATPase using ABA-insensitive mutants, including abi1-1, abi2-1, and ost1-2. The results indicated that an early ABA signaling pathway, the PYR/PYL/RCAR-PP2Cs-SRK2/SnRK2s pathway (Ma et al., +-ATPase (Hayashi and Kinoshita, +-ATPase of the rtl1 mutant and found that ABA-induced inhibition of BL-induced H+-ATPase phosphorylation was greatly suppressed in the rtl1 mutant (Figures rtl1 mutant exhibited a higher level of phosphorylation of guard cell H+-ATPase prior to BL illumination than the WT. These results suggest that CHLH affects ABA-induced inhibition of the BL-induced phosphorylation of the penultimate Thr of guard cell H+-ATPase, and that this inhibition is mediated by both the PYR/PYL/RCARs-PP2Cs-SRK2/SnRK2s pathway and CHLH. Further investigation is needed to clarify the relationship between the PYR/PYL/RCARs-PP2Cs-SRK2/SnRK2s pathway and CHLH in this context.We recently established an immunohistochemical method for detecting BL-induced and phototropin-mediated phosphorylation of plasma membrane H Figures . In addiCHLH-GFP transgenic plants in which the CER6 promoter drives the CHLH-GFP exhibited a closed-stomata phenotype under light conditions (Figures CHLH overexpression driven by the CaMV35S promoter, which induces overexpression of CHLH in whole plant, was found to cause development of an ABA-hypersensitive phenotype in terms of stomatal movement and increased resistance to dehydration (Shen et al., CHLH overexpression causes drought tolerance. In addition, it is worthy of note that expression of Vicia faba VfPIP, a putative aquaporin gene, driven by the CaMV35S promoter in A. thaliana has been shown to improve their drought tolerance at least in part through lower transpiration phenotype, although the VfPIP transgenic plants had longer primary and lateral roots, and a greater number of lateral roots than the control plants (Cui et al., CHLH in guard cells confers drought tolerance via manipulation of the stomatal aperture. Our results suggest that manipulation of the stomatal aperture via overexpression of CHLH in guard cells may prove very useful to improve drought tolerance in plants of economic importance.In the present study, we found evidence that  Figures , and dro Figures . PreviouThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In vitro kinase assays demonstrated that piceatannol significantly inhibited phosphatidylinositol 3-kinase (PI3K) activity and suppressed phosphatidylinositol (3) expression in the H-ras MCF10A cells. Ex vivo pull-down assays revealed that piceatannol directly bound to PI3K, inhibiting PI3K activity. Data from molecular docking suggested that piceatannol is a more tight-binding inhibitor than resveratrol due to the additional hydrogen bond between the hydroxyl group and the backbone amide group of Val882 in the ATP-binding pocket of PI3K.Metastasis is one of the most threatening features of the oncogenic process and the main cause of cancer-related mortality. Several studies have demonstrated that matrix metalloproteinases (MMPs) are critical for tumor invasion and metastasis. Resveratrol , a phenolic compound of red wine, has been reported to be a natural chemopreventive agent. However, the cancer preventive effects of piceatannol , a metabolite of resveratrol and the underlying molecular mechanisms have not yet been fully elucidated. In this study, we report that piceatannol inhibits H-ras-induced MMP-2 activity and the invasive phenotype of MCF10A human breast epithelial cells harboring mutated H-ras (H-ras MCF10A cells) more effectively than resveratrol. Piceatannol attenuated the H-ras-induced phosphorylation of Akt in a time- and dose-dependent manner, whereas resveratrol, at the same concentrations, did not exert an inhibitory effect.  Tumor invasion and metastasis are among the most threatening features of the oncogenic process and are the cause of 90% of human cancer-related deaths . Tumor cras signaling pathway is one of the most frequent defects in human cancers , from phosphatidylinositol (2), thereby affecting the activation of phosphoinositide-dependent kinases (PDKs) and Akt signaling  and the protein assay kit was from Bio-Rad Laboratories .Piceatannol was obtained from A.G. Scientific, Inc. . Resveratrol was purchased from Sigma-Aldrich . LY294002 and triciribine were obtained from Calbiochem . Dulbecco’s modified Eagle’s medium (DMEM)/F12, horse serum, L-glutamine and the penicillin/streptomycin/fungizone mixture were purchased from Gibco-BRL . Protein A/G sepharose beads were purchased from Santa Cruz Biotechnology, Inc. . The antibodies against phosphorylated Akt (Ser473) and total Akt were obtained from Cell Signaling Technology . The antibodies against PI3K p85 and p110 were obtained from Upstate Biotechnology . CNBr-Sepharose 4B and  for 40 min on ice followed by centrifugation at 14,000 rpm for 10 min. The protein concentration of the supernatant was determined using a dye-binding protein assay kit (Bio-Rad Laboratories) as described in the manufacturer’s manual. Lysate protein (30 μg) was subjected to 10% SDS-PAGE and electrophoretically transferred onto a polyvinylidene difluoride (PVDF) membrane . After blotting, the membrane was blocked in 5% fat-free dry milk for 1 h and then incubated with the specific primary antibody for 2 h at room temperature. Protein bands were detected by using an enhanced chemiluminescence (ECL) detection kit (Amersham) after hybridization with the HRP-conjugated secondary antibody.2, 250 μM ATP containing 10 μCi of [γ-32P]ATP] for an additional 10 min at 30°C. The reaction was terminated by the addition of 15 μl of 4 N HCl and 130 μl of chloroform/methanol (1:1). After vortexing, 30 μl of the lower chloroform phase was spotted onto a 1% potassium oxalate-coated silica gel plate, which was previously activated for 1 h at 110°C. The resulting 32P-labeled phosphatidylinositol-3-phosphate (PIP) was separated by thin layer chromatography (TLC) and radiolabeled spots were visualized by autoradiography.An active PI3K protein (80 ng) was incubated with piceatannol or LY294002 at the indicated concentrations for 10 min at 30°C. The mixtures were then incubated with 20 μl of 0.5 mg/ml phosphatidylinositol . After 5 min at room temperature, the mixtures were incubated with reaction buffer . Following incubation with gentle rocking overnight at 4°C, the beads were washed five times with buffer  and proteins bound to the beads were analyzed by immunoblotting.http://www.rcsb.org/pdb/).Insight II  was used for molecular docking and structure analysis; the crystal coordinates of PI3K in the complex with ATP (accession code 1E8X) are available on the Protein Data Bank  and catalytic subunits (p110) that are encoded by different genes ,41. Prevhibitors ,45. PI3Khibitors , we dockhibitors . The B rhibitors . The hydhibitors . Therefohibitors . In thisIn conclusion, piceatannol inhibits H-ras-induced MMP-2 expression, invasion, migration and anchorage-independent growth, whereas resveratrol has relatively minor inhibitory effects in MCF10A human breast epithelial cells. These inhibitory effects were mediated mainly through the blocking of the PI3K/Akt pathway. Piceatannol markedly suppressed PI3K activity by directly binding to PI3K. Our computer modeling data suggest that piceatannol is a more tight-binding inhibitor than resveratrol due to the additional hydrogen bond between the hydroxyl group and the backbone amide group of Val882 in the ATP binding pocket of PI3K.


However, the molecular targets and mechanisms of luteolin are still poorly understood. In this study, we sought to investigate the effects of luteolin on UVB-induced photoageing and the molecular mechanisms involved, using HaCaT human keratinocytes and SKH-1 hairless mice. Luteolin was found to inhibit UVB-induced MMP-1 expression in HaCaT cells, as well as UVB-induced activation of AP-1, a well-known transcription factor targeting the MMP-1 promoter region, as well as c-Fos and c-Jun, which comprise the AP-1 complex. In contrast, Western blot data showed that UVB-induced phosphorylation of JNK, ERK and p90RSK was not inhibited by luteolin. In vitro kinase assay data revealed that luteolin significantly suppressed JNK1 and p90RSK activity, but not that of JNK2 and ERK2. Pull-down assays showed that luteolin binds JNK1 in an ATP-competitive manner and p90RSK2 in an ATP-independent manner. Luteolin also inhibited UVB-induced wrinkle formation and MMP-13 expression, a rodent interstitial collagenase in mouse skin, in vivo. Taken together, our observations suggest that luteolin exhibits anti-photoageing effects in vitro and in vivo and may have potential as a treatment for the prevention of skin ageing.Multiple lines of evidence suggest that natural compounds can prevent skin ageing induced by ultraviolet light. Luteolin, a bioactive compound found in chilli, onion, broccoli, celery and carrot, has been reported to exhibit anti-photoageing effects  Skin, is the most exposed organ of the body and in direct contact with sources of environmental stress including ultraviolet light (UV) The matrix metalloproteinases (MMPs) are a family of enzymes that can degrade components of the extracellular matrix (ECM). MMPs play important roles in morphogenesis, angiogenesis, arthritis, tumour invasion and photoageing Previous studies have uncovered some of the molecular mechanisms underlying UV-induced MMP expression. UV activates growth factor receptors on the cell surface, including EGFR, followed by receptor-coupled signal transduction through pathways involving mitogen-activated protein kinases (MAPKs), Jak/STAT, protein kinase-C and PI3kinase/Akt in vitro and in vivo, using HaCaT human keratinocytes and a mouse model of skin ageing.Luteolin  is a flavonoid found in diverse fruits and vegetables such as chilli, onion, broccoli, celery and carrot A 21. It Luteolin (98%) was purchased from Indofine Chemical . Anti-human MMP-1 and antimouse MMP-13 antibodies were purchased from Epitomics  and Chemicon  respectively. Antibodies to detect phosphorylated JNK (Thr183/Tyr185), total JNK, phosphorylated p90RSK (Thr359/Ser363), total p90RSK and phosphorylated c-Jun (Ser63) were purchased from Cell Signaling Biotechnology . Antibodies against phosphorylated ERK1/2 (Thr202/Tyr204), total ERK, total c-Jun were obtained from Santa Cruz Biotechnology . Recombinant active JNK1 and JNK2 proteins were obtained from Upstate Biotechnology  and recombinant active RSK2 was obtained from Signalchem . CNBr-Sepharose 4B, [γ-32P]ATP and the chemiluminescence detection kit were purchased from GE Healthcare , and the protein assay kit was obtained from Bio-Rad Laboratories . G418 and the luciferase assay substrate were purchased from Promega .2 incubator in 10% FBS–DMEM with 2 mM l-glutamine and 25 μg/ml gentamicin. The HaCaT human keratinocyte cell line was stably transfected with the AP-1 and c-Fos luciferase reporter plasmid and maintained in 10% FBS–DMEM containing 200 μg/ml G418 The HaCaT human keratinocyte cell line was cultured in monolayers at 37°C in a 5% COSKH-1 hairless mice  were purchased from the Institute of Laboratory Animal Resources at Seoul National University . Animals were acclimated for 1 week prior to the study and had free access to food and water. The animals were housed in climate-controlled quarters (24°C at 50% humidity) with a 12-hrs light/12-hrs dark cycle.3 viable cells were suspended in 100 μl of 10% FBS/DMEM and added to each well of a 96-well plate. Plates were incubated at 37°C in a 5% CO2 incubator. When cells reached 80–90% confluence, they were starved by culturing in serum-free FBS–DMEM for another 24 hrs. The cells were treated for 1 hr with luteolin (5 and 10 μM) before exposure to UVB (0.01 J/cm2), and then incubated for 5 or 3 hrs respectively. The cells were disrupted with 100 μl lysis buffer  and luciferase activity was measured using a luminometer .Confluent monolayers of HaCaT cells stably transfected with the AP-1 or c-Fos luciferase plasmid were harvested, and 8 × 10Western blotting was performed as described previously in vivo Western blotting, mice received topical application of luteolin (10 and 40 nmol) dissolved in 200 μl acetone on their backs 1 hr before UVB (0.18 J/cm2) irradiation. To isolate protein from mouse skin, the treated dorsal skin region of each mouse was excised and placed on ice. Fat was removed and the skin was placed in liquid nitrogen and immediately pulverized with a mortar and pestle. The pulverized skin was blended on ice with a homogenizer , and skin lysates were centrifuged at 16,128 × g for 20 min. After the protein content was determined using a Bio-Rad protein assay kit, 100 μg of mouse skin lysate was subjected to 10% SDS-PAGE and transferred to a PVDF membrane (Amersham Pharmacia Biotech). Membranes were processed and proteins were analysed as described for the in vitro Western blot assay.For in vitro JNK1 and JNK2 kinase assays were performed in accordance with the instructions provided by Millipore . About 3 mM of the ATF2 substrate peptide was included. A 2.5 μl aliquot was removed from the reaction mixture containing 2.5 μl of each substrate and 10 μl diluted [γ-32P]ATP solution, and incubated at 30°C for 10 min. Fifteen microlitres aliquots were then transferred onto p81 paper and washed three times with 0.75% phosphoric acid for 5 min. per wash and once with acetone for 5 min. For RSK2, every reaction solution contained 25 μl of assay reaction buffer and a magnesium-ATP cocktail buffer. A 2.5 μl aliquot was removed from the reaction mixture containing 2.5 μl of each substrate and 10 μl diluted [γ-32P]ATP solution, and incubated at 30°C for 10 min.; then 15 μl aliquots were transferred onto p81 paper and washed three times with 0.75% phosphoric acid for 5 min. per wash and once with acetone for 5 min. The in vitro ERK2 kinase assays were performed in accordance with the instructions provided by Upstate Biotechnology. A total of 0.33 mg/ml of myelin basic protein substrate peptide was included. Four microlitres aliquots were removed after incubating the reaction mixture at 30°C for 15 min., to which 10 μl of diluted [γ-32P]ATP solution was added. This mixture was incubated for 10 min. at 30°C, and then 25 μl aliquots were transferred onto p81 paper and washed three times. The radioactive incorporation was determined using a scintillation counter. Each experiment was performed three times.The 3 (pH 8.3) and 0.5 M NaCl] was mixed. The mixture was rotated at 4°C overnight. The medium was transferred to 0.1 M Tris–HCl buffer (pH 8.0) and rotated end over end at 4°C overnight. The medium was washed three times with 0.1 M acetate buffer (pH 4.0) containing 0.5 M NaCl followed by a wash with 0.1 M Tris–HCl (pH 8.0) containing 0.5 M NaCl.Luteolin–Sepharose 4B freeze-dried powder (0.3 g) was suspended in 1 mM HCl and coupled solution . After incubation with gentle rocking overnight at 4°C, the beads were washed five times with buffer , and proteins bound to the beads were analysed by Western blotting2) irradiation. The mice in the third and fourth groups received topical application of luteolin (10 or 40 nmol) in 200 μl acetone 1 hr before UVB (0.18 J/cm2). These treatments were performed three times per week for 15 weeks.The animal experimental protocol (SNU-060512-1) was approved and experimental animals were maintained under specific pathogen-free conditions based on the guidelines established by the Animal Care and Use Committee of Seoul National University. Skin photoageing was induced in mice utilizing a UVB irradiation system. The UVB radiation source  emitted at wavelengths of 254, 312 and 365 nm, with peak emission at 312 nm. SKH-1 mice were divided into four groups of three animals each. In the control group, the dorsal skin of the mice was topically treated with 200 μl acetone alone. In the UVB group, the dorsal skin of the mice was topically treated with 200 μl acetone 1 hr before UVB . MMP-1 levels in the culture media collected after the indicated durations were determined by Western blotting. UVB radiation induced MMP-1 expression after 48 hrs  and In addition to c-Fos, activation of the AP-1 complex is dependent on phosphorylation of c-Jun, and we investigated whether luteolin has an effect on UVB-induced c-Jun phosphorylation. Pretreatment with luteolin significantly inhibited UVB-induced phosphorylation of c-Jun in HaCaT cells A. HoweveIn vitro kinase assay data shows that JNK1 activity was significantly suppressed by luteolin, but there was no significant change in JNK2 activity . We further investigated the binding activity of luteolin with JNK1 in the presence of ATP. The binding activity of luteolin with JNK1 was reduced by ATP in a dose-dependent manner, suggesting that luteolin competes with ATP to bind JNK1 (lower panel). In vitro and ex vivo pull-down assays indicated that p90RSK2 binds luteolin–Sepharose 4B beads (upper and middle panels) and the binding activity of luteolin with p90RSK2 was not affected by the presence of ATP (lower panel), suggesting that luteolin suppresses p90RSK activity in an ATP-independent manner.in vivo, we examined the outcome of treatment using a well-developed SKH-1 hairless mouse model 2) 1 hr after luteolin treatment, three times per week for 15 weeks. Representative photographs of the mice at the conclusion of the treatment duration show that luteolin inhibits UVB-induced wrinkle formation  in vitro kinase assay results indicate that luteolin significantly inhibits JNK1 and p90RSK2 activity, but not that of JNK2 and ERK2. Furthermore, pull-down assays indicated that luteolin bound directly to JNK1 (in an ATP-competitive manner), but not JNK2 . p38 has also been reported to regulate UVB-induced c-Fos gene expression in HaCaT cells AP-1 is a well-known transcription factor that binds to the TPA-response element (TRE) with the consensus sequence TGACTCA and activates the transcription of target genes including MMP-1, MMP-3 and MMP-9 et al., have reported that JNK2 is a negative regulator of c-Jun et al., contend that JNK2 is a positive regulator of c-Jun The c-Jun N-terminal kinases (JNKs) are members of the MAPK family and are heavily involved in apoptosis, cell survival and proliferation in vitro and in vivo. Our observations indicate that JNK1 and p90RSK2 are novel molecular targets of luteolin for the inhibition of UVB-induced signal transduction, leading to MMP-1 up-regulation (In summary, we have evaluated the preventive effects of luteolin on UVB-induced photoageing. Luteolin potently inhibited UVB-induced MMP-1 expression and wrinkle formation gulation . We ther


Inhibition of the proteasome is a widely used strategy for treating multiple myeloma that takes advantage of the heavy secretory load that multiple myeloma cells (MMCs) have to deal with. Resistance of MMCs to proteasome inhibition has been linked to incomplete disruption of proteasomal endoplasmic-reticulum (ER)-associated degradation (ERAD) and activation of non-proteasomal protein degradation pathways. The ATPase p97 (VCP/Cdc48) has key roles in mediating both ERAD and non-proteasomal protein degradation and can be targeted pharmacologically by small molecule inhibition. In this study, we compared the effects of p97 inhibition with Eeyarestatin 1 and DBeQ on the secretory apparatus of MMCs with the effects induced by the proteasome inhibitor bortezomib, and the effects caused by combined inhibition of p97 and the proteasome. We found that p97 inhibition elicits cellular responses that are different from those induced by proteasome inhibition, and that the responses differ considerably between MMC lines. Moreover, we found that dual inhibition of both p97 and the proteasome terminally disrupts ER configuration and intracellular protein metabolism in MMCs. Dual inhibition of p97 and the proteasome induced high levels of apoptosis in all of the MMC lines that we analysed, including bortezomib-adapted AMO-1 cells, and was also effective in killing primary MMCs. Only minor toxicity was observed in untransformed and non-secretory cells. Our observations highlight non-redundant roles of p97 and the proteasome in maintaining secretory homeostasis in MMCs and provide a preclinical conceptual framework for dual targeting of p97 and the proteasome as a potential new therapeutic strategy in multiple myeloma. Multiple myeloma (MM) is a tumour of transformed plasma cells, terminally differentiated B cells that are highly specialised to synthesise and secrete large amounts of immunoglobulins (Ig) . The proPlasma cells and MM cells (MMCs) have a well-developed secretory apparatus to accommodate Ig secretion but are nevertheless highly sensitive to agents that interfere with intracellular protein metabolism -7. This N2,N4-dibenzylquinazoline-2,4-diamine (DBeQ), a reversible inhibitor of the p97 ATPase activity, was found to be a potent activator of caspases in cancer cells and to inhibit both ERAD and autophagosome maturation [Prior to proteasomal degradation, misfolded proteins are exported from the ER lumen to the cytosolic side of the ER, where they are earmarked for degradation by poly-ubiquitination . This acturation ,27. Furtturation ,31. Thusturation ,33.The essential roles of p97 and the proteasome in regulating protein degradation, which are closely linked in ERAD but also show a considerable degree of non-redundancy, open up the possibility that simultaneous inhibition of p97 and the proteasome could be highly efficient in disrupting secretory homeostasis in MMCs. Here we demonstrate that inhibitors of either p97 or the proteasome have quite different effects on the secretory apparatus in MMCs, and that these effects vary substantially between different MMC lines, and between MMCs and non-secretory or non-transformed cells. Moreover, we show that dual inhibition of p97 and the proteasome has dramatic effects on ER morphology and protein turnover that are selective for MMCs and are very different from the effects of single inhibition with either drug. Our results provide a conceptual preclinical framework for simultaneous targeting of p97 and the proteasome as a potential new therapeutic strategy in multiple myeloma.Bone marrow myeloma cells, bone marrow mesenchymal stroma cells, and skin fibroblasts were collected at the Imperial College London Centre for Haematology after approval by the National Research Ethics Service Committee East of England – Cambridge Central and obtaining signed informed consent in accordance with the Declaration of Helsinki.The human MMC lines OPM-2, RPMI 8226, U-266, KMS-11, and parental/bortezomib-adapted AMO-1 have been described previously ,15. PrimApoptosis was assessed by staining with Annexin V-FITC and propidium iodide  as previously described . Cells nIPK, GRP94, eIF2α, phospho-eIF2α (Ser51), S6 ribosomal protein, phospho-S6 ribosomal protein (Ser235/236), β-tubulin , and ubiquitin .Whole cell lysates were prepared as previously described . PrimaryCells were incubated with 100nM BODIPY FL brefeldin A  at 37°C for 40 minutes in the dark, washed and resuspended in phosphate-buffered saline (PBS), and analysed immediatedy on a BD LSR II flow cytometer. For morphological ER analysis, cells were incubated in medium with 1µM ER Tracker Blue-White DPX (Molecular Probes) at 37°C for 15 minutes, pelleted, and resuspended in 50µl medium. The cell suspension was applied to an imaging chamber , and ima7 cells were fixed, washed, post-fixed and embedded as previously described [1x10escribed . UltrathWe first determined the apoptotic sensitivity of a panel of human MMC lines to bortezomib and the p97 inhibitor Eer1 . The resIncreased intracellular levels of ubiquitinated proteins are considered to indicate impaired protein degradation by the proteasomal machinery ,15,41. TInhibitors of the proteasome and of p97 cause ER stress in MM and other tumour cells, and ER stress is known to induce changes in ER structure ,26,42-44To further investigate the changes in ER configuration we used transmission electron microscopy (TEM) of OPM-2 cells , which sIPK (DNAJC3) and GRP94, after proteasome inhibition, and a decrease after dual ERAD inhibition, while Eer1 had no detectable effect on the levels of either protein. Together, these observations indicate that bortezomib increases ER content in MMCs, whereas inhibition of p97 with Eer1 causes moderate alterations to ER structure. In contrast, dual p97/proteasome inhibition drastically alters the configuration of the ER.We next investigated how dual p97/proteasome inhibition affects cellular ER content by staining OPM-2 cells with the fluorescent brefeldin A derivative, BFA-BODIPY , and immNext, we investigated the impact of single  and dual p97/proteasome inhibition on key signalling pathways in human MMC lines, and compared the effects with those induced in untransformed primary human fibroblasts . ConsistActivation of the Akt pathway, as indicated by phosphorylation of Akt, is an important survival signal that has been shown to occur in a proportion of MMCs . In OPM-The observed changes in ER configuration  and Akt In the light of the activation of caspase-3 that we observe and the previously described role of JNK in bortezomib-induced cell death , we nextDepletion of p97 by conditional gene knockout or RNAi knockdown has limited utility for predicting the effects of pharmacological p97 inhibition due to slow and incomplete impact on p97 function . To inveThe key finding of this paper is that all of the MMCs that we have tested show dramatic changes to the ER in response to dual inhibition of p97 and the proteasome, and that this is accompanied by disruption of intracellular protein metabolism and cell death. Importantly, the toxic effects of dual p97 and proteasome inhibition are also observed for cell lines that are insensitive to bortezomib. The effects of dual inhibition are quite different from the effects of single inhibition of p97 or the proteasome, adding significant information on the non-redundant roles of p97 and the proteasome in maintaining the integrity of the secretory apparatus.Dual p97/proteasome inhibition causes changes in key protein translation pathways in MMCs that are consistent with a terminal translational shut-down. This interpretation is based on the observation of proteolytic processing of eIF2α, and loss of both phosphorylated and total S6P and Akt -50. It iER staining and electron microscopy showed that proteasome inhibition had a very mild impact on the appearance of the ER in MMCs, while p97 inhibitors caused clear alterations to ER structure. The effect of p97 inhibition on ER structure could reflect a hitherto unrecognised role for p97 in the metabolism of protein components of the ER membrane or of proteins that play a role in maintaining ER integrity. Such functions are suggested by the requirement of a p97/p37 complex for Golgi and ER maintenance in interphase . The draAnother important finding of this study is that the deleterious effects of dual inhibition of p97 and the proteasome occur preferentially in MMCs. In fibroblasts, dual p97/proteasome inhibition failed to induce activation of caspase-3 and did not affect levels of phosphorylated or overall Akt and S6P. Moreover, dual inhibition did not induce proteolytic processing of eIF2α in fibroblasts but increased its phosphorylation, indicating PERK-dependent adaptive UPR signalling. Furthermore, dual p97/proteasome inhibition was only moderately toxic to primary PBMNCs. Taken together, these findings indicate that dual p97/proteasome inhibition shows a considerable degree of selectivity towards MMCs.in vivo analysis will be required to elucidate potential toxic and anti-myeloma effects of pharmacological p97 inhibition, alone and in combination with bortezomib. Systemic in vivo studies are currently not possible due to the limited solubility of Eer1 and DBeQ. However, the findings presented here form a conceptual preclinical framework for combined targeting of p97 and the proteasome as a potential novel therapeutic approach in MM and provide a strong incentive for developing p97 inhibitors that can be administered systemically [It is also noteworthy that the toxic effects of dual p97/proteasome inhibition on MMCs were achieved at bortezomib concentrations similar to steady-state plasma concentrations maintained in patients after the early peak . This isemically .In contrast to the clinical effectiveness of proteasome inhibitors in MM, they have shown disappointing results in most other haematological malignancies and solid cancers. The potent activity of p97 inhibitors in various cancer cells suggests that p97 might be a more broadly useful target for antineoplastic therapy than the proteasome ,26,55. OFigure S1Effect of single and dual p97/proteasome inhibition on ER structure in MMC lines.OPM-2, U-266, and KMS-11 cells were stained with ER Tracker Blue-White DPX following treatment for 24h (14h for KMS-11) with BTZ (5nM), Eer1 (5µM), or both. Representative confocal microscopic images are shown.(TIF)Click here for additional data file.Figure S2ER vacuolisation caused by dual ERAD inhibition is not caused by overwhelming ER stress or apoptotic signalling.OPM-2 cells were stained with ER Tracker Blue-White DPX following treatment for 24h with high doses of BTZ, Eer1, and tunicamycin, or BTZ+Eer1 after pretreatment with the pan-caspase inhibitor zVADfmk.(TIF)Click here for additional data file.Figure S3Representative transmission electron miscrocopic images of OPM-2 cells treated with Bortezomib (5nM) and Eer1 (5µM) for 24h.(TIF)Click here for additional data file.


FSHR gene revealed a heterozygous novel missense mutation c. 1536G>A resulting in an amino acid substitution M512I. We asked whether this mutant FSHR affected FSHR-mediated signaling pathways involving cAMP/protein kinase A (PKA), phosphatidylinositol-3 kinase (PI3K)/protein kinase B (AKT) and v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog kinase (SRC)/ p42/p44 extracellular signal-regulated protein kinases (ERK1/2). Thus, 293T cells expressing wild-type (FSHRwt), the mutant FSHR (FSHRmt), or both (FSHRwt/mt) were treated with FSH and subjected to measurements of intracellular cAMP, cAMP-induced CRE (cAMP response element)-mediated luciferase assays and immunoblot analyses of phosphorylated PI3K and ERK1/2. There were no differences in luciferase activities or phosphorylation levels of ERK1/2 among FSHRwt, FSHRmt cells and FSHwt/mt cells. However, FSHRmt cells showed a significant reduction in both cAMP production and PI3K phosphorylation levels with unchanged phosphorylation of ERK1/2 upon FSH stimulation in comparison to FSHwt cells. Also, FSH treatment did not provoke PI3K phosphorylation in FSHwt/mt cells. These results indicate that the novel missense M512I FSHR mutation identified herein did not participate in hyperactivation of FSHR-mediated signaling pathways but rather in hypoactivation of the FSH-mediated PI3K/AKT pathway. Thus, this study demonstrates a new functional property of this novel mutatnt FSHR, which, however, might not be involved in the pathogenesis of sOHSS in this FSHoma patient.Spontaneous ovarian hyperstimulation syndrome (sOHSS) is a rare event that may result from a FSH-producing pituitary adenoma (FSHoma), activating mutations of the FSH receptor (FSHR), and cross-reactivity of the FSHR to elevated hCG and TSH in the setting of pregnancy or hypothyroidism. The objective of this study was to investigate whether an aberrant FSHR was present in a woman with sOHSS and a non-surgically diagnosed FSHoma whose serum FSH levels and FSH bioactivity were nearly normal. Sequencing of the patient’s  FSHR , leading to the phosphorylation of key substrates, one of which is CREB (cAMP-regulatory element binding protein) ,37.FSHR mutation was first identified in a patient with sOHSS in 2003 ,7,39FSHRin vitro, in particular, in the presence of the patient’s serum. The wild-type and mutant FSHR, however, exhibited very similar cAMP/PKA activation in response to the FSH contained in the patient’s serum.In addition to this patient, her mother and two sisters had the same heterozygous mutation but had no symptoms of sOHSS. These findings make it unlikely that this mutation is causative for sOHSS. FSHoma is an extremely rare disease , and it The series of negative results prompted us to perform molecular analysis of the FSH/FSHR-mediated signaling pathways other than FSH/FSHR/cAMP/PKA/CREB pathway. First, we focused on intracellular cAMP production, one of the most upstream events of FSH/FSHR signaling pathways. We found that cAMP levels were not increased but were in fact decreased in FSHRmt cells when treated with FSH . This reWe then investigated the FSH/FSHR/PI3K/AKT pathway and found that mutant FSHR inhibited the phosphorylation of PI3K. It has been reported that cAMP activates PKA, which, in turn, induces the phosphorylation of PI3K through GAP2 inactivation in granulosa cells . Given tThe other conflicting result is that CRE-mediated luciferase activity was increased without an elevation of cAMP production upon FSH treatment of FSHwt/mt cells. Genua et al. reported an alternative pathway in which ERK1/2 phosphorylated CREB in a cAMP-independent manner and thereby activated CRE-mediated gene transcription . Given tIn summary, we obtained novel findings regarding the missense M512I FSHR mutation that is newly identified here. This mutant appears to be inactive in that it inhibited FSH-induced cAMP production and PI3K activation. This finding, however, does not support but rather contradicts our initial hypothesis that the mutant FSHR might be an active mutant and, therefore, might hyper-stimulate ovaries in response to the nearly normal or slightly elevated levels of FSH produced from FSHoma in this patient. Although the mechanism underlying OHSS observed in this patient remains to be clarified, we have for the first time demonstrated the presence of a novel mutation of FSHR together with its new properties as an inactive mutant.


KH-CB19, a potent and highly specific inhibitor of the CDC2-like kinase isoforms 1 and 4 (CLK1/CLK4). Cocrystal structures of KH-CB19 with CLK1 and CLK3 revealed a non-ATP mimetic binding mode, conformational changes in helix αC and the phosphate binding loop and halogen bonding to the kinase hinge region. KH-CB19 effectively suppressed phosphorylation of SR (serine/arginine) proteins in cells, consistent with its expected mechanism of action. Chemical inhibition of CLK1/CLK4 generated a unique pattern of splicing factor dephosphorylation and had at low nM concentration a profound effect on splicing of the two tissue factor isoforms flTF (full-length TF) and asHTF .There is a growing recognition of the importance of protein kinases in the control of alternative splicing. To define the underlying regulatory mechanisms, highly selective inhibitors are needed. Here, we report the discovery and characterization of the dichloroindolyl enaminonitrile  ► We report a highly selective nanomolar inhibitor KH-CB19 for the kinases CLK1/4 and DYRK1 ► KH-CB19 cocrystal structures revealed an ATP competitive but not ATP mimetic binding mode ► KH-CB19 formed halogen bonds with the kinase hinge region ► KH-CB19 led to dephosphorylation of SR proteins and effected splicing of TF isoforms in cells There are about 23,000 protein-coding genes in the human genome. However, the human proteome consists of a far larger number of unique protein sequences. In fact, some 90% of all transcribed genes may undergo alternative splicing and more than 80% may have at least 15% abundance of minor splicing forms . In manyThe regulation of alternative splicing is highly complex. In addition to the essential enzymatic step of RNA breakage and ligation, the spliceosome must recognize the exon and intron boundaries precisely and in a controlled fashion. Not surprisingly, the splicing machinery involves hundreds of auxiliary factors that control splice site selection, spliceosome assembly and the splice reaction . Indeed,TG003) with 20 nM and 15 nM potency for CLK1 and CLK4, respectively. However, more comprehensive profiling of this compound revealed strong inhibition of TG003 for all CLK family members except for CLK3 but also cross reactivity with casein kinase (CK1δ and CK1ɛ), dual-specificity tyrosine phosphorylation-regulated kinase (DYRK1B), Yeast Sps1/Ste20-related kinase (YSK4) and proviral insertion site in Moloney Murine Leukemia Virus (PIM) kinase isoforms , with high specificity for CLK1/CLK4 isoforms and a unique binding mode to the kinase hinge region. The lead compound shows single-digit nanomolar activity in modulating alternative splicing in human endothelial cells.Dichothrix baueriana and has been reported to have antiproliferative as well as antiviral properties (3), an easy-to-functionalize derivative of the alkaloid bauerine C, as a basis for structural diversification  inhibitors, which are structural hybrids between a common kinase inhibitor scaffold and the β-carboline alkaloid bauerine C A, havingoperties . In thisfication B.3) we started from ethyl 3-cyanomethyl-6,7-dichloro-1-methyl-1H-indole-2-carboxylate (1) (tert-butoxy-bis(dimethylamino)methane) to give the tertiary enaminonitrile 2 as a mixture of E/Z isomers. This intermediate was then heated with ammonium acetate and glacial acetic acid in a microwave reactor to give 4-cyano-bauerine C (3). Both 3 and the intermediate tertiary enaminonitrile 2 were screened against a panel of 106 kinases using a thermal shift assay and showed only interaction with CLK family members (see KH-CB20 (as an E/Z mixture) was originally isolated as a side product in the synthesis of 3 and also screened against the kinase panel. Serendipitously, the kinase assay revealed KH-CB20 to be a potent and selective inhibitor of CLK1 and the closely related isoform CLK4, with significantly reduced affinity to CLK2 and CLK3 (KH-CB20 was optimized. Addition of sulfuric acid to the reaction mixture and shorter reaction time largely prevented cyclisation to 4-cyano-bauerine C (3) and led to predominant formation of the primary enaminonitrile KH-CB20 as a 71:29 mixture of E- and Z-isomers. Separation of the E-isomer KH-CB19 could be achieved by selective recrystallization from toluene. The pure E-isomer KH-CB19 and the E/Z-mixture KH-CB20 had similar kinase binding activity (For the preparation of 4-cyano-bauerine C (late (1) , which wand CLK3 . Thus thactivity .KH-CB20 and KH-CB19 showed potent inhibition of CLK1 with an IC50 of 20 nM, and for the pure isomer KH-CB19, almost 100-fold selectivity against the CLK3 isoform -1H-1,2,4-triazole-1-carbothiamide), which has been published as a potent CDK1 and CDK2 inhibitor  of carbon-bound chlorine and sp2-hybridized oxygen. The linear C-Cl⋯O geometry also fulfilled the geometrical criteria for a chlorine halogen bond to the kinase backbone  cocrystal structure . Pretreatment of HMEC-1 with 10 μM TG003, a previously identified CLK inhibitor, only reduced the phosphorylation of SRp20, but had no effect on the phosphorylation state of other SR proteins under normal conditions (lane 3). Stimulation of HMEC-1 with TNF-α led to an increase in the phosphorylation of all detected SR proteins only 2 min post induction (lane 4). Pretreatment of cells with KH-CB19 or TG003 led to a reduction of the TNF-α-induced increase in phosphorylation of all analyzed SR proteins (lanes 5 and 6) compared with TNF-α-stimulated controls (lane 3). However, the effect of 10 μM KH-CB19 was far greater under both normal and proinflammatory conditions as compared to cells treated with 10 μM TG003. Dose response of KH-CB19 was tested using SRp75 and SRp55. As shown in TG003 had little effect on SRp75 and SRp55 phosphorylation at the tested concentrations.To assess the phosphorylation state of SR proteins, western blotting was performed 2 min poststimulation of human microvascular endothelial cells (HMEC-1) with TNF-α A. SRp75,KH-CB19 significantly reduced the basal expression of flTF as well as asHTF (lane 4). Pharmacologic inhibition of CLKs using KH-CB19 also lowered TNF-α-induced expression of both TF mRNA splice variants to baseline (lane 5). Treatment of HMEC-1 cells with 10 μM TG003 also reduced the basal expression of flTF and asHTF mRNA (lane 6), and showed only slightly reduced mRNA expression in TNF-α-induced cells 1 hr post stimulation (lane 7).Unstimulated HMEC-1 constitutively express both tissue factor (TF) isoforms, the soluble asHTF as well as the membrane bound full-length TF (flTF) at the mRNA level B, lane 2KH-CB19 when compared with typical ATP mimetic ligands.Despite the substantial effort in developing targeted kinase inhibitors, the task of selective inhibitor design remains highly challenging . As a reKH-CB19 binding mode is the presence of a halogen bond formed with one CLK hinge region backbone carbonyl. Halogen bonds are short-range molecular interactions involving polarized halogens. Such contacts occur frequently in inhibitor target complexes but have only recently been recognized as intermolecular interactions that may favorably contribute to ligand affinity  splicing factors in cells and significantly altered splicing of the two tissue factor isoforms flTF (full-length TF) and asHTF . The discovered inhibitor class is therefore a useful model and an excellent probe compound for the development of inhibitors that target protein splicing. Furthermore, the described binding mode of the discovered dichloroindolyl enaminonitrile inhibitors may serve as a template for the development of selective inhibitors for other kinase targets that explore non-ATP mimetic interactions with the kinase active site and halogen bonding with the hinge backbone.CLK1 and CLK3 were prepared as described . In brieThermal denaturation experiments were carried out in an Mx3005p real-time PCR machine (Agilent) using a protein concentration of 2 μM and an inhibitor concentration of 10 μM. Samples were buffered in 10 mM HEPES (pH 7.5), 500 mM NaCl and a 1:1000 dilution of SyproOrange (Invitrogen). The assay and data evaluation were carried out as described K00546 is CDK1/2 inhibitor III purchased from Merck Biosciences (cat. # 217714). TG003 was purchased from Merck Biosciences (cat. #219479).Phosphorylation reactions were monitored using a coupled-enzyme assay in which ADP production is coupled to NADH oxidation by pyruvate kinase (PK) and lactate dehydrogenase (LDH) as described . The rea3VO4, 10 mM beta-glycerophosphate) and 4.5 μl enzyme solution in assay buffer. The peptide concentration was 2 μM. Concentrations for the enzyme, as well as for MgCl2 and MnCl2 were adjusted specifically to the requirements of the individual enzyme. ATP concentrations were adjusted to the KM values of the specific enzyme. After incubation for 60 min at 30°C the kinase reactions were stopped by addition of 16 μl stop solution . Stopped kinase reactions were analyzed in a LC3000 reader . Compounds were tested from 0.1 nM to 10 μM in eight steps.Enzymatic kinase selectivity screening was carried out using the Caliper mobility shift assay which is based on the difference in capillary electrophoresis mobility of a fluorescent tagged peptide as a result of the addition of a phosphate moiety by the studied kinase. The kinase reactions were started by addition of 4.5 μl substrate mix consisting of ATP and peptide substrate in assay buffer  were grown by mixing 100 nl protein (8.0 mg/ml) with 50 nl reservoir solution containing 2.1 M sodium malate pH 7.0. CLK3 crystals with K00546 or KH-CB19 were grown by mixing 75 nl of protein  with an equal volume of reservoir solution containing 0.2 M (NH4)2SO4, 0.1 M Bis-Tris (pH 5.5), and 25% PEG3350.Aliquots of the purified proteins were set up for crystallization using a Mosquito crystallization robot . Coarse screens were prepared in Greiner 3-well plates using three different drop ratios of precipitant to protein per condition . Initial hits were optimized using Greiner 1-well plates with an increase in the drop size. All crystallizations were carried out using the sitting drop vapor diffusion method at 4°C. CLK1 crystals with KH-CB19), at the Diamond Light Source on beamline I02 using a ADSC Q315 detector at 0.9050 Å (CLK3/K00546), or in-house on a Bruker system equipped with a Microstar generator and a Pt135 detector at 1.54 Å. Indexing and integration was carried out using MOSFLM  or XPREP (K01762) and scaling was performed with SCALA  were recorded using a Hewlett Packard 5989 A Mass Spectrometer with a 59980 B Particle Beam LC/MS-interface . High-resolution mass spectra were obtained using a Jeol Mstation 700. Melting points were determined with a Büchi B-540 apparatus  and are uncorrected. Microwave reactor: CEM Discover . Purification by flash column chromatography (FCC) was done using Silica gel 60 . All solvents and chemicals were purchased from Sigma-Aldrich, Fluka, and Acros.NMR spectra were recorded using a Jeol JNMR-GSX 400 and Jeol JNMR-GSX 500 . tert-butoxy-bis(dimethylamino)methane) were added to a solution of 2.00 g (6.43 mmol) ethyl 3-(cyanomethyl)-6,7-dichloro-1-methyl-1H-indole-2-carboxylate (1)  δ 7.60 , 7.47 , 7.27 , 7.24 , 6.93 , 6.57 , 4.39 , 4.35 , 4.28 , 3.21 2), 1.42 ; 13C NMR  δ 162.0 , 161.7 , 153.6 , 151.4 , 135.2 , 134.9 , 131.1 , 130.8 , 130.5 , 129.6 , 129.4 , 128.4 , 123.7 , 123.4 , 122.9 , 121.5 , 120.9 , 120.6 , 119.2 , 116.6 , 116.5 , 115.7 , 68.2 , 65.5 , 61.9 , 61.8 , 42.5 (2′-N(CH3)2), 35.6 (1-CH3), 14.4 (CH2CH3); E/Z ratio (%) 18:82; MS EI m/z  369 [M•+] (10), 367 [M•+] (68), 365 [M•+] (100), 292 (27).Under nitrogen, 2.00 g (11.57 mmol) Bredereck's reagent (late (1)  in 10 mlE/Z)-ethyl 6,7-dichloro-3-[1-cyano-2-(dimethylamino)vinyl]-1-methyl-1H-indole-2-carboxylate (2), 8.0 g ammonium acetate and 2 ml glacial acetic acid were irradiated in a microwave reactor at 112°C and 150 W power for 45 min. The mixture was poured into ice-water and the precipitate filtered off. The crude product was resuspended in toluene and the solvent removed to give 200 mg (28%) as a beige solid. Mp 410°C (decomp); 1H NMR  δ 11.97 , 8.16 , 8.01 , 7.52 , 4.61 ; 13C NMR  δ 155.6 (C=O), 136.6 (C-3), 136.6 (C-8a), 131.7 (C-7), 127.4 (C-9a), 122.9 (C-6), 121.2 (C-4b), 120.1 (C-4a), 120.0 (C-6), 116.7 (CN), 115.9 (C-8), 84.0 (C-4), 34.3 (N-CH3); MS EI m/z  295 [M•+] (10), 293 [M•+] (68), 291 [M•+] (100).890 mg (2.43 mmol) (E/Z)-ethyl 6,7-dichloro-3-[1-cyano-2-(dimethylamino)vinyl]-1-methyl-1H-indole-2-carboxylate (2), 4.0 g (52 mmol) ammonium acetate, 5 ml glacial acetic acid and 2 ml conc. sulfuric acid were irradiated in a microwave reactor at 112°C and 150 W power for 15 min. After cooling, the mixture was poured into 100 ml cold aqueous ammonia followed by extraction with ethyl acetate (3 × 50 ml). The organic layer was dried over MgSO4 and the solvent evaporated. The crude product was purified by FSC (methylene chloride/ethanol 20:1), followed by recrystallization from ethanol to give 260 mg (72%) as yellowish crystals. Mp 390°C (decomp); 1H NMR  δ 7.53 , 7.41 , 7.28 , 7.24 , 7.17 , 7.05 , 5.02 , 4.42 , 4.40 , 4.39-4.37 , 4.38 , 4.31 , 1.44 , 1.42 ; 13C NMR  δ 161.8 , 161.6 , 150.4 , 146.9 , 135.5 , 135.2 , 131.2 , 131.0 , 130.3 , 129.5 , 128.2 , 126.5 , 123.5 , 123.1 , 121.5 , 120.6 , 120.3 , 118.2 , 116.9 , 116.6 , 115.9 , 112.3 , 75.0 , 73.0 , 62.2 , 61.9 , 35.6 (1-CH3), 14.4 , 14.3 ; E/Z ratio (%) 71:29; MS EI m/z  341 [M•+] (74), 339 [M•+] (47), 337 [M•+] (8), 292 (100), 229 (31).390 mg (1.06 mmol) ; 1H NMR  δ 7.41 , 7.28 , 7.17 , 4.46-4.41 , 4.41 , 4.38 , 1.44 ; 13C NMR  δ 161.6 (C=O), 146.9 (C-2′), 135.5 (C-7a), 131.1 (C-6), 130.2 (C-2), 126.5 (C-3a), 123.5 (C-5), 121.6 (CN), 120.6 (C-4), 116.9 (C-7), 112.3 (C-3), 74.9 (C-1′), 62.2 (CH2), 35.6 (1-CH3), 14.3 (CH2CH3); MS EI m/z  341 [M•+] (7), 339 [M•+] (50), 337 [M•+] (69), 292 (100), 229 (32), 149 (64).Recrystallization of the 2, 95% air). Cells from passages 2 to 6 were used. For inhibition experiments, HMEC-1 endothelial cells were switched to EC basal medium  for 1 hr. After that, cells were pretreated with the CLK inhibitor KH-CB19 (1 nM to 100 μM) or TG003 , respectively, for 1 hr and then stimulated with 10 ng/ml TNF-α . Analysis of the TF isoform mRNA expression was done 1 hr post stimulation with TNF-α and assessment of the phosphorylation state of SR proteins by western blotting was performed 2 min post induction of the cells. Positive controls were stimulated only with TNF-α, and negative controls were untreated.Human microvascular cells (HMEC-1) were cultured in endothelial cell (EC) growth medium containing 5% fetal calf serum at 37°C in a humidified incubator , hTF_right_1 (5′-TGCAGTAGCTCCAACAGTGC-3′), GAPDH_For (5′-GAGTCAACGGATTTGGTCGT-3′) and GAPDH_Rev (5′-GACAAG CTTCCCGTTCTCAG-3′). PCR conditions were as follows: 94°C, 2 min, and 36 cycles of 94°C, 30 sec; 58°C, 30 sec; and 72°C, 1 min. PCR products were separated on 1.5% agarose gels, excised, and purified and their identity was confirmed by automated sequencing.Western blot analysis of samples from cell lysates of inhibited, stimulated, and unstimulated HMEC-1 cells were performed as previously described . For detAll data were expressed as mean ± SEM. Data were analyzed by Student's t test or 1-way ANOVA. A probability value ≤ 0.05 was deemed significant.


It has been suggested that the interaction between Gβγ and the PH domain of PKD, or the Gβγ-induced PLCβ/PKC activity is critical for the induction of PKD activation. However, the relative contribution of these two apparently independent events to Gβγ-mediated PKD activation has yet to be addressed.Protein kinase D (PKD) constitutes a novel family of serine/threonine protein kinases implicated in fundamental biological activities including cell proliferation, survival, migration, and immune responses. Activation of PKD in these cellular activities has been linked to many extracellular signals acting through antigen receptor engagement, receptor tyrosine kinases, as well as G protein-coupled receptors. In the latter case, it is generally believed that the Gα subunits of the Gq family effectively activate all the three PKD isoforms (PKD1/2/3), while Gα subunits of other G protein families  are ineffective. Though the Gα subunits of Gi family are unable to stimulate PKD, receptors linked to Gi proteins are capable of triggering PKD activation in cell lines endogenously expressing (HeLa cells and Jurkat T-cells) or exogenously transfected with (HEK293 cells) Gβγ-sensitive PLCβ2/3 isoforms. This indicates that the Gi-mediated PKD activation is dependent on the released Gβγ dimers upon stimulation. Further investigation on individual Gβγ combinations (i.e. Gβ1 with Gγ1–13) revealed that, even if they can stimulate the PLCβ activity in a comparable manner, only those Gβ1γ dimers with γ2, γ3, γ4, γ5, γ7, and γ10 can serve as effective activators of PKD. We also demonstrated that Gi-mediated PKD activation is essential for the SDF-1α-induced chemotaxis on Jurkat T-cells.In this report, we demonstrate that among various members in the four G protein families, only the Gα subunits of the Gi proteins may activate PKD in a PLCβ2/3-dependent manner, and the specific identities of Gγ components within Gβγ dimers may determine this stimulatory action.Our current report illustrates that Gβγ dimers from the G Protein kinase D (PKD) constitutes a novel family of diacylglycerol (DAG)-responsive serine/threonine protein kinases with different structural, enzymological and regulatory properties from the protein kinase C (PKC) family members -3. To da2 termini that display high affinity for DAG or phorbol ester, a pleckstrin homology domain (PH domain) for negative regulation of kinase activity  inositol was purchased from DuPont NEN . M2 affinity gels and protein A-agarose were obtained from Sigma . HA-PKD1 and FLAG-PKD2 constructs were gifts from Dr. J. Van Lint , and Myc-PKD3 constructs were kindly provided by Dr. Q. J. Wang .HEK293 and Jurkat T-cells were obtained from American Type Culture Collection . Pertussis toxin (PTX) was purchased from List Biological Laboratories . Cell culture reagents including Dulbecco’s phosphate-buffered saline (PBS), trypsin, fetal bovine serum (FBS), penicillin-streptomycin mixture, RPMI 1640 medium, minimum essential medium (MEM), Dulbecco’s modified Eagle’s medium (DMEM) and Lipofectamine PLUS™ were obtained from Invitrogen . The cDNAs encoding PLCβ2R, 293/β2AR and 293/fMLPR), exponentially growing HEK293 cells were transfected with cDNA of BK2R, β2AR or fMLPR in pcDNA3.1-zeo using Lipofectamine PLUS™. The cells were then selected with Zeocin (50 μg/mL). 293/fMLPR-Gα16 cells were established by transient transfection of 293/fMLPR stable cell lines with Gα16 in pcDNA3.HEK293 cells were cultured in MEM supplemented with 10% (v/v) FBS, 50 units/ml penicillin, and 50 μg/ml streptomycin. Jurkat T-cells were cultured in RPMI1640 containing 10% (v/v) FBS. For PLC assays and co-immunoprecipitation assays, HEK293 cells were seeded at 60% confluency into 12-well plates or 6-well plates, respectively. Transfection was performed on the following day using Lipofectamine PLUS™ reagents. For the establishment of stable cell lines . Lysates obtained were subjected to in vitro PKD kinase assay. Fifty μl of each supernatant was used for the detection of PKD isoform expression and stimulatory phosphorylation, and the remaining lysate (450 μl) was incubated overnight at 4°C with specific affinity gels to immune-precipitate the corresponding PKD isoform . The resulting immunoprecipitates were washed twice with lysis buffer and twice with kinase assay buffer . Washed immunoprecipitates were resuspended in 40 μl of kinase assay buffer containing 2.5 mg/ml of Syntide-2 (PLARTLSVAGLPGKK), and the kinase reactions were initiated by the addition of 10 μl of ATP buffer containing 1 μCi of [γ-32P]-ATP per sample. After 10-min incubation at 30°C with occasional shaking, the reactions were terminated by adding 100 μl of 75 mM H3PO4 and spotting 75 μl of the reaction mix onto P-81 phosphocellulose paper. Free [γ-32P]-ATP was separated from the labelled substrate by washing the P-81 paper four times (5 min each) in 75 mM H3PO4. The papers were dried and the radioactivity incorporated into Syntide-2 was determined by scintillation counting.Twenty-four hours after transfection, HEK293 cells were serum-starved overnight and then treated with 500 μl (per well) of ice-cold detergent-containing lysis buffer  was added to the samples and then electroporated using the Nucleofector®. Electroporated cells were then incubated at room temperature for 10 min before transferring them into the 12-well plate with culture medium. The knock down of PLCβ1, PLCβ2 and PLCβ3 was performed in similar manner, with the corresponding isoform-specific siRNA obtained from Santa Cruz Biotechnology .The knock down of PKD1, PKD2 and PKD3 was performed by introducing the corresponding PKD isoform-specific siRNA from Invitrogen  using Nucleofector® Kit V from Lonza . Briefly, 1×102O7, 1% Triton X-100, 1 mM dithiothreitol, 200 μM Na3VO4, 100 μM phenylmethylsulfonyl fluoride, 2 μg/ml leupeptin, 4 μg/ml aprotinin and 0.7 μg/ml pepstatin). Clarified lysates were resolved on 1 μ2% SDS-polyacrylamide gels and then transferred to nitrocellulose membranes . Stimulatory phosphorylation of PKD1, PKD2, ERK and CREB were detected by their corresponding antisera and horseradish peroxidase-conjugated secondary antisera. The immunoblots were visualized by chemiluminescence with the ECL kit (Amersham Biosciences). Antibodies sources are as follows: anti-phospho-PKD1-Ser744/748, anti-phospho-PKD1-Ser916 , anti-phospho-ERK-Thr202/Tyr204, anti-PKD1 were obtained from Cell Signaling Technology . Anti-phospho-PKD2-Ser876 and anti-PKD2 were purchased from R & D Systems . Anti-PKD3 was obtained from Bethyl Laboratories .Cells in 12-well plate were lysed in 300 μl of ice-cold lysis buffer  were preloaded with Fluo-4 (AM) followed by incubation at 37°C for 1 h. These labeled cells were then transferred to a black-walled and clear-bottomed 96-well plate (1×105 cells/well) placed in the Fluorometric Imaging Plate Reader (FLIPR), and 50 μl of HBSS (with or without agonists) was added to each well. The resulting fluorescent signals that reflect the intracellular Ca2+ transients were monitored by an excitation wavelength of 488 nm and detection with the emission wavelength from 510 to 570 nm.Jurkat T-cells were serum-starved overnight in the absence or presence of PTX (10 ng/ml) and then washed with Hank’s balanced salt solution (HBSS). Washed cells  to remove cellular debris. Lysates were incubated at 4°C overnight with M2 affinity gels (20 μl/ sample) for the binding with Flag-tagged Gβ subunits. The resulting immunoprecipitates were collected by centrifugation at 1,000 g, 4°C, for 3 min and then washed three times with 500 μl lysis buffer. Bound proteins were eluted by 50 μl of lysis buffer and 10 μl of 6× SDS-containing sample buffer, and boiled μfor 5 min prior to separation by 12% SDS-polyacrylamide gel electrophoresis (PAGE). Flag-tagged Gβ, HA-tagged Gγ subunits, PLCβ6/ml were added to the top chamber of a 24-well transwell  and incubated for 4 h at 37°C. The cells which passed through the membranes and migrated to the lower chambers were quantified under microscopy.The chemotactic ability of Jurkat T cells was evaluated using transwell plates  with polycarbonate inserts with 5-μm pores (Costar 3421). Lower chambers were loaded with 600 μl of migration media alone or containing SDF-1α at the concentration of 100 nM. Cells (0.1 ml) at 1 × 10The values shown in each figure represent mean ± SEM from at least three individual experiments. Statistical analyses were performed by ANOVA, followed by the Bonferroni’s post test. Differences with a value of P < 0.05 were considered statistically significant.q-kinase assays using syntide-2 as an exogenous substrate for PKD. As shown in Figure 738/742 exhibits some cross-reactivity with PKD2 and PKD3, anti-phospho-PKD1910 was also employed to detect PKD1 phosphorylation. Likewise, anti-phospho-PKD2876 was used for PKD2. As PKD3 lacks the phosphorylation site equivalent to phospho-PKD1910, only the phosphorylation at PKD3731/735 was monitored. In agreement with the results from the in vitro kinase assay, stimulatory PKD phosphorylation for all three PKD isoforms was enhanced in the presence of constitutively active Gα mutants from the Gq subfamily , Gs (GαsL and Gαolf) and G12 (G12 and G13) families  rapidly and potently stimulated PKD1 phosphorylation  and GLP receptor (glucagon-like peptide) failed to activate PKD, even when stimulatory phosphorylation of ERK was clearly detected  and CXCR4 receptor (by SDF-1α) led to observable PKD activation. This is in contrast to the result presented in Figure i-coupled fMLP receptor in HEK293 cells failed to promote PKD activation.Next, we investigated whether PKD phosphorylation can be induced upon activation of Gp Figure A. Agonisd Figure B. Unexper by UK1404 and CXi-coupled receptors to stimulate PKD phosphorylation in HeLa cells was contrary to the results obtained with either GαiQL  was abundantly expressed in HeLa cells  is more difficult to achieve in transient transfections, HEK293 cells stably expressing Gγ2 were employed in these assays. As expected, co-expression of various combinations of Gβγ alone did not induce any stimulatory phosphorylation as compared to the vector control in HEK293 cells  and one of the twelve combinations of Gβ1γx dimer, with or without PLCβ2. As shown in Figure 1γx combinations tested, Gβ1γ2, Gβ1γ3, Gβ1γ4, Gβ1γ5, Gβ1γ7 and Gβ1γ10 consistently triggered strong and significant PKD1 phosphorylation upon co-expression with PLCβ2, however, there was no significant change in PKD1 phosphorylation in other Gβ1γx/PLCβ2-overexpressing cells , resulting in elevated levels of IP3 formation . In agreement with the Gβ1γx/PLCβ2-induced PKD1 phosphorylation profile, Gβ1γ2/PLCβ2 and Gβ1γ7/PLCβ2 induced significant PKD kinase activity with all three PKD isoforms, while Gβ1γ9/PLCβ2 failed to do so . As expected, Gβ1γx failed to induce PKD phosphorylation with PLCβ1 which is insensitive to Gβγ  effectively stimulated PKD phosphorylation in the presence of PLCβ2/3 , Gβ1γx  with or without PLCβ2. FLAG-tagged Gβ1 was immunoprecipitated from the lysates of the transfectants, and the immune complexes were subjected to SDS-PAGE, followed by Western blotting for any PKD co-immunoprecipitated with Gβ1. As shown in Figure 1γ7 dimer and PLCβ2, but not when PLCβ2 was absent. Despite comparable expressions of the various constructs  for chemotaxis [2 and PLCβ3 are endogenously expressed in Jurkat T cells. Indeed, Jurkat T-cells endogenously express both PLCβ2 and PLCβ3 isoforms, with the former being more abundant  to confirm that SDF-1α-induced signaling and chemotaxis in Jurkat T-cells are mediated via Gi proteins. Both SDF-1α-induced intracellular Ca2+ mobilization  are capable of inducing the kinase activity of all PKD isoforms  but not others  could effectively activate PKD in the presence of PLCβ2/3  could be immunoprecipitated with PKD  are compatible with the PH domain of PKDs for productive conformation changes, which result in functional activation of PKDs. In fact, our unpublished data showed that PKD activation triggered by Gi-coupled receptors is sensitive to inhibitors for PLCβ (e.g. U73122) as well as to Gβγ subunit scavengers (e.g. transducin). Since only specific Gβγ dimers are capable of stimulating PKD in the presence of PLCβ2/3, our results actually suggest a dual requirement of functional PLCβ activity and compatible Gβγ dimers for Gi-mediated PKD activation. It remains unclear if all the members in the Gq family  also activate PKD in a similar manner. However, it should be noted that another scaffold protein named PAR3 have been suggested as a Gq-specific signaling component with selective recruitment of PLCβ1, while PLCβ2/3 isoforms may have high preferences towards NHERF members in Gi-mediated signaling [q family (much stronger activators for PLCβ isoforms as compared to Gβγ dimers) are capable of stimulating PKD in a Gβγ-independent manner.The assembly of a Gβγ/PLCβed NHERF . Coincid members ,54. Hencignaling ,54. The i-coupled receptor leads to the liberation of free Gβγ dimers, which then interact with PLCβ2/3 and activate PKD. This may help to elucidate part of the mechanism regarding secretory activities regulated by receptor-induced Gβγ translocation between the Golgi and plasma membrane [i-coupled chemotactic response may be mediated by the Gβγ/PLCβ/PKD axis  are activated equally well by Gα subunits from the Gethality . As for ethality , lymphoiethality , and monethality . Recent ethality  and insuethality . Furtherq family effectively activate all three PKD isoforms (PKD1/2/3), while Gα subunits of other G protein families  are inefficient in these kinase activations. However, receptors linked to Gi proteins are capable of triggering PKD activation in cell lines endogenously expressing (HeLa cells and Jurkat T-cells) or exogenously transfected with (HEK293 cells) Gβγ-sensitive PLCβ2/3 isoforms, indicating the involvement of Gβγ dimers for the Gi-mediated PKD activation. Although the presence of PLCβ2/3 is highly important, only those Gβ1γ dimers with γ2, γ3, γ4, γ5, γ7, and γ10 are effective activators of PKD, and the specific interaction between Gβγ, PKD and PLCβ2/3 may play a pivotal role in this Gβγ-mediated PKD signaling pathway. Furthermore, the biological significance of Gi-mediated PKD activation is illustrated by SDF-1α-induced chemotaxis on Jurkat T-cells, in which the chemotaxic activity is abolished by pretreatment with PTX and knockdown of PKD. Taken together, our current report illustrates that Gβγ dimers from Gi proteins may activate PKD in a PLCβ2/3-dependent manner, and the identity of Gγ of the Gβγ dimer being a determinant.Collectively, among various members of G proteins, only the Gα subunits of the G2-adrenergic receptor; DAG: Diacylglycerol; fMLPR: N-formyl-methionyl-leucyl-phenylalanine receptor; GPCR: G protein-coupled receptor; PLCβ: Phospholipase Cβ; PKD: Protein kinase D; PTX: Pertussis toxin.BK2R: Bradykinin type II receptor; β2AR: βThe authors declare that they have no competing interests.WWIL and ASLC carried out the experiments participated in the design of the study and wrote the manuscript. LSWP and JZ carried out some of the experiments. YHW participated in the design of the study and revised the manuscript. All authors read and approved the final manuscript.i subfamily failed to induce PKD activation. (A) HEK293 cells were transiently transfected with pcDNA3 or WT/QL forms of Gα subunits from the Gi subfamily. Cell lysates were subjected to SDS-PAGE. Gα subunits, phospho-PKD1738/742, phospho-PKD1910, total PKD1, tag of PKD1 (HA) were analyzed by Western blotting using respective specific antibody. (B) HA-PKD1, FLAG-PKD2 and Myc-PKD3 were immunoprecipitated from cell lysates described in (A), and subjected to in vitro PKD kinase assays in terms of syntide-2 phosphorylation. Results are the average of at least three independent experiments, and represented as fold increase over pcDNA3 control (±S.E.M.).Constitutively active Gα subunits from the GClick here for files and G12 subfamilies failed to induce PKD activation. (A) HEK293 cells were transiently transfected with pcDNA3 or WT/QL forms of Gα subunits from the Gs and G12 subfamilies. Cell lysates were subjected to SDS-PAGE. Gα subunits, phospho-PKD1738/742, phospho-PKD1910, total PKD1, tag of PKD1 (HA) were analyzed by Western blotting using respective specific antibody. (B) HA-PKD1, FLAG-PKD2 and Myc-PKD3 were immunoprecipitated from cell lysates described in (A), and subjected to in vitro PKD kinase assays. Results are the average of at least three independent experiments, and represented as fold increase over pcDNA3 control (±S.E.M.).Constitutively active Gα subunits from the GClick here for file2 and specific Gγ subunits are required in Gβγ-induced PKD activation in HEK293 cells. (A) HEK293 cells stably transfected with pcDNA3 or HA-Gγ2 were transfected with pcDNA3, FLAG-Gβ1 or FLAG-Gβ2. Cell lysates were subjected to SDS-PAGE. FLAG-Gβ, HA-Gγ, phospho-PKD1738/742, phospho-PKD1910 and total PKD1 were analyzed by Western blotting using respective specific antibody. (B) HEK293 cells were transiently transfected with vector control, PLCβ2, various Gβγ dimers and tagged PKD isoforms . The cultures were then lysed, and the tagged PKD isoforms were immunoprecipitated for in vitro PKD kinase assay. (C) HEK293 cells transiently transfected with pcDNA3, Gβ, Gγx, Gβγ combinations with or without PLCβ1 or PLCβ2 were lysed, and analyzed by Western blotting for PKD1 phosphorylation.PLCβClick here for file2. HEK293 cells were transfected with pcDNA3, PLCβ2, various combinations of Gβγ with or without PLCβ2. Transfectants were lysed, and the lysates were subjected to Western blotting using antibodies against phosphorylated PKD1, PKD, PLCβ2, Flag-tagged Gβ1 and HA-tagged Gγ subunits.The expression profiles of Gβγ dimers and the corresponding PKD activation in the presence of PLCβClick here for file2, but not PLCβ1, can be co-immunoprecipitated with various PKD isoforms. HEK293 cells were transiently transfected with pcDNA3, PLCβ1/2 with tagged PKD1/2/3 as indicated. HA-PKD1, FLAG-PKD2 and Myc-PKD3 were immunoprecipitated from cell lysates with their respective affinity gels and further analyzed by Western blotting for the possible interaction with PLCβ1/2.PLCβClick here for file


Epidermophyton floccosum revealed susceptibility to more tested agents than those of Trichophyton mentagrophytes. Non-steroidal anti-inflammatory drugs (NSAIDs) are the most common pharmacological group that has three primary therapeutic effects including anti-inflammatory, anti-pyrexia, and analgesia. In this study, seven of NSAIDs were tested against two species of skin pathogenic fungi (dermatophytes). Percentage inhibition was determined for effective agents. Diclofenac, Aspirin and Naproxen showed much more ability to inhibit dermatophytes growth. In conclusion, many of NSAIDs may have the ability to inhibit pathogenic fungi. Others may also have potential activity toward fungal growth. Candida albicans  or Diclofenac revealed strong and dose-dependent inhibition of colonies growth  are the centerpiece of pharmacotherapy for most rheumatological disorders and are used in large numbers as analgesic and antipyretics . In addialbicans . Furthers growth . Sulindais agent . Herman  Aspirin . Trichophyton, Microsporum, and Epidermophyton . Naproxen was supplied from Medical Bahri Company, (Damascus (AI-Gassoulaeh), Syria). Piroxicam was supplied from Shaghai Pharmaceutical Co. LTD, . Celecoxib was supplied from Micro-Labs, Limited, Sipcot Industrial Complex . Mefenamic acid was supplied from Brown and Burk Pharmaceutical Limited, Spicot, . Aspirin was supplied from SDI, .Antifungal assay°C for 1 week. Perpendicular colony diameters (mm) of grown strains were measured and percentage inhibition calculated according the formula: The colony diameter method recorded by Kücüc and Kivan  was usedPercentage inhibition=(C-T)×100CWhere: C = colony diameter (mm) of the control; T = colony diameter (mm) of the test plate.Minimal inhibitory concentrations determination (MIC)4 cfu/mL. MIC was performed according to Santos and Hamdan . Serial °C and were read visually after 7 days of incubation.Microdilution plates (96 wells) were set up in accordance with the NCCLS reference method . Each miT. mentagrophytes and at 280 μg/mL for E. floccosum. Aspirin and Naproxen also ceased fungal development through decreased colonies diameter of growing fungi, but less than Diclofenac  as the first NSAID in 1897 , NSAIDs Candida albicans did not influence by Mefenamic acid, Meloxicam or Naproxen , a hemeprotein that exists in two isoforms (COX-1 and COX-2), and converts arachidonic acid to prostanoids such as prostaglandin . NSAIDs COX-1 is important in the protection of human gastrointestinal mucosa. Inhibition of this enzyme can result in formation of ulcers. On the other hand, stimulation of COX-2 results in pain and inflammation . TherefoDipodascopsis uninucleata fungus can use its COX to incorporate arachidonic acid within lipids synthesis  In addition to mammalian, COX can be found in several species of fungi. ynthesis . Cells oCandida albicans and Cryptococcus neoformans can secrete prostaglandins de nova or via conversion of exogenous arachidonic acid   , otherwioformans .Aspergillus nidulans (Dermatophytes, the main subject of this study, also have the ability to produce prostaglandin that responsible for chronic fungal colonization . Therefonidulans  or functnidulans .The usage of NSAIDs at therapeutic doses could cause many adverse effects. Some of NSAIDs cause less damage in human than others, especially Celecoxib or Rofecoxib  and coatIn conclusion, NSAIDs contain many promising agents within their group which can be used against dermatophytes. Diclofenac, Aspirin and Naproxen are more effective on two main species of dermatophytes. Other NSAID agents have variable actions on dermatophytes, based on fungal species.