In hormone receptor-positive breast cancers, most tumors in the early stages of development depend on the activity of the estrogen receptor and its ligand, estradiol. Anti-estrogens, such as tamoxifen, have been used as the first line of therapy for over three decades due to the fact that they elicit cell cycle arrest. Unfortunately, after an initial period, most cells become resistant to hormonal therapy. Peptidylprolyl isomerase 1 (Pin1), a protein overexpressed in many tumor types including breast, has been demonstrated to modulate ERalpha activity and is involved in resistance to hormonal therapy. Here we show a new mechanism through which CDK2 drives an ERalpha-Pin1 interaction under hormone- and growth factor-free conditions. The PI3K/AKT pathway is necessary to activate CDK2, which phosphorylates ERalphaSer294, and mediates the binding between Pin1 and ERalpha. Site-directed mutagenesis demonstrated that ERalphaSer294 is essential for Pin1-ERalpha interaction and modulates ERalpha phosphorylation on Ser118 and Ser167, dimerization and activity. These results open up new drug treatment opportunities for breast cancer patients who are resistant to anti-estrogen therapy. In the normal mammary gland, estrogen receptor alpha  and its ligand, estradiol (E2), primarily control the ductal outgrowth and side branching that occur during pregnancy and the menstrual cycle ERalpha belongs to the superfamily of steroid nuclear receptor transcription factors and shares four main functional domains with other nuclear hormone-responsive receptors: the N-terminal transcriptional activation function (AF-1) domain, DNA-binding domain (DBD), hinge region and ligand-binding domain (LBD) (AF-2). When estradiol binds to the AF-2 domain, it induces structural changes that facilitate ERalpha dimerization, nuclear translocation and the binding of DNA to regulate gene transcription The hinge region, although initially characterized as merely a flexible connection between the DBD and LBD regions, has recently been discovered to have a more complex function. Indeed, it comprises the nuclear translocation signal and includes estrogen-independent regulatory sequences: selective mutation studies showed that the binding of ERalpha to c-Jun and Sp-1 transcription factors requires an intact hinge domain cis and transThe Ser/Thr-Pro motifs exist in two distinct conformations: In the present study, we show that Pin1 regulates the functions of ERalpha under basal conditions through its interaction with Ser294. We demonstrate that a mutation of Ser294 reduces the levels of Ser118 and Ser167 phosphorylation with important implications for ERalpha dimerization and activation. Finally, we propose a new working model for Pin1 and ERalpha and suggest a novel pathway that can be inhibited to overcome resistance to hormonal therapy.MCF7 breast cancer cell lines were purchased from American Type Culture Collection , 293FT from Invitrogen . Cells were grown at 37°C, in a 5% CO2/95% atmosphere. Hormone-free medium was prepared with phenol red–free EMEM with 2 mmol/L L-glutamine, 0.1 mmol/L nonessential amino acids, 50 units/mL penicillin, 50 µg/mL streptomycin, and 3% charcoal-stripped FBS.Antibodies were purchased from: Pin1 (600-401-A20), 6XHis (600-401-382) from Rockland Immunochemicals, Gilbertsville, PA, USA; ERalpha (sc-8002), ERalphaS-118 (sc-101675), p-AKT (sc-7985R), from Santa Cruz Biotechnology, Santa Cruz, CA, USA;ERalphaS-167 (31478), from ABCAM Cambridge, MA, USA; α-tubulin (T-6074) from Sigma Inc., St Louis, MO, USA; AKT (9272), MAPK (9102), p-MAPK (9101) from Cell Signaling, Beverly, MA, USA. Kinase inhibitors were purchased from: flavopiridol (F3055) from Sigma Inc., St Louis, MO; AG825 (1555) and U0126 (1144) from Tocris Bioscience, Ellisville, Missouri 63021, USA; LY294002 (9901) from Cell Signaling, Beverly, MA, USA. Estradiol was purchased from Sigma Inc., St Louis, MO, USAshRNA plasmids Pin1 (SHCLNG-NM_006221), CDK1 (SHCLNG-NM_001786), CDK2 (SHCLNG-NM_001798) and CDK4 (SHCLNG-NM_000075) were obtained from Sigma Inc., St Louis, MO, USA. Scrambled shRNA (17920), psPAX2 packaging plasmid (12260) and pMDG.2 envelope plasmid (12259) were obtained from Addgene Inc, Cambridge, MA, USA.GCGGATCCGCGGCAGGAGGGAAGATGG at the 5′ end and PIN1-EcoRIR GCGAATTCCTGGGCTCCCCACCCTCAC at the 3′ end with BamHI and EcoRI adaptor sequences, respectively. The PCR generated products were ligated in the pGEX-2T plasmid for the prokaryotic expression vector .For GST pull-down experiments, the IMAGE: 3941595 clone was utilized to amplify the Pin1 human gene with the oligonucleotide primers PIN1-BamHIF ATG GAT CCA CCA TGA CCA TGA CCC TCC-3′, AB-EcoRI-Rev 5′-ATG AAT TCT CCT TGG CAG ATT CCA TAG-3′, CD-BamHI-F 5′-ATG GAT CCA CCA TGG CCA AGG AGA CTC GCT ACT GTG-3′, CD-EcoRI-Rev 5′-ATG AAT TCT TCT TAG AGC GTT TGA TCA TG-3′. EF-BamHI-F 5′-ATG GAT CCA CCA TGT CTA AGA AGA ACA GCC TGG CC-3′, EF-EcoRI-Rev 5′-ATG AAT TCC AGA CCG TGG CAG GGA AAC-3′. After BamHI/EcoRI double digestion, fragments were ligated in pcDNA6 His/Myc vector. ERalpha-His-CDSer294Ala was generated by site-directed mutagenesis with the QuickChange mutagenesis kit .ERalpha- His-AB, His-CD and His-EF plasmids were derived from VP16-ERalpha (ADDGENE:11351) following amplification with primers: AB-BamHI-F To obtain pM-GAL4DBD-ERalpha, we digested VP16-ERalpha with EcoRI. The pM destination vector was digested with BamHI and XbaI. The ERalpha fragment and pM vector ends were filled with Klenow polymerase and ligated.5′-GCTGCCAACCTTTGGCCAGCCCCGCTCATGATCAAACGC-3′ and ERS294A-r1 5′-GCGTTTGATCATGAGCGGGGCTGGCCAAAGGTTGGCAGC-3′ERalphaSer294Ala was generated by site-directed mutagenesis with the QuickChange mutagenesis kit  and confirmed by direct sequencing. VP16-ERalpha was used as the template for the mutagenesis. The primers used were: ERS294A-f1 All the plasmids were sequence verified.http://www.broadinstitute.org/genome_bio/trc/publicProtocols.html) and Rizzolio et al. To generate knock down cells, lentiviral particles were produced as described  was performed with SYBR Green PCR Master Mix  in a LightCycler® 480 Real-Time PCR System instrument . Samples were run in triplicates and the efficiency of each primer was calculated utilizing an internal standard control GAPDH.Total RNA was prepared from tissues using the RNA extraction kit RNAeasy . One µg of total RNA was reverse transcribed in a 20 µl reaction using M-MLV reverse transcriptase . Primers to amplify GST and GST-Pin1 proteins were produced in BL21 bacteria cells. Cells were grown to mid log phase and then induced to express protein by adding 0.25 mM of isopropyl-1-thio-b-D-galactopyranoside . The cultures were shaken for 4 h; bacteria were then pelleted and resuspended in NENT buffer (20 mM Tris (pH 8), 100 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 0.5% NP-40). Cell suspensions were sonicated and pelleted so that the supernatant could be collected. The supernatant was incubated with glutathione agarose beads  overnight at 4°C. The agarose beads were pelleted and washed three times in NENT buffer. The GST protein was analyzed by electrophoresis gel and blue coomassie staining. 1 mg of protein was pulled down with 10 ug of GST or GST-Pin1.Sub-confluent MCF7 cells were harvested and proteins were prepared as follows: the cell pellet was resuspended in lysis buffer . 1 mg of proteins was immunoprecipitated, utilizing 4 µg of Pin1, ERalpha antibody or mouse IgG overnight at 4°C. The immunoprecipitated protein complex was collected with agarose protein A/G beads (Pierce) for 3 h at 4°C. After washing, the protein immunocomplex was run on SDS-PAGE followed by immunoblot analyses to detect Pin1 or ERalpha proteins.GST-D WT or mutant constructs were purified from E. Coli, 1 µg of recombinant protein incubated with 100 U of CDK2/CycA2, CDK4/CycD1, CDK6/CycD1  in the kinase reaction buffer  for 30 minutes at RT. The reaction was analyzed with the Kinase Glo luminescence assay  according to the manufacturer's protocol.4 cells/well and grown in hormone-free medium. After three days, cells were transfected  with 300 ng of VP16ERalpha and pM-GAL4DBD-ERalpha wild-type or Ser294Ala mutant, 300 ng of GAL4-LUC-pGL2Basic (Promega E1641) and 50 ng of renilla plasmid . After 24 hours, cells were treated with 10 nM of 17-β-estradiol for 12 hours. The luminescence of each sample was measured in a single tube luminometer  with the Dual-Luciferase Reporter Assay System . Each transfection was performed three times in order to overcome the variability inherent in the experiment.293FT cells were seeded in 96 multiplate wells at 2×10t-test. Quantification of western blot was carried out with Adobe Photoshop.Statistical analyses were performed using GraphPad software by applying unpaired Student's in vivo. To understand which mechanism is involved in the interaction between Pin1 and ERalpha under basal conditions, we then decided to carry out all experiments in the absence of FBS and estradiol unless indicated.Previous studies demonstrated that Pin1 indirectly controls ERalpha activity Pin1 is a short protein that contains two known functional domains, a WW interacting domain (amino acids 6–37) and a PPIase domain (amino acids 54–163). Total cellular lysate was incubated with GST, GST-Pin1 protein, GST-WW and GST-PP domains . GST pulTo clarify which ERalpha domain is involved in the binding of Pin1, ERalpha protein was split into three regions   and fuseSince Pin1 activity requires prior phosphorylation of its target site and the Ser/Thr-Pro motif is the minimal consensus sequence recognized by Cyclin Dependent Kinases (CDKs) In order to further demonstrate the involvement of the CDK pathway in Pin1-ERalpha binding, we decided to use flavopiridol (FLV), a potent CDKs-inhibitor. MCF7 cells cultured in phenol-red-free and hormone-free medium were treated for 16 hours with 0.25-0.125 µM concentrations of FLV. As expected, inhibition of CDK activity blocked Pin1 binding on ERalpha .in vivo, a full-length ERalpha expression plasmid bearing the mutation Ser294Ala was generated. 293FT cells were co-transfected with ERalpha WT or Ser294Ala and GFP plasmid as controls. Two days before treatment, cells were grown in phenol-red-free medium containing 3% charcoal-stripped FBS. After 45 min of E2 treatment, cellular pellets were collected and lysed for Western blot analysis. We could note that phosphorylation on Ser118 and Ser167, both implicated in ERalpha transcriptional activation, was reduced in cells transfected with the Ser294Ala ERalpha plasmid. The Ser294Ala is located in the D domain, a flexible region that lies between two distinct dimerization interfaces in the DBD and LBD regions Phosphorylation of ERalpha on serine 118 and 167 is required for the full activity of protein function To confirm that Pin1 modifies ERalpha activity, we carried out a real-time PCR (qRT-PCR) on two well-known estrogen-induced genes (CTSD and TFF1) in Pin1 kd MCF7 cells treated with E2 for 6 h . We wereERalpha is regulated by multiple signaling pathways including the PI3K/AKT pathway and the extracellular-regulated kinase pathway (MAPK). Neither pathway excludes the other, and they can activate ERalpha in a ligand-dependent or -independent manner Evaluation of the ERalpha status is an essential component in the pathological classification of breast cancers and determines the success of endocrine therapies cis conformation. After Pin1 binding, the AF1 domain is switched to the trans conformation by promoting a slight local structural change A number of studies suggest that the benefit of endocrine therapy is proportional to the level of ERalpha expression Here we report that under basal conditions, ERalpha Ser294 is the target of Pin1. We demonstrated by GST pulldown and coimmunoprecipitation assays on MCF7 cell extracts that Pin1 interacts with ERalpha and this interaction was reduced when serine 294 was mutated to alanine.The hinge region is a relatively short domain spanning 51 amino acids (263–314), and with several important functions in ERalpha regulation. Complete deletion of the hinge region impairs ligand-induced downregulation of the receptor On the basis of results in the literature Although Ser294 has a proven role in the activity of ERalpha, there is a lack of information regarding the pathway involved in Ser294 phosphorylation and its functional significance. Serines residues located in the N-terminal domain  and in the Hinge region (Ser294) match the consensus sequence recognized by families of Serine/Threonine proline directed kinases, including cyclin-dependent kinases. ERalpha transcriptional activity is virtually abolished under conditions where CDK2 activity is suppressed by the kinase inhibitor p27 or by a dominant negative CDK2 mutant, regardless the presence or absence of hormones Overall, our results suggest a new pathway that involves PI3K/AKT, CDK2 and Pin1 and open up new opportunities for treating ERalpha-positive breast cancer patients who are resistant to hormonal therapy.


Thus, identifying an iPLA2βinhibitor that can be reliably and safely used in vivo is warranted. Currently, the mechanism-based inhibitor bromoenol lactone (BEL) is the most widely used to discern the role of iPLA2β in biological processes. While BEL is recognized as a more potent inhibitor of iPLA2 than of cPLA2 or sPLA2, leading to its designation as a “specific” inhibitor of iPLA2, it has been shown to also inhibit non-PLA2 enzymes. A potential complication of its use is that while the S and R enantiomers of BEL exhibit preference for cytosol-associated iPLA2β and membrane-associated iPLA2γ, respectively, the selectivity is only 10-fold for both. In addition, BEL is unstable in solution, promotes irreversible inhibition, and may be cytotoxic, making BEL not amenable for in vivo use. Recently, a fluoroketone (FK)-based compound (FKGK18) was described as a potent inhibitor of iPLA2β. Here we characterized its inhibitory profile in beta-cells and find that FKGK18: (a) inhibits iPLA2β with a greater potency (100-fold) than iPLA2γ, (b) inhibition of iPLA2β is reversible, (c) is an ineffective inhibitor of α-chymotrypsin, and (d) inhibits previously described outcomes of iPLA2β activation including (i) glucose-stimulated insulin secretion, (ii) arachidonic acid hydrolysis; as reflected by PGE2 release from human islets, (iii) ER stress-induced neutral sphingomyelinase 2 expression, and (iv) ER stress-induced beta-cell apoptosis. These findings suggest that FKGK18 is similar to BEL in its ability to inhibit iPLA2β. Because, in contrast to BEL, it is reversible and not a non-specific inhibitor of proteases, it is suggested that FKGK18 is more ideal for ex vivo and in vivo assessments of iPLA2β role in biological functions.Ongoing studies suggest an important role for iPLA Of these, the Group VI iPLA2s are the most recently described and the least well characterized. The iPLA2 was first purified from macrophages in 1994 2β. Subsequently, a membrane-associated enzyme was identified and designated as iPLA2γ. More comprehensive reviews of the iPLA2 enzymes can be found elsewhere Phospholipases A2β has been proposed to participate in membrane phospholipid remodeling, signal transduction, cell proliferation, inflammation, and apoptosis 2β contributes to these abnormalities, reagents that more specifically impact iPLA2β in vitro and in vivo are needed. Of the currently used inhibitors, iPLA2β is targeted by arachidonyl trifluoromethyl ketone (AACOCF3), methyl arachidonyl fluorophosphonate (MAFP), and palmitoyl trifluoromethyl ketone (PACOCF3); inhibitors that are sometimes used for “selective” inhibition of cPLA2Since its original description in heart and pancreas, the iPLA2βand now available iPLA2β-KO and Tg mice 2β, the majority of studies to assess the role of the iPLA2β isoform, have utilized the only available specific inhibitor of iPLA22 enzymes and has little or no effect on cPLA2 or sPLA2 activity While siRNAs directed at iPLA2 in biological processes and, to date, is still considered the only available specific irreversible inhibitor of iPLA2. Recently, the S- and R-enantiomers of BEL have been demonstrated to exhibit specific inhibition of iPLA2β and iPLA2γ, respectively 2 enzymes by BEL have been described 22β 2 acts on the inhibitor Over the years, BEL has been used to discern the involvement of iPLAin vivo use: (a) irreversible inhibition of iPLA2, (b) inactivation of other serine proteases, and (c) high toxicity due to its interaction with cysteines. For these reasons, recent efforts were directed towards synthesizing alternative compounds that can specifically inhibit iPLA2. Assays for PLA2 activity in the presence of these compounds have led to the identification of fluoroketone (FK)-based compounds as potential inhibitors of the iPLA2 enzyme group 2s. However, modification of the FK group along with addition of a hydrophobic terminus connected by a medium-length carbon chain to mimic the fatty acid chain conferred selectivity of the FK compounds for iPLA2 versus sPLA2 or cPLA22 and was 195 and >455 times more potent for GVIA iPLA2 than for GIVA cPLA2 and GV sPLA2, respectively.However, several features of BEL decrease its feasibility for 2β, the biochemical assays were performed using human Group VIA enzyme purified from Sf9 cells 2β in biological systems. Recently, earlier generation of FK compounds (FKGK11 and FKGK2) were found to be effective in ameliorating experimental autoimmune encephalomyelitis, however, there was no demonstration of iPLA2β inhibition by these compounds 2β is predominantly expressed in pancreatic islet beta-cells 2β as a means to preventing beta-cell apoptosis. Though FKGK11 in now commercially available, it has been reported to be 7-fold less potent than FKGK18 in inhibiting iPLA22β, it would allow us to utilize it to prevent beta-cell apoptosis in vivo. In the present study, we therefore set out to characterize the ability of FKGK18 to inhibit iPLA2β using INS-1 cells overexpressing iPLA2β (OE), rodent myocardial preparations, and human pancreatic islets.While the above study demonstrated the potential of FKGK18 in inhibiting iPLA2β activity, INS-1 insulinoma cells overexpressing iPLA2β (OE) were used. Cytosol from the OE cells was prepared and activity in 30µg protein aliquots was assayed in the presence of varying concentrations of FKGK18. As seen in 2+-independent PLA2 activity in a concentration-dependent manner, similar to S-BEL, which preferentially inhibits cytosol-associated iPLA2β −8M) for FKGK18 was similar to that of S-BEL R-BEL, which inhibits membrane-associated iPLA2γwas a weaker inhibitor of iPLA2 enzymatic activity, as reflected by an estimated IC50 of 3×10−6M. These findings suggest that FKGK18 is equipotent to S-BEL as an in vitro inhibitor of cytosol-associated Ca2+-independent PLA2 activity, which is manifested by iPLA2β.To test the ability of FKFK18 to inhibit iPLA2β, cytosol was prepared from myocardial tissues isolated from WT and iPLA2β-KO mice 2+-independent PLA2 activity in the WT group is stimulated by ATP, a characteristic of iPLA2β 2+-independent PLA2 activity ± FKGK18 in the iPLA2β-KO group was barely above background. These findings suggest that the myocardial cytosol-associated activity is manifested by iPLA2β and that it is inhibitable by FKGK18.To confirm FKGK18 ability to inhibit iPLA2, we examined whether FKGK18 also exhibited a similar inhibitory profile against iPLA2γ. We chose the myocardial membrane preparation because it is predominantly enriched in iPLA2γ activity R-BEL and FKGK18 on iPLA2γ activity were compared. As seen in R-BEL and FKGK18 with an IC50 of ∼1–3µM. To confirm that the inhibition is of activity manifested by iPLA2γ, membrane fractions were prepared from hearts of iPLA2β-KO mice. As shown in 2 activity similar to R-BEL, with an IC50∼1µM. These findings suggest that iPLA2γ is also inhibitable by FKGK18.Because the putative mechanism of FKGK18 inhibition is through interaction with the lipase consensus sequence of iPLA2β and iPLA2γ, there was a distinct separation in the potency of the drug to inhibit the two activities. To verify localization that the cytosol and membrane preparations contained the expected isoform of iPLA2, cytosol and membrane preparations were processed for immunoblotting analyses using antibodies directed against iPLA2β or iPLA2γ. As shown in 2β was predominantly localized in the cytosol (Top Panel) and iPLA2γ in the membrane (Middle Panel). Thus, the activities measured in cytosol and membrane fractions are expected to be manifested by iPLA2β and iPLA2γ, respectively. As illustrated in 2β activity was shifted nearly two log-units to the left of membrane-associated iPLA2γactivity. The IC50 of FKGK18 for inhibition of cytosol-associated activity was nearly 100-fold lower than for membrane-associated activity, suggesting that FKGK18 is a more potent inhibitor of iPLA2β than iPLA2γ.Though FKGK18 exhibited an ability to inhibit both iPLAR-BEL is more potent than S-BEL S-BEL. This was done by monitoring α-chymotrypsin-catalyzed digestion of BSA in the presence of saturating concentrations of S-BEL or FKGK18. The peptide digests were resolved by Bis-Tris gel electrophoresis and peptide fragments derived from α-chymotrypsin activity were visualized by colloidal blue staining. As shown in  (Lanes 1), in the presence of inhibitors alone a protein band corresponding to full-length BSA (70 kDa) is evident. However, in the presence of enzymes alone, BSA digestion in complete, as reflected by the absence of intact BSA (Lanes 2). Inclusion of FKGK18 with the enzymes also resulted in near completed digestion of BSA (Lanes 3). In contrast, inclusion of S-BEL with the enzymes, inhibited BSA digestion, as evidenced by visualization of the full length BSA (Lanes 4). These findings suggest that, unlike S-BEL, FKGK18 is not a non-specific inhibitor of non-iPLA2 proteases.It has been reported that BEL inhibits other proteases 2β is irreversible 2βinhibition following a single exposure to FKGK18. INS-1 OE cells were treated with FKGK18 (10−5 M) for up to 48 h and cytosol was prepared and assayed for residual iPLA2β activity. As shown in h was similarly stimulated by ATP, indicating preservation of a viable iPLA2β activity. However, neither basal nor ATP-stimulated activity in cytosol prepared from cells treated with FKGK18 from 2 to 48hours was inhibited. These findings raise the possibility that the interaction of FKGK18 with the enzyme is disturbed during preparation of cytosol, thus restoring native enzyme activity, suggesting that FKGK18 is not an irreversible inhibitor of iPLA2β.Because BEL inhibition of iPLAS-BEL. We previously reported that GSIS and hydrolysis of arachidonic acid (AA) from beta-cell membrane phospholipids, reflected by increases in medium content of PGE2, a metabolized product of AA, are inhibited by BEL. We therefore treated human islets with glucose in the absence or presence of FKGK18 and measured insulin secretion and PGE2 release into the media. As illustrated in mM glucose (20G), relative to basal concentration of glucose (5mM). In the presence of FKGK18, there was no change in basal insulin secretion. In contrast, stimulated insulin secretion was significantly decreased to basal levels. In parallel with GSIS, PGE2 content in the medium was significantly increased in the presence of 20G  group have been synthesized as potential inhibitors of the PLA2 enzymes. This led to the identification of FKGK18  and the membrane-associated iPLA2 (iPLA2γ) activities in iPLA2βINS-1 OE cells and myocardial preparations from WT and iPLA2β-KO mice reveal that the potency of FKGK18 to inhibit iPLA2β is similar to the S-enantomer of BEL whereas the potency of FKGK18 to inhibit iPLA2γ is similar to the R-enantomer of BEL. However, FKGK18 inhibits iPLA2β with a greater potency than iPLA2γ; as reflected by the nearly 100-fold lower IC50 value for iPLA2β vs. iPLA2γ.Our findings comparing the cytosol-associated iPLA2βrevealed that even following exposure of INS-1 OE cells to the drug for 48 h, both basal and ATP-stimulated iPLA2β activities in cytosol prepared from these cells were similar to that measured in vehicle-treated cells. This is in contrast to the observation of concentration-dependent inhibition of iPLA2β activity when FKGK18 is added directly to cytosol preparations. Thus unlike with BEL, whose inhibition of iPLA2β activity in islets and INS-1 cells is close to 10–20% of control activity even after 24h of exposure 2β is reversible. This most likely is a consequence of disassociation of FKGK18 from iPLA2β during cell lysis in the cellular fractionation process. That this is not due to an inability of the FKGK18 to enter the cell is supported by the accompanying functional analyses in islets and cells, as discussed below.Attempts to determine the duration of FKGK18 inhibition of iPLAR-BEL was determined to be a more potent inhibitor of chymotrypsin than its chiral counterpart S-BEL S-BEL nearly completely inhibited α-chymotrypsin-catalyzed digestion of BSA. In contrast, α-chymotrypsin-catalyzed digestion of BSA was nearly complete in the presence of FKGK18. These findings suggest that FKGK18 is not an effective inhibitor of α-chymotrypsin and imply that in contrast to BEL, it is not a non-specific inhibitor of proteases.We also examined for evidence of FKGK18 inhibition of α-chymotrypsin, which has been identified as a suitable target for aromatic haloenol lactones resulting in its mechanism-based inhibition 2 appears to be distinct from that of BEL. With respect to BEL, Cys651 alkylation is the covalent modification of iPLA2β that is responsible for loss of activity, and the alkylating species is a diffusible hydrolysis product of BEL rather than a tethered acyl-enzyme intermediate 2 favoring high affinity of the inhibitor presumably confers the greater specificity of FK compounds for iPLA2 versus non-iPLA2 enzymes.The mechanism of FKGK18 inhibition of iPLA2β inhibition. These included: (a) GSIS, (b) hydrolysis of arachidonic acid (AA), as reflected by PGE2 generation, (c) ER stress-induced neutral sphingomyelinase 2 (NSMase2) expression, and (d) ER stress-induced beta-cell apoptosis. As discussed below, during our descriptions of iPLA2β role in beta-cell function and survival, we observed that these outcomes are all inhibited by BEL.In view of these findings, we next examined whether FKGK18 can inhibit biological processes in intact insulinoma cells and human pancreatic islets. Because FKGK18 appears to be a reversible inhibitor, we assessed its impact on biological outcomes that would reflect dynamic iPLA2β-catalyzed hydrolysis of AA from the sn-2 position of beta-cell membranes, which are enriched in AA-containing phospholipids mM) promoted significant increases in both insulin secretion and PGE2 release, relative to basal (5mM) glucose condition. Initial studies followed the protocol previously used with BEL min prior to the replacement of media containing higher glucose concentrations (20mM). With this protocol, we found that both GSIS and PGE2 release were not affected (data not shown), further supporting the reversible nature of FKGK18 inhibition. However, when FKGK18 was present during the entire stimulatory period, both GSIS and PGE2 release were reduced significantly. It therefore seems plausible to deduce that the inhibitory effects of FKGK18 on iPLA2β, and thus effects on GSIS and PGE2 release, are conditional upon the constant presence and exposure to FKGK18.We demonstrated that GSIS from pancreatic islets parallels iPLA2β, which induces NSMase2 and accumulation of ceramides as a consequence of increased hydrolysis of sphingomyelins h and remained higher at 24h. Presence of FKGK18 promoted a concentration-dependent decrease in NSMase2 message at both time points. Because NSMase2 increase is an early event and apoptosis is a later event, the incidence of apoptosis was assessed at 24h. Thapsigargin, in the absence of FKGK18 promoted INS-1 OE beta-cell apoptosis, as reflected by increased TUNEL staining. However, in the presence of FKGK18 a concentration-dependent decrease in the incidence of apoptosis was evident. These findings are analogous to those observed in insulinoma cells and islet beta-cells, where both ER stress-induced NSMase2 expression and apoptosis were inhibited by BEL treatment We also reported that prolonged ER stress promotes beta-cell apoptosis via activation of iPLA2βthan iPLA2γ and because, unlike BEL, it inactivates iPLA2β reversibly and appears to not be a non-specific inhibitor of proteases, FKGK18 may be the inhibitor of choice for in vitro, ex vivo and more importantly, in vivo studies. It is recognized that FKGK18 may have effects on other classes of non-PLA2 enzymes and continued studies with this and future FK derivatives will reveal other, if any, non-specific effects of these compounds. The ability to identify an inhibitor of iPLA2β that can be used in vivo is timely and warranted because of the ever going recognition of roles for iPLA2β-derived products in biological processes in the context of diabetes, inflammation, and neurodegenerative and myocardial disorders. It is plausible that FKGK18 or newer generations of FK compounds could be used to prevent or delay abnormalities associated with these disease states.In summary, our studies reveal that FKGK18 is a more potent inhibitor of iPLA2β-KO breeding pairs), generously provided by Dr. John Turk (Washington University School of Medicine) were bred, maintained, and prepared for experiments according to the protocols approved by University of Alabama at Birmingham IACUC (APN# 120809160).Mice  ; PNPLA8 antibody ; Apoptosis Detection Kit [APO-DirectTM] ; S- and R-BEL, prostaglandin E2 EIA Kit – Monoclonal # 514010 ; MIAMI Medium 1A ; Bis-tris, colloidal blue staining kit, RPMI medium 1640, Fast SYBR® Green PCR Master Mix, SDS sample buffer, and Superscript III First-strand Synthesis System ; Human Insulin ELISA , chymotrypsin and trypsin ; RNeasy kit ; Primary antibodies  and secondary antibodies  ; and BSA and Pierce ECL Western Blotting Substrate .Human islets were acquired from Islet Cell Resource Centers for Islet Distribution Program. FKGK18 hexan-2-one) was synthesized, as previously described 2β (INS-1 OE) were generated and cultured as previous described mM glucose, 10% fetal calf serum, 10mM HEPES buffer, 2mM glutamine, 1mM sodium pyruvate, 50mM mercaptoethanol (BME), and 0.1% (w/v) each of penicillin and streptomycin in cell culture conditions , as described 2/95% air) for two days prior to use. Islets were also isolated from WT and iPLA2β-KO mice, as described 2/95% air) overnight prior to use. All inhibitors tested were prepared in DMSO vehicle and all experimental protocols included DMSO-only treated replicates. Of note, FKGK18 concentrations ≥5×10−5M caused cells to detach from the bottom of wells and die and for this reason, the concentrations of drug used were below these.INS-1 cells overexpressing iPLA2+-independent PLA2 activity in aliquots of cytosol or membrane fractions (30µg of protein) was assayed by ethanolic injection (5µl) of the substrate 16:0/[14C]20:4 GPC  in assay buffer . Assay mixtures were incubated , and the assay reaction was terminated with butanol (100µl) addition and vigorous vortexing. The reaction mixture was centrifuged , and products in the upper butanol layer were analyzed by silica gel G thin-layer chromatography (TLC) in petroleum ether-ethyl ether-acetic acid (80/20/1). The TLC plate region containing free fatty acid was identified with iodine vapor, scraped, and the released 14C fatty acid was quantitated by liquid scintillation spectrometry. Specific iPLA2 activity was calculated from the dpm of released fatty acid and protein content, as described S- BEL, R-BEL, or FKGK (10−11–10−5M). In some studies, to verify that the measured activity reflected that of iPLA2β, the ability of ATP (10 mM) to stimulate activity was used as a positive control.INS-1 OE cell or islet cytosol and membrane fractions were prepared and protein concentration determined using Coomassie reagent. Enzymatic Ca2γ 1:200; and GAPDH 1:500) and then with secondary antibodies (1:1000). Immunoreactive bands were visualized by enhanced chemiluminescence.Protein from cytosolic and microsomal membrane fractions was analyzed by SDS-PAGE; an equal protein load of 30 μg. The samples were run on a 10% acrylamide gel and then transferred onto Immunobolin-P PVDF membranes. Target proteins were probed with primary antibodies  containing 5mM D-glucose (5G) in the presence of vehicle only or FKGK18 (10−6 or 10−5 M). Following pre-treatment, the medium was aspirated and islets were further treated for 1h with 5G or 20G in the presence of vehicle or FKGK18. The medium was then collected and the contents of insulin and PGE2 were determined by ELISA. Briefly, collected medium was titrated against internal standard controls of known concentrations and standard curves were then generated to calculate sample unknown concentrations for insulin (using a log standard curve) and for PGE2 (using a 4-parameter logistics curve generated by ReaderFit software). The values were normalized to protein per sample (i.e. 30 islets) and are presented as mU/μg islet protein for insulin and pg/μg islet protein for PGE2.Human islets (30/condition) were incubated in 96-well round bottomed plates in MIAMI medium, supplemented with 10% fetal calf serum and 0.1% (w/v) each of penicillin and streptomycin, as described µg) was reconstituted in 3 ul of 6 M Urea/50mM Tris-HCl (pH 8). Reduction and alkylation was performed by adding 0.15µl of 200 mM DTT/50 mM Tris-HCl (pH 8) for 1h, and 0.6µl of 200mM iodoacetamide/50mM Tris-HCl (pH 8) for 45min in dark, respectively, at room temperature. Alkylation was quenched by adding 0.6µl of 200mM DTT/50mM Tris-HCl (pH 8), and incubating for 15min at room temperature. Twenty microliters of 1 mM CaCl2/50mM Tris-HCl (pH 7.5) was added to dilute the urea concentration down to 0.6M. Protease inhibitors FKGK18 and/or S-BEL were added to appropriate tubes. Samples were incubated with 0.2µg of trypsin and 0.2µg of chymotrypsin at 37°C for 15min, and immediately quenched by denaturing in SDS sample buffer, as per manufacturers instructions. Even volumes of each samples were loaded and separated on a 10% Bis-tris gel and peptide fragments were separated for 35min at 200V constant and visualized by overnight colloidal blue staining.Bovine serum albumin  for 8 or 24h in the presence of vehicle or FKGK18 (10−7–10−5 M). Total RNA was prepared using the RNeasy kit as previous ® Green PCR Master Mix in a plate-based LightCycler® 480 System (Roche Life Sciences). The primers were designed based on published sequences for rat NSMase2 and 18S, Gene Bank #AB047002 and #X01117, respectively. Primer sets (sense/antisense) were as follows: NSMase2, ccggatgcacactacttcagaa/ggattgggtgtctggagaaca and 18S, agtcctgccctttgtacaca/gatccgagggcctcactaaac.INS-1 OE cells were treated with thapsigargin (1h in the absence or presence of FKGK18 (10−7–10−5 M). The cells were then processed for apoptosis as described TM kit, according to manufacturer’s protocol. Briefly, cells were harvested, spun at 800g and washed in PBS preceding the addition of fixation buffer (1% (w/v) paraformaldehyde in PBS (pH 7.4). Following a 30min incubation period on ice, the cells were washed in PBS, resuspended in 70% ethanol, and incubated for a further 30min on ice. DNA labeling solution  was then added and the cells were incubated for 1h at 37°C. After rinsing with rinse buffer and resuspension in PI/RNase staining buffer (provided in kit), cells were incubated in the dark for 30min at room temperature prior to flow cytometry analysis at excitation wavelength 623nm and 520nm for PI  and FITC-dUTP (staining apoptotic cells), respectively. Of note, FKGK18 concentrations ≥5×10−5M caused cells to detach and die and for this reason, the concentrations of drug used in the various analyses were ≤5×10−5M.INS-1 OE cells were treated with vehicle or thapsigargin for 24Data were converted to mean ± standard error of the means and the Students’ t-test was applied to determine significant differences between two samples (p<0.05).


Members of the Mps1 kinase family play an essential and evolutionarily conserved role in the spindle assembly checkpoint (SAC), a surveillance mechanism that ensures accurate chromosome segregation during mitosis. Human Mps1 (hMps1) is highly phosphorylated during mitosis and many phosphorylation sites have been identified. However, the upstream kinases responsible for these phosphorylations are not presently known.in vivo phosphorylation sites in hMps1. While in vivo analyses indicate that Aurora B and hMps1 activity are required for mitotic hyper-phosphorylation of hMps1, in vitro kinase assays show that Cdk1, MAPK, Plk1 and hMps1 itself can directly phosphorylate hMps1. Although Aurora B poorly phosphorylates hMps1 in vitro, it positively regulates the localization of Mps1 to kinetochores in vivo. Most importantly, quantitative mass spectrometry analysis demonstrates that at least 12 sites within hMps1 can be attributed to autophosphorylation. Remarkably, these hMps1 autophosphorylation sites closely resemble the consensus motif of Plk1, demonstrating that these two mitotic kinases share a similar substrate consensus.Here, we identify 29 hMps1 kinase is regulated by Aurora B kinase and its autophosphorylation. Analysis on hMps1 autophosphorylation sites demonstrates that hMps1 has a substrate preference similar to Plk1 kinase. The purpose of mitosis is to equally distribute the duplicated genome amongst dividing cells. Defects in chromosome segregation can lead to aneuploidy, which in turn is implicated in tumorgenesis in vitroin vivoin vitro autophosphorylation of the catalytic domain of hMps1 on at least 16 residues was described and phosphorylations at both T676 and T686 were shown to be important for catalytic activity in vivo phosphorylation sites outside the catalytic domain have been described SAC function requires several proteins that localize to the outer KT. Of these, Bub1, Bub3, Mad1, Mad2, Mad3/BubR1 and Mps1 are conserved from yeast to humans in vivo phosphorylation sites within hMps1, including 4 sites that have not previously been reported. While Cdk1, MAPK, Plk1 and hMps1 itself can phosphorylate hMps1 in vitro, the kinase activities of Aurora B and hMps1 are required for the hyper-phosphorylation induced upshift (retardation of the gel electrophoretic mobility) of hMps1 during mitosis. Using quantitative mass spectrometry, we demonstrate that numerous phosphorylation sites, including several sites matching the Plk1 consensus, are in fact hMps1 autophosphorylation sites. This indicates that hMps1 and Plk1 are able to phosphorylate similar motifs.Here, we have explored which upstream kinases might be responsible for phosphorylation of hMps1 during mitosis. We identified 29 2 based enrichment of phosphopeptides and analysis by mass spectrometry. A total of 29 phosphorylation sites were identified  ur study  [30], .in vivo phosphorylation sites not only conform to the proposed hMps1 consensus motif but are also phosphorylated by hMps1 in vitro. These include MCAK (T87), Nup98 (S591) and Cep215 (S613)  .in vivo hMps1 phosphorylation by mass spectrometry and investigated candidate upstream kinases for their possible involvement in hMps1 phosphorylation. Although a number of the hMps1 phosphorylation sites analyzed here were also identified independently by other groups in vivo evidence for phosphorylation of several sites that had previously been identified only in vitro. Our biochemical analysis of hMps1 in mitotic cells indicates that Aurora B kinase and hMps1 itself are required for hMps1 hyper-phosphorylation during mitosis. Given that the mitotic upshift of hMps1 correlates with increased kinase activity in vivo activity of hMps1. While hMps1 readily phosphorylated itself in vitro, only marginal in vitro phosphorylation of hMps1 by Aurora B was observed. Thus, it appears plausible that Aurora B causes mitotic hyperphosphorylation of hMps1 through an indirect mechanism. In line with evidence from XenopusIn this study we examined Saccharomyces cerevisiae revealed a preference for D/E at the −2 position for yeast Mps1 In addition to encompassing most of the previously identified phosphorylation sites , our stuIn vivo, substrate specificity of any kinase is strongly influenced by subcellular localization. Moreover, the interactions between kinases and their substrates are influenced also by molecular features (in either kinase and/or substrate) that reside outside of the interaction domain between consensus motif and catalytic site. Nevertheless, our present data suggest that hMps1 and Plk1 share at least some common physiological substrate(s). Thus, the access of the two kinases to these substrates is expected to be tightly regulated in time and space. In future studies, it will be interesting to explore the possibility that hMps1 and Plk1 converge on a set of common physiological substrates during mitotic progression.Although both hMps1 and Plk1 are important for mitotic progression and the fidelity of chromosome congression, the known roles of these kinases are widely different. While disruption of hMps1 function interferes with SAC activity and thus accelerates traverse through mitosis, inhibition of Plk1 causes a SAC-dependent mitotic arrest. Thus, it may appear surprising that at least some substrates of hMps1 and Plk1 share a similar consensus motif. HeLa S3 cells were routinely maintained in DMEM (Invitrogen) supplemented with 10% FBS and penicillin-streptomycin . Thymidine was used at 2 mM, Nocodazole at 100 ng/ml, SP600125 at 20 µM, Reversine at 0.5 µM, Mps1-IN-1 at 1.0 µM (kindly provided by Nathanael S. Gray), ZM447439 at 5 µM, TAL at 1 µM, Roscovitine at 100 µM, U0126 at 20 µM, and MG132 was used at 20 µM.For expressing recombinant hMps1, wild type and kinase dead hMps1 cDNA was amplified and cloned into the pVL1393 baculovirus transfer vector (Pharmingen) with an N-terminal GST tag, and then expressed in Sf9 cells following the manufacturer's instructions. Recombinant His-Plk1 Monoclonal anti-hMps1-N1 In vitro phosphorylation of recombinant kinase dead GST-hMps1 was carried out at 30°C using 300 ng of recombinant hMps1-KD and 100 ng of each kinase in 40 µl of kinase reaction buffer . Reactions were stopped after 30 min by addition of sample buffer. Samples were then resolved by SDS-PAGE and visualized by autoradiography. Recombinant active Cdk1/Cyclin B, MAPK (Erk2) and Aurora B kinase were purchased from Invitrogen.13C615N4 -L-arginine and 13C615N2-L-lysine (Cambridge Isotope Laboratories) at the concentration of 44 and 86 µg/ml respectively, and supplemented with 10% dialyzed fetal bovine serum, 50 units/ml penicillin, and 50 µg/ml streptomycin. Unlabeled and labeled HeLa cells were synchronized in mitosis using a sequential Thymidine block/release, Nocodazole block protocol. Unlabelled mitotic cells were collected and released into MG132 plus DMSO; in parallel, labeled mitotic cells were collected and released into MG132 plus TAL or SP600125. After 40 minutes, both sets of cells were collected and equal amount of cell lysates (∼15 mg each) were mixed together. Endogenous hMps1 was then immunoprecipitated using the anti-hMps1-N1 mAb. Samples were separated by a 4–12% NuPAGE gel (Invitrogen).HeLa S3 cells were cultured in DMEM formulated with either unlabeled L-lysine and L-arginine or labeled 2 affinity purification with glycolic acid as a modifier http://www.ebi.ac.uk/IPI/IPIhelp.html) with carbamidomethyl cysteine as a fixed modification and oxidized methionine, phosphorylation , 13C615N4 -L-arginine, and 13C615N2-L-lysine as variable modifications. Searches were performed with a precursor mass tolerance of 5 ppm and fragment ion tolerances of 0.7 Da and identified phosphorylation sites were further validated by visual inspection of MS/MS spectra. For phosphopeptide quantification, the ratios between the monoisotopic peaks of labeled and unlabeled forms of phosphopeptides were calculated by MSQuant Coomassie Brilliant Blue (CBB)-stained protein bands were in-gel digested by trypsin  essentially as described http://rsb.info.nih.gov/ij/) on non-deconvolved images. Quantification of KT intensities was performed as previously described HeLa S3 cells grown on coverslips were fixed with a pre-extraction-fixation method using PHEM buffer 2, 100 mM NaCl, 1 mM DTT, 100 µM NaF, 1 mM Sodium Ortho-Vanadate) for 1 h, followed by overnight blocking in kinase buffer with 0.5 mg/ml BSA. The next day, the membrane was blocked again with kinase buffer containing 1 mg/ml BSA and 50 µM cold ATP at RT for 1 h. The blocking buffer was subsequently replaced with kinase reaction buffer containing 0.2 mg/ml BSA, 45 µCi/ml [γ-32P]-ATP and 50 µM cold ATP in the presence of recombinantly expressed GST-Mps1-WT or GST-Mps1-KD (kinase dead) for 3 h on a shaker at 30°C (5.0 ml reaction volume per membrane). Membranes were then washed extensively: 10×15 min in 1 M NaCl, 3×5 min in H2O, 3×15 min 5% H3PO4 , 3×5 min in H2O, and then sonicated overnight in 8 M urea, 1% SDS (w/v), and 0.5% (v/v) ß-mercaptoethanol to remove residual nonspecific radioactivity. The membranes were washed again with H2O, followed by ethanol and dried before being visualized by autoradiography.Peptide arrays were generated using standard F-moc chemistry on a MultiPep robotic spotter (Intavis) following the manufacturer's instructions. Peptides synthesized and immobilized on cellulose membranes were tested for phosphorylation by human GST-Mps1. Dried membranes were first washed in ethanol and then hydrated in kinase buffer . The left panel shows CBB staining of the gel. The right panel shows the result of autoradiography.(TIF)Click here for additional data file.Table S1Summary of identified hMps1 phosphorylation sites. The first column summarizes phosphorylation sites, with novel in vivo sites identified in this study shown in bold. The second column shows short sequences adjacent to the phosphorylation sites (underlined residues). In the absence of definitive information on a particular phosphorylation site, potential alternative positions are shown. The third column shows the MASCOT score for each phosphopeptide identified in this study.(PDF)Click here for additional data file.Table S2Relative phosphorylation levels after treatment with Mps1-IN-1. Sites on hMps1 identified by mass spectrometry after treatment with Mps1-IN-1 are listed together with the corresponding peptide sequences and relative phosphorylation levels. The phosphoacceptor is shown in bold and residues matching the proposed hMps1 consensus motif are underlined.(PDF)Click here for additional data file.Table S3Autophosphorylation sites for weblogo analysis. Autophosphorylation sites identified in previous studies and in the present work were analyzed by Web logo software. 12 sites  were demonstrated as autophosphorylation by our SILAC analysis. As reported by other groups in vivo autophosphorylation sites. Although S436 and S821 were also reported as autophosphorylation sites (PDF)Click here for additional data file.


However, the ALDH2 hypothesis has not been reconciled with early studies demonstrating potent inhibition of the GTN response by diphenyleneiodonium (DPI) [Aldehyde dehydrogenase-2 (ALDH2) catalyzes vascular bioactivation of the antianginal drug nitroglycerin (GTN) to yield nitric oxide (NO) or a related species that activates soluble guanylate cyclase (sGC), resulting in cGMP-mediated vasodilation . Accordi50 of 0.20±0.03 µM, whereas cGMP formation induced by the NO donor DEA/NO was not affected. DPI (10 µM) caused a pronounced right-ward shift of the GTN concentration response . DPI also inhibited the established dehydrogenase and esterase activities of ALDH2 with similar potency, again in a substrate-competitive manner. This was confirmed by molecular modeling, suggesting overlapping binding sites of DPI and GTN in the catalytic site of the enzyme  inhibited the high affinity component of aortic relaxation to GTN without affecting the response to NO, indicating that the drug interfered with GTN bioactivation. As shown in Figure e Figure , indicatThe identification of DPI as potent ALDH2 inhibitor may have implications beyond the cardiovascular pharmacology of organic nitrates. A well established function of ALDH2 is detoxification of reactive aldehydes in liver and heart. NADPH oxidase activation has been implicated in both alcohol-induced liver injury and cardiac dysfunction. In view of the present results, showing that DPI potently inhibits the various enzymatic functions of ALDH2, it may be necessary to revise some of the earlier conclusions that are based on inhibitory effects of aryliodonium compounds.


Glycine soja transcriptional profile, and identified three PPCK genes  as potential alkali stress responsive genes. In this study, we confirmed the induced expression of GsPPCK3 under alkali stress and investigated its tissue expression specificity by using quantitative real-time PCR analysis. Then we ectopically expressed GsPPCK3 in Medicago sativa and found that GsPPCK3 overexpression improved plant alkali tolerance, as evidenced by lower levels of relative ion leakage and MDA content and higher levels of chlorophyll content and root activity. In this respect, we further co-transformed the GsPPCK3 and SCMRP genes into alfalfa, and demonstrated the increased alkali tolerance of GsPPCK3-SCMRP transgenic lines. Further investigation revealed that GsPPCK3-SCMRP co-overexpression promoted the PEPC activity, net photosynthetic rate and citric acid content of transgenic alfalfa under alkali stress. Moreover, we also observed the up-regulated expression of PEPC, CS (citrate synthase), +-ATPaseH and NADP-ME genes in GsPPCK3-SCMRP transgenic alfalfa under alkali stress. As expected, we demonstrated that GsPPCK3-SCMRP transgenic lines displayed higher methionine content than wild type alfalfa. Taken together, results presented in this study supported the positive role of GsPPCK3 in plant response to alkali stress, and provided an effective way to simultaneously improve plant alkaline tolerance and methionine content, at least in legume crops.So far, it has been suggested that phosphoenolpyruvate carboxylases (PEPCs) and PEPC kinases (PPCKs) fulfill several important non-photosynthetic functions. However, the biological functions of soybean PPCKs, especially in alkali stress response, are not yet well known. In previous studies, we constructed a  Medicago sativa L.) is used for pasture, hay, silage and green-chop, and acts in crop rotation through its positive effects on soil fertility and soil structure As a versatile crop, alfalfa  and 434 (53%) million hectares, respectively Phosphoenolpyruvate carboxylase  is a kind of tightly controlled cytosolic enzyme which functions in carbon fixation during photosynthesis SCMRP gene according to the maize methionine-rich 10 ku zein protein Besides high stress tolerance, another important desired trait for alfalfa is high nutritional value in terms of essential amino acids. As we know, legume plants are deficient in the sulfur-containing amino acids, namely, methionine and cysteine Glycine soja (G07256) microarray data, we isolated and characterized an alkali stress responsive gene GsPPCK3. Our results demonstrated that GsPPCK3 overexpression in alfalfa improved plant tolerance to alkali stress. In this case, we co-transformed the GsPPCK3 and SCMRP genes into alfalfa, and demonstrated that the transgenic alfalfa displayed not only higher alkali tolerance but also higher methionine content. Taken together, results presented in this study demonstrated the biological function of GsPPCK3 under alkali stress, and provided an effective way to simultaneously improve plant alkaline tolerance and methionine content, at least in legume crops.For these reasons, in this study, we aimed to generate the transgenic alfalfa not only with higher alkali tolerance but also with higher methionine content. Based on the Glycine soja (G07256) roots and leaves in response to salt-alkali stress , by using the Affymetrix® Soybean Genome Array GsPPCK1, GsPPCK2 and GsPPCK3 were identified as potential stress responsive genes  of 915 bp encoding 304 amino acids with an estimated molecular weight (Mr) of 34 000 and a theoretical pI of 5.35. A BLASTP search at NCBI showed that GsPPCK3 shared 90%, 84%, 69% and 61% sequence identity with the Glycine max GmPPCK3 (GeneBank Accession: NP_001238645), GmPPCK2 (NP_001236660), GmPPCK1 (NP_001241581) and GmPPCK4 (NP_001237016), respectively. It is worth noting that GsPPCK3 contained a 30 amino acid length sequence (KLLLASVFLFDIIFGGFCVDGIFGCFVFVG) at its C-terminus, which did not exist in any soybean and Arabidopsis PPCKs. By comparing the DNA and mRNA sequences of GsPPCK3 and GmPPCK3, we found that this 30 aa sequence was encoded by the 90 bp length intron of GmPPCK3.In this study, we obtained the full length coding region of Glycine max and Arabidopsis thaliana (AtPPCK1 and AtPPCK2)  . ProteinAtPPCK2) , along wGsPPCK3 under alkali stress, we carried out the quantitative real-time PCR analyses. Consistent with the microarray data, GsPPCK3 expression in both leaves and roots of Glycine soja seedlings was greatly and rapidly induced by alkali stress (3 (pH8.5) treatment, GsPPCK3 displayed an obvious increase and reached a maximum point at 1 h. It is noteworthy that the induction degree of GsPPCK3 in roots was significantly higher than that in leaves. The possible reasons for the stronger response might be that plant roots were the exact sites of perception and injury for stresses, or there were different response mechanisms for GsPPCK3 expression between roots and leaves. Anyway, it is obvious that GsPPCK3 expression was greatly induced by alkali stress, suggesting an important role of GsPPCK3 in plant response to alkali stress.In an attempt to verify the induced expression of i stress . After 5GsPPCK3 expression patterns, we further investigated the spatial specific expression of GsPPCK3 in Glycine soja seedlings  M. Sativa through the Agrobacterium tumefaciens-mediated transformation strategy . These results suggested that GsPPCK3 and SCMRP co-transformation promoted seedling growth of the transgenic alfalfa under alkali stress.We firstly compared the alkali tolerance between WT and GsPPCK3 overexpression in alfalfa, PEPC activity, which was regulated by PPCK phosphorylation To further elucidate the influence of 3 concentration 3 , +-ATPaseH and NADP-ME after 100 mM NaHCO3 treatment. The real-time PCR analysis indicated that expression of PEPC, CS, +-ATPaseH and NADP-ME was induced by alkali stress in both WT and transgenic lines. Expectedly, the expression levels in transgenic plants were significantly higher than that in WT , without obvious changes for other amino acids. Specifically speaking, the methionine contents were 0.97±0.04% in WT plants, 2.27±0.89% (2.34 folds to WT) in transgenic line PS-2 and 2.23±0.67% (2.29 folds) in line PS-31. Taken together, these results suggested that GsPPCK3-SCMRP co-overexpression not only enhanced the alkaline tolerance, but also increased the methionine content of transgenic alfalfa.In addition to the enhanced alkali tolerance, we also determined the content of 16 amino acids in leaves of both WT and transgenic plants, to verify if  content . As expe3/Na2CO3 content, caused injury to plants not only through salt stress, but also through alkali stress Saline-alkaline stress, as a kind of widespread environmental stress with significantly negative impact on plant growth, severely reduces crop productivity and affects agricultural production worldwide. It has been suggested that alkaline soil, characterized by high NaHCOGlycine soja, the wild ancestor of cultivated soybean (Glycine max), could normally germinate and grow in the alkaline soil with a pH value at 9.02 Glycine soja (G07256) under alkali stress , and three of the four PPCK genes GsPPCK1, GsPPCK2 and GsPPCK3 were identified as putative stress responsive genes  seeds, obtained from Jilin Academy of Agricultural Sciences , were treated with 98% sulfuric acid for 10 min, washed five times with sterile water, and then kept in complete darkness with humidity for 2–3 days to promote germination. The seedlings were transferred and grown in 1/4 Hoagland solution for 3 weeks at 24–26°C and 16 h light/8 h dark cycles. To explore the expression profiles of GsPPCK3 under alkali stress, the 3-week-old seedlings were treated with 1/4 Hoagland solution containing 50 mM NaHCO3 (pH 8.5) for 0, 1, 3, 6, 12 and 24 h, respectively. Equal amounts of leaves and roots were harvested and stored at −80°C after snap-frozen in liquid nitrogen.Medicago Sativa L. was kindly obtained from Heilongjiang Academy of Agricultural Sciences , and grown in green house under controlled environmental conditions . To investigate the expression patterns of stress-responsive genes, the 4-week-old seedlings after shoot cottage were treated with 1/4 Hoagland solution containing 50 mM NaHCO3 (pH 8.5) for 0, 3, 6, 12 and 24 h, respectively. Samples were harvested and stored as described above.Glycine soja seedlings, by using RNeasy Plant Mini Kit , and subjected to cDNA synthesis by using SuperScript™ III Reverse Transcriptase kit . The full-length coding region of GsPPCK3 was PCR amplified with gene specific primers (5′-AAGATAGAATGTGCACAGCCCTAAAG-3′ and 5′-TTCTCAAGTGAGTTCAGCCGTGTC-3′), and cloned into pGEM-T vector  for sequencing.Total RNA was extracted from the 3-week-old http://blast.ncbi.nlm.nih.gov/Blast.cgi). Homology searches were executed by BLASTP at Phytozome (http://www.phytozome.net/soybean). TMHMM (http://www.cbs.dtu.dk/services/TMHMM/) and Tmpred (http://www.ch.embnet.org/software/TMPRED_form.html) were used to predict the transmembrane domains.Sequence similarity was examined by using the on-line BLASTP program at NCBI (in G. soja (Accession: DQ355800) and M. sativa (Accession: Medtr3g085850) were used as internal references, respectively. Expression levels for all candidate genes were calculated by using the 2-ΔΔCT method, and the relative intensities were normalized as described previously Total RNA extraction and cDNA synthesis were operated as described above. Quantitative real-time PCR analyses were performed by using SYBR Premix ExTaq™ II Mix  on an ABI 7500 sequence detection system . The glyceraldehyde-3-phosphate dehydrogenase genes GsPPCK3 and SCMRP on plant stress tolerance and methionine content, we constructed the expression vectors carrying the GsPPCK3 gene alone (GsPPCK3 and SCMRP genes together (GsPPCK3 gene was inserted to the bone vector pBEOM (made in our lab) under the control of the cauliflower mosaic virus (CaMV) 35S promoter, with the binding enhancers E12 and omega. The SCMRP gene was under the control of double CaMV35S promoter and omega sequence. The Bar gene was used as the selectable marker. The recombinant vectors were introduced into A. tumefaciens strain EHA105, and then transformed into M. sativa by using the cotyledonary node method. The transformants were selected by using 0.5 mg L−1 glufosinate ammonium, and regenerated shoots were rooted on 1/2 Murashige and Skoog (MS) medium. At last, the glufosinate-positive seedlings were transplanted into soil and grown in green house under controlled conditions.In order to investigate the influence of ne alone , and thetogether , respectGsPPCK3 and SCMRP genes in the glufosinate-positive plants was confirmed by PCR analysis using CaMV35S promoter specific forward primer and Bar gene specific reverse primer (5′-CCTGTGCCTCCAGGGAC-3′ and 5′-GCGGTCTGCACCATCGTC-3′). GsPPCK3 transcript levels in the PCR-positive plants were analyzed by semi-quantitative RT-PCR analysis using gene specific primers (5′-CCCTCCTTTCACCTCACC-3′ and 5′-GAACCGAAGTCCGCCAGT-3′). Expression levels of the SCMRP gene were examined by using a pair of specific primers (5′-CAGCAGGGTCTCGCTTCACT-3′ and 5′-GCAGATTCCAATGCCACAAT-3′). The alfalfa GAPDH gene was used as an internal control. And the PCR- and RT-PCR positive seedlings were subjected to western blot analysis with specific polyclonal antibody to the C-terminus of GsPPCK3 protein (CHPWILSAGDTAELT).Presence of the GsPPCK3 transgenic alfalfa, the 3-week-old WT and transgenic plants with similar sizes were exposed to alkali stress by irrigating with 1/8 Hoagland solution containing either 0, or 50, or 100 mM NaHCO3 every 3 days for a total of 12 days. For phenotypic analysis of GsPPCK3-SCMRP transgenic alfalfa, the 4-week-old plants were exposed to alkali stress by irrigating with 1/8 Hoagland solution containing either 0, or 100, or 150 mM NaHCO3 every 3 days for 15 days.The lignified WT and transgenic alfalfa plants were propagated by stem cuttings, and about 2 weeks later, adventitious roots appeared. The seedlings were transplanted into plastic culture pots filled with a mixture of peat moss: soil , irrigated with 1/8 Hoagland nutrient solution and grown in green house under controlled conditions. For phenotypic analysis of The total chlorophyll content was determined in 80% (v/v) acetone extract according to the method of Arnon −2 s−1, and the CO2 concentration was in the range of 350 to 400 µmol mol−1. The flow rate was adjusted to 500 µmol s−1, and the leaf temperature was maintained at 25°C. For statistical analysis, three independent experiments were conducted with at least 5 independent plants per genotype and experiment.The net photosynthesis rate was determined by using the LI-6400 chamber . During the experiments, the light intensity was 500 µmol photons m2, 10 µg mL−1 chymostatin, 10 µg mL−1 bestatin, 10 µg mL−1 leupeptin, 1 mM PMSF, 1 µg mL−1 microcystin-L/R (L and R are two variable amino acids in the structure of microcystin), and 14 mM β-mercaptoethanol. Protein concentration was determined according to the method of Bradford (1976) PEPC activity was measured by the coupled spectrophotometric method at 340 nm and 30°C as previously described by Gonzalez Leaves of WT and transgenic alfalfa were fixated at 105°C for 15 min, and then dried to constant weight at 80°C. About 100 mg dried leaves were crushed, suspended in 6 M HCl, and then hydrolyzed at 110°C for 22 h with nitrogen replacement. The hydrolysate was dried under reduced pressure, dissolved in 0.02 M HCl, and subjected to the amino acid analyzer .Figure S1Schematic representation of the constructs for Agrobacterium tumefaciens-mediated transformation into Medicago sativa. a, Schematic representation of the GsPPCK3 overexpression construct. b, Schematic representation of the GsPPCK3-SCMRP overexpression construct.(TIF)Click here for additional data file.


Eukaryotic protein kinases are generally classified as being either tyrosine or serine-threonine specific. Though not evident from inspection of their primary sequences, many serine-threonine kinases display a significant preference for serine or threonine as the phosphoacceptor residue. Here we show that a residue located in the kinase activation segment, which we term the “DFG+1” residue, acts as a major determinant for serine-threonine phosphorylation site specificity. Mutation of this residue was sufficient to switch the phosphorylation site preference for multiple kinases, including the serine-specific kinase PAK4 and the threonine-specific kinase MST4. Kinetic analysis of peptide substrate phosphorylation and crystal structures of PAK4-peptide complexes suggested that phosphoacceptor residue preference is not mediated by stronger binding of the favored substrate. Rather, favored kinase-phosphoacceptor combinations likely promote a conformation optimal for catalysis. Understanding the rules governing kinase phosphoacceptor preference allows kinases to be classified as serine or threonine specific based on their sequence.  •A single active site residue can determine kinase phosphoacceptor specificity•Favored and disfavored substrates promote distinct kinase-bound conformations•A simple rule predicts kinase phosphoacceptor preference from its DFG+1 residue Proper signal transmission by protein kinases requires that they phosphorylate specific substrates at defined sites. Multiple mechanisms can act in concert to provide substrate specificity for kinases, including kinase-substrate colocalization, compartmentalization through the use of scaffold proteins, substrate recruitment through kinase adaptor subunits, and direct physical interactions between kinases and their substrates . In ordeOne important aspect of the kinase phosphorylation site motif is the identity of the phosphorylation site residue itself. Almost all kinases in eukaryotes phosphorylate protein substrates on Ser, Thr, or Tyr residues. Ser-Thr kinases, which make up approximately 80% of the human kinome, comprise several groups phylogenetically distinct from Tyr-specific kinases . AccordiHere, we show that phosphoacceptor preference of Ser-Thr kinases is determined largely by the identity of a single residue, which we term “DFG+1.” This residue is located within the kinase activation segment, a conformationally flexible loop important for kinase regulation. Mutagenesis of this residue is sufficient to change the phosphoacceptor preference for kinases from distinct groups in a predictable manner. Identification of this residue therefore establishes a simple rule that can be used to predict phosphorylation site preference for a kinase of unknown specificity. We go on to show by X-ray crystallography of kinase-peptide complexes that conformation, rather than binding affinity, drives phosphoacceptor preference. Overall, these studies explain how a protein kinase is able to discriminate between Ser and Thr, two residues that differ only by a single methyl group.S. cerevisiae  and examined their phosphorylation specificity alongside their wild-type (WT) counterparts using optimized synthetic peptides that differ only at the phosphosite. As anticipated, WT MST4 preferentially phosphorylated the Thr peptide, while WT PAK4 preferentially phosphorylated the Ser peptide  group. Most kinases in this group, including those in the mammalian Ste20-like (MST) kinase family, are Thr selective , while m peptide . Further peptide A and S2. on kcat . These r2+/calmodulin-dependent protein kinase-related group) and PKA . We generated a series of Snf1 mutants in which the DFG+1 residue (Leu198) was changed to residues most commonly found in other kinases at this position and evaluated the ability of these mutants to phosphorylate Ser- and Thr-containing peptides  to Val inverted its selectivity from Ser to Thr specific  preference for Thr as the phosphoacceptor D.S112T). Coexpression of WT PAK4 catalytic domain with BAD in HEK293 cells yields robust phosphorylation at Ser112  such that the hydroxyl group is pointed away from the active site. Additionally, two alternate conformations are observed for the DFG+1 Val residue. The PAK4F461V cocrystal structures therefore suggest that Val at the DFG+1 residue favors Thr over Ser because the additional methyl group allows interplay between the DFG+1 and phosphoacceptor residues, correctly positioning them to promote an optimal conformation for phosphate transfer.To understand why kinases with a β-branched DFG+1 residue favor Thr, we examined the cocrystal structures of PAK4 complex D. The ph residue E. AlthouKm values. However, because the Km value for an enzyme reaction is not necessarily equivalent to the substrate dissociation constant, substrate binding, conformation, and product release could all theoretically contribute to phosphoacceptor discrimination. We note, however, that Ser and Thr peptides appear to bind with equal affinity to the Ser-selective kinase PKA  was expressed in E. coli, purified by one-step glutathione affinity chromatography, and then activated by phosphorylation with recombinant Elm1 kinase, as described  were expressed with N-terminal hexahistidine tags in escribed . Descrip33P]ATP at 30°C for varying times, and aliquots were spotted on to P81 filter discs. Filters were quenched in 75 mM phosphoric acid and then washed three times for 4 min in 75 mM phosphoric acid, washed once briefly in acetone, air-dried, and analyzed by liquid scintillation counting. The amount of product formed was calculated from a standard curve made using [γ-33P]ATP at the same specific activity. Initial rates were determined by fitting the data to a line with Excel, and catalytic constants were calculated by fitting the initial velocity data to the Michaelis-Menten equation using KaleidaGraph software. Detailed procedures, including buffer compositions for each kinase reaction, are provided in Kinase activity toward peptide substrates was determined by filter binding assay using radiolabeled ATP. Kinases were incubated with peptides and [γ-2 in Dulbecco’s modified Eagle’s medium containing 4.5 g/l D-glucose and L-glutamine (Invitrogen), 10% fetal bovine serum (FBS), and 1% penicillin/streptomycin. Cells in 6-well plates were cotransfected with 0.4 μg pEGFP-PAK4 catalytic domain and 3.6 μg pEBG-BAD using Lipofectamine 2000 (Invitrogen). After 24 hr, cells were exchanged into reduced serum medium (0.1% FBS) for 16 hr and treated with 250 nM wortmannin for 30 min prior to lysis. Cell lysates were prepared by washing briefly with PBS and extracting into lysis buffer  for 10 min at 4°C with rotation and centrifuging . GST-tagged BAD was purified from the supernatant with 20 μl glutathione Sepharose 4B suspension . Samples were subjected to SDS-PAGE followed by immunoblotting with α-BAD pSer112 antibody , α-GST antibody  and α-GFP antibody (Clontech #632381), and detection by enhanced chemiluminescence.HEK293 cells were cultured at 37°C and 5% COF461V catalytic domain were obtained with both PAKtide-T (GGRRRRRTWYFGGGK) and PAKtide-S (RRRRSWY) using the vapor diffusion hanging drop method. For WT PAK4 with peptides, we grew WT PAK4 crystals against 0.1 M Tris-HCl (pH 7.5) and 1.5–2.0 M NaOAc at room temperature and soaked with peptides. For PAK4F461V cocrystals with peptide, we grew against conditions of 0.1 M Tris-HCl (pH 7.5) and 1.5–2.0 M NaOAc at room temperature. For cocrystals of PAK4 in complex with PAKtide-S(L) (RRRRRSWYFDG), we grew against 1.7 M ammonium sulfate, 15% PEG400, and 0.1 M Tris-HCl (pH 8.0). Crystallographic data were collected at Advanced Photon Source (APS) beamline 24-ID-E, National Synchrotron Light Source (NSLS) beamline X25, or the Swiss light source beamline SLS-X10. Following structure solution, good electron density was observed for each of the kinase domains and their bound peptides.Crystals of WT PAK4 and PAK4


Homologous recombination (HR) plays an important role in the maintenance of genome integrity. HR repairs broken DNA during S and G2 phases of the cell cycle but its regulatory mechanisms remain elusive. Here, we report that Polo-like kinase 1 (Plk1), which is vital for cell proliferation and is frequently upregulated in cancer cells, phosphorylates the essential Rad51 recombinase at serine 14 (S14) during the cell cycle and in response to DNA damage. Strikingly, S14 phosphorylation licenses subsequent Rad51 phosphorylation at threonine 13 (T13) by casein kinase 2 (CK2), which in turn triggers direct binding to the Nijmegen breakage syndrome gene product, Nbs1. This mechanism facilitates Rad51 recruitment to damage sites, thus enhancing cellular resistance to genotoxic stresses. Our results uncover a role of Plk1 in linking DNA damage recognition with HR repair and suggest a molecular mechanism for cancer development associated with elevated activity of Plk1. ► Plk1 phosphorylates Rad51 recombinase at S14 in a highly specific manner ► Rad51 phosphorylation at S14 triggers subsequent phosphorylation at T13 by CK2 ► T13-phosphorylated Rad51 binds to the Nijmegen breakage syndrome gene product, Nbs1 ► Rad51 binding to Nbs1 is important for cellular resistance to genotoxic stresses Precise repair of DNA double-strand breaks (DSBs) that are caused during DNA replication and by exogenous stresses such as ionizing radiation (IR) is critical for the maintenance of genome integrity. Accurate regulation of homologous recombination (HR), which repairs DSBs using the replicated sister chromatid as a repair template, is important during S and G2 phases of the cell cycle. Downregulation of HR results in chromosomal rearrangements due to the engagement of alternative error-prone DSB repair mechanisms such as nonhomologous end-joining (NHEJ), whereas hyperrecombination also causes various genome instability phenotypes including loss of heterozygosity, gene amplification, and gene deletion .NBS1 gene exhibit increased sensitivity to IR  is an autosomal recessive chromosomal instability syndrome, and cells defective in the ty to IR . Nbs1, tty to IR . These ety to IR  or, whenty to IR . The ssDty to IR . Recruitty to IR . BRCA2 w cancers . BRCA2-dS and G2 . StudiesS and G2 . Nonetheve cells , and eleve cells . A recenve cells . TogetheHR processes are temporally controlled by the central cell-cycle regulators, cyclin-dependent kinases (CDKs)  but a coHere, we report a role of Plk1 in the DNA damage response wherein it directly phosphorylates Rad51 at residue S14 in its N-terminal regulatory domain. Significantly, S14 phosphorylation stimulates subsequent phosphorylation of Rad51 at T13 by casein kinase 2 (CK2), creating a phospho-binding motif for the Nbs1 Forkhead-associated (FHA) domain. Cells expressing Rad51 variants that retain binding to Nbs1, but not those with impaired Nbs1 binding, were rendered resistant to genotoxic stresses independently of BRCA2 function, whereas dynamic interaction between Nbs1 and Rad51 was important for precise HR repair of DSB. These findings demonstrate a mechanism for genome integrity control by Plk1-dependent phosphorylation of Rad51.32P-ATP, efficient incorporation of 32P into full-length Rad51 and the N-terminal Rad51 domain  but not the ATPase core domain (residues 84–339), was detected   near itsdetected B. No phodetected C. S14 phdetected D.Next, we examined whether Rad51 is also phosphorylated in vivo at S14. First, N-terminally FLAG-tagged Rad51 and its S14 variants were transiently expressed in HEK293T cells, and FLAG-purified Rad51 fusions were blotted with the phospho-specific antibody. The pS14 antibody detected signals from wild-type (WT) Rad51 but not from the S14 variants A, demonsIn an attempt to identify the molecular function of Plk1-mediated S14 phosphorylation, we carried out a short sequence BLAST analysis using the Plk1 phosphorylation motif in Rad51 and its surrounding sequence (hereafter designated as Rad51 DTSV motif). This analysis revealed a remarkable similarity to regions of Mdc1 containing SDTD motifs, a known target of CK2, where CK2 phosphorylation of the threonine between two acidic aspartate residues creates a binding site for Nbs1 A Chapma. TherefoKm and Vmax values for a CK2 peptide substrate with an alanine substitution at the pS/T +1 position  was more susceptible to CK2 phosphorylation, whereas a NTD with S14A substitution was less efficiently phosphorylated D. These position . We furt. Nbs1 contains two separate phospho-binding domains, namely the FHA domain and the BRCA1 C-terminus (BRCT) repeat domain, both of which interact with CK2-phosphorylated Mdc1 B Lloyd . TitratiTo examine further whether the S14 residue plays physiologically important roles, U2OS cell lines stably expressing nontagged versions of Rad51 S14 variants were generated A. Flow cOur biochemical analyses shown in BRCA2-defective cancer cells, exhibit profound sensitivity to inhibitors of poly(ADP-ribose) polymerase (PARP); hence, cancers harboring mutations in BRCA2 can be treated effectively with PARP inhibitors  and a truncated GFP (GFPTr) (U2OS-SCR18)  . FollowiS-SCR18) G and S4FBRCA2-defective EUFA423 cells stably expressing Rad51 variants. We exploited a zinc-finger nuclease (ZFN) that introduces a DSB at a native AAVS1 locus within the PPP1R12C gene and a donor plasmid containing a promoterless GFP gene between sequences homologous to those flanking the AAVS1 site  Rad51 recombinase is directly phosphorylated by Plk1 at S14 in a cell cycle- and DNA damage-responsive manner; 2) Plk1-mediated phosphorylation stimulates subsequent CK2-mediated phosphorylation at T13; 3) T13 phosphorylation of Rad51 by CK2 triggers a direct interaction with the FHA domain of the MRN component, Nbs1; and 4) Rad51 phosphorylation at either S14 or T13 is important for accurate HR and for cellular resistance to IR and to PPAR inhibition. Collectively, these data support the model illustrated in Mounting evidence points toward active roles of Plk1 in DNA damage responses  but therDespite the emerging importance of CK2-mediated phosphorylation in DNA damage signaling , the molThis phospho-dependent interaction between Nbs1 and Rad51 also reveals a direct link between DNA damage recognition and HR repair. It is noteworthy that Nbs1 is recruited not only to chromatin, through interactions between the Nbs1 FHA domain and CK2-phosphorylated Mdc1 but also directly to ssDNA regions . The FHAXenopus systems; Rad51 depletion caused G2 arrest with accumulated ssDNA lesions or DSBs  or phospho-mimetic aspartate (S14D) were less proficient in HR than WT Rad51. This observation suggests that both impaired and unduly stable interaction between Rad51 and Nbs1 have negative effects on canonical error-free HR, highlighting the importance of dynamic quality control of HR proteins during DSB repair . Intrigureatment . Clinicareatment . It is tIn summary, we have established a direct link between DNA damage recognition and HR repair, mediated through a phospho-dependent interaction between Nbs1 and Rad51. A primary cell-cycle regulator, Plk1, plays a critical role in the regulation of this interaction, which can modulate the BRCA2 dependency of HR repair. Our findings represent a significant step toward a comprehensive understanding of HR regulation by cell-cycle regulators, which may be exploited in the further development of effective cancer treatments.2 in Dulbecco's modified Eagle's medium supplemented with streptomycin (0.1 mg/ml), penicillin (100 units/ml), and 10% v/v fetal bovine serum. Where indicated, cells were treated with 0.2 mM nocodazole (Sigma-Aldrich) or 9 μM RO-3306 (Enzo Life Sciences) for 20 hr, or 50 μM BTO-1 (Sigma-Aldrich) or 0.1 μM BI-2536 (Axon Medchem) for 2.5 hr. The 137Cs-source of an IBL 637 , siBRCA2 (100 nM), or control siRNA with DharmaFECT1 (100 nM) (Dharmacon); cells were further incubated for 24 hr before analyses.HeLa, HEK293T, and U2OS cells were cultured at 37°C with 5% CO2, 50 mM NaF, 0.1% NP40, 10% glycerol, 1 mM Na3VO4, 20 mM β-glycerophosphate, 1 mM dithiothreitol, 10 mM benzamidine HCl, 25 units/ml Benzonase nuclease [Novagen]) supplemented with Protease inhibitor cocktail . For immunoprecipitation, the extract was precleared with 10 μl of control IgG beads, followed by incubation with antibody cross-linked to beads. After extensive washing, immune complexes were separated by SDS-PAGE and analyzed by western blotting, following standard protocols. Where indicated, the membrane was treated with Re-Blot Plus Mild Solution (Millipore) before incubating with another antibody; for Rad51 phosphorylation analyses, pT13/pS14, pS14, and Rad51 antibodies were applied in this order.Cell extract was prepared using extraction buffer  by the addition of 1 μl of recombinant Plk1 or CK2. Following incubation at 30°C for 30 min, reactions were stopped by heating at 95°C for 5 min in SDS sample buffer. Proteins were then resolved by SDS-PAGE and visualized by staining with InstantBlue (Expedeon). After drying gel with DryEase (Invitrogen), 32P-labeled products were detected by autoradiography. For far-western analysis, kinase reaction was carried out in kinase buffer supplemented with 250 μM ATP, with no γ32P-ATP. The reaction mixture was then resolved by SDS-PAGE and transferred to a Protran nitrocellulose membrane , followed by incubation with recombinant full-length FLAG-Nbs1 (∼ 2 μg). Anti-Nbs1 antibody was then applied to the membrane to detect Nbs1 protein.Protein substrates  were phosphorylated in kinase buffer  at 18°C. Protein samples were dialyzed extensively into 50 mM HEPES (pH 7.5), 150 mM NaCl, 2 mM β-mercaptoethanol and peptides were desalted and buffer exchanged using NAP-5 purification columns  into the relevant buffer . In gene137Cs-source of a GRAVITRON RX 30/55 irradiator (Gravatom) and incubated further for 14 days. Cells were then fixed and stained with Coomassie stain, and colonies of > 50 cells were counted. The mean surviving fraction was calculated as a percentage of the mean seen in the nonirradiated control. To assess sensitivity to the PARP1 inhibitor Olaparib, cells treated with siRNA were seeded at a density of 5,000 cells  . For HR-mediated gene targeting assay, EUFA423 Flp-In cells stably expressing Rad51 variants were seeded at a density of 1.9 × 10(800 μg)  with or


By contrast, the current study establishes a clear correlation between the IC50in vitro growth inhibitory concentration in human cancer cells and the Ki for the inhibition of activity of purified human α1β1 NaK.Surface-expressed Nain vitro growth inhibitory effects of seven cardiac glycosides including five cardenolides  and two bufadienolides  were determined by means of the MTT colorimetric assay and hellebrigenin-induced cytotoxic effects were visualized by means of quantitative videomicroscopy. The binding affinity of ten of the 14 compounds under study was determined with respect to human α1β1, α2β1 and α3β1 NaK complexes. Lactate releases and oxygen consumption rates were also determined in cancer cells treated with these various cardiac glycosides.The in vitro anticancer activity than the corresponding glycoside, the current study demonstrates that the hellebrin / hellebrigenin pair is at odds with respect to this rule. In addition, while some cardiac steroid glycosides , but not the aglycones, display a higher binding affinity for the α2β1 and α3β1 than for the α1β1 complex, both hellebrin and its aglycone hellebrigenin display ~2-fold higher binding affinity for α1β1 than for the α2β1 and α3β1 complexes. Finally, the current study highlights a common feature for all cardiotonic steroids analyzed here, namely a dramatic reduction in the oxygen consumption rate in cardenolide- and bufadienolide-treated cells, reflecting a direct impact on mitochondrial oxidative phosphorylation.Although cardiotonic steroid aglycones usually display weaker binding affinity and in vitro cancer cell growth.Altogether, these data show that the binding affinity of the bufadienolides and cardenolides under study is usually higher for the α2β1 and α3β1 than for the α1β1 NaK complex, excepted for hellebrin and its aglycone form, hellebrigenin, with hellebrigenin being as potent as hellebrin in inhibiting  B refers to the 3H-ouabain bound at a particular concentration of the cardiac glycoside [CG] and BCG=0 refers to the 3H-ouabain bound at 1-2nM 3H-ouabain in the absence of other cardiac glycosides. The KD was calculated from K0.5 by taking into account ouabain-CG competition as KD = K0.5/ (1 + [Ouf]/ KDOu). With KDOu values α1β1 9.2 nM, α2β1 21.5 nM and α3β1 11 nM, respectively [f]/ KDOu were 1.06, 1.03, and 1.06, respectively. Binding of each cardiac glycoside was estimated in 3 separate experiments.Expression of the human isoforms α1β1, α2β1 and α3β1 in is, 1 mM . Binding2, 25 mM histidine, pH 7.4, 1 mM EGTA, 0.01 mg/ml SOPS, 0.001 mg/ml cholesterol, and 0.005 mg/ml C12 E8 in 48-well plates. The reaction (37°C for 1 h) was started by the addition of 1mM ATP. Pi release was measured with a malachite green dye to detect the phosphor-molybdate . The percent inhibition V /V0 was calculated for each cardiac glycoside concentration, and Ki values were obtained by fitting the data to the function V /V0 = Ki /([CG] + Ki ) . V0 and V represent the control rate and rate of NaK-ATPase activity at particular concentrations of cardiac glycosides, [CG], respectively.The inhibition of NaK activity of the purified detergent-soluble α1β1, α2β1, and α3β1 complexes by cardiotonic steroids and an analysis of the inhibition data  were also performed as previously described . The inhD or Ki values ± SEM for each isoform were calculated. Statistical significance was calculated by the unpaired Students t-test. P values <0.05 were considered significant.Average K2 consumption was determined using the MitoXpress assay according to the manufacturer’s instructions . Briefly, after adding the oxygen probe to tumor cells cultured for 24 hours in 96-well microplates with or without the tested compounds, the wells were sealed with mineral oil, and respiration was evaluated using time-resolved fluorescent plate readers. Under these conditions, O2 depletion was translated into an increase in probe phosphorescence signal, and the slope of this signal was used to derive the O2 consumption rate.For enzymatic determination of lactate production, confluent tumor cells were incubated with 10 mM glucose for 24 hours. Culture medium was then filtered through centrifugation columns with a 10 KDa cutoff, and L-lactate concentrations were determined on an ISCUSflex analyzer . OIn general, cardenolides and bufadienolides share a steroid backbone, but these compounds differ from each other on the basis of an unsaturated butyrolactone (cardenolides) versus an α-pyrone (bufadienolides) moiety Figure A. The chin vitro growth inhibition of human cancer cells . However, one clear exception was noticed with hellebrin and hellebrigenin (Table 50 for hellebrigenin (mean IC50 ± SEM: 16 ± 5 nM) compared to hellebrin (mean IC50 ± SEM: 28 ± 7 nM), although the difference was not statistically significant  of the cell lines under study influence the cardiotonic steroid-mediated in vitro growth inhibitory effects of gamabufotalin-rhamnoside / gamabufotalin and hellebrin / hellebrigenin. The data obtained were gamabufotalin-rhamnoside: 1,840 nM; gamabufotalin: > 10,000 nM; hellebrin: > 10,000 nM, and hellebrigenin: > 10,000 nM. Thus, these four compounds display weaker growth inhibitory effects in normal fibroblasts than in cancer cells, as already observed previously with other cardiotonic steroids al cells ,12. ThisThe glycosylation patterns of cardiotonic steroids markedly influence their anticancer activity profiles. For example, Langenhan et al.  demonstr3H-ouabain displacement compared to the aglycone, and in NaK inhibition assays the aglycone is even somewhat superior to the glycoside  also demonstrate the association of cancer cell growth inhibition and inhibition of the low affinity cardiac glycoside-binding rodent α1β1 complex. The gamabufotalin-rhamnoside is therefore a useful tool to check this association in rodent cells because even a “low affinity” effect is in the μM concentration range.The features of binding and inhibition of the human α1β1 complex of the different cardiac glycosides are reflected in the ICNaK α1β1 ,53. It hNaK α1β1 . The par50 growth inhibitory concentration and Ki for inhibition of NaK activity of purified human α1β1 suggests that the inhibition of α1β1 is the first step in the cancer growth inhibition effects of cardiac glycosides, provided that NaK α3β1 is not more present in vitro (as in the case of the current study). This feature is also true for some cancer types that overexpress the NaK α1β1 but not the α3β1 complex, such as gliomas [2+ handling and NAD/NADH ratios [The clear correlation between the IC gliomas , melanom gliomas , NSCLCs  gliomas  and rena gliomas . Anomali gliomas ,4,55, thH ratios -58. ThisH ratios .+/K+-ATPase; NSCLC: Non-small-cell lung carcinoma; QVM: Quantitative videomicroscopy.ATCC: American type culture collection; DSMZ: Deutsche Sammlung von Mikroorganismen and Zellkulturen; ECACC: European Collection of Cell Culture; GBM: Glioblastoma; GGR: Global growth ratio; MDR: Multidrug resistance; MTT: 3--2,5-diphenyltetrazolium bromide; NaK: NaNone of the listed authors have competing interests related to the publication of this manuscript.in vitro growth inhibition measurements and quantitative videomicroscopy analyses. WM performed the MDR assays. AC, DMT, MZ and EU performed the analytical chemistry analyses and compound purification. AK and EA performed the NaK binding studies. AE, BK, WB, OF, SK and RK wrote and edited the manuscript. All authors read and approved the final manuscript.AE, BK, WB, OF, SK and RK conceived of the study and designed the assays. LMYB performed