The mitochondrial signaling complex PKA/AKAP1 protects neurons againstmitochondrial fragmentation and cell death by phosphorylating and inactivatingthe mitochondrial fission enzyme Drp1. Mitochondrial shape is determined by fission and fusion reactions catalyzed bylarge GTPases of the dynamin family, mutation of which can cause neurologicaldysfunction. While fission-inducing protein phosphatases have been identified,the identity of opposing kinase signaling complexes has remained elusive. Wereport here that in both neurons and non-neuronal cells, cAMP elevation andexpression of an outer-mitochondrial membrane (OMM) targeted form of the proteinkinase A (PKA) catalytic subunit reshapes mitochondria into an interconnectednetwork. Conversely, OMM-targeting of the PKA inhibitor PKI promotesmitochondrial fragmentation upstream of neuronal death. RNAi and overexpressionapproaches identify mitochondria-localized A kinase anchoring protein 1 (AKAP1)as a neuroprotective and mitochondria-stabilizing factor in vitro and in vivo.According to epistasis studies with phosphorylation site-mutant dynamin-relatedprotein 1 (Drp1), inhibition of the mitochondrial fission enzyme through aconserved PKA site is the principal mechanism by which cAMP and PKA/AKAP1promote both mitochondrial elongation and neuronal survival. Phenocopied by amutation that slows GTP hydrolysis, Drp1 phosphorylation inhibits thedisassembly step of its catalytic cycle, accumulating large, slowly recyclingDrp1 oligomers at the OMM. Unopposed fusion then promotes formation of amitochondrial reticulum, which protects neurons from diverse insults. Mitochondria, the cellular powerhouse, are highly dynamic organelles shaped byopposing fission and fusion events. Research over the past decade has identifiedmany components of the mitochondrial fission/fusion machinery and led to thediscovery that mutations in genes coding for these proteins can cause humanneurological diseases. While it is well established that mitochondrial shapechanges are intimately involved in cellular responses to environmentalstressors, we know very little about the mechanisms by which cells dynamicallyadjust mitochondrial form and function. In this report, we show that thescaffold protein AKAP1 brings the cAMP-dependent protein kinase PKA to the outermitochondrial membrane to protect neurons from injury. The PKA/AKAP1 complexfunctions by inhibiting Drp1, an enzyme that mechanically constricts andeventually severs mitochondria. Whereas active, dephosphorylated Drp1 rapidlycycles between cytosol and mitochondria, phosphorylated Drp1 builds up ininactive mitochondrial complexes, allowing mitochondria to fuse into aneuroprotective reticulum. Our results suggest that altering the balance ofkinase and phosphatase activities at the outer mitochondrial membrane mayprovide the basis for novel neuroprotective therapies. In addition,fragmentation of neuronal mitochondria is necessary for their transport to andproper development and function of synapses. Moreover, mitochondrial fission is anearly step in the mitochondrial apoptosis pathway, and inhibiting fission can blockor delay apoptosis in a variety of cell types, including neurons Opposing fission and fusion events determine the shape and interconnectivity ofmitochondria to regulate various aspects of their function, including ATPproduction, CaFission and fusion processes are catalyzed by large GTPases of the dynaminsuperfamily. Mitochondrial fission requires dynamin-related protein 1 (Drp1), which,similar to the “pinchase” dynamin, is thought to mechanically constrictand eventually sever mitochondria. Normally a largely cytosolic protein, Drp1 isrecruited to the outer mitochondrial membrane (OMM) by a poorly characterizedmultiprotein complex that includes the transmembrane proteins Fis1 and Mff CDK  isphosphorylated by the cyclin-dependant kinase 1/cyclin B complex, leading tofragmentation of the mitochondrial network during mitosis. Phosphorylation ofSerPKA  is mediatedby both PKA and Ca2+/calmodulin-dependent protein kinase I (CaMKI).Three laboratories, including ours, found that SerPKA phosphorylation byPKA promotes mitochondrial elongation presumably through Drp1 inhibition Our understanding of the signaling events that regulate this group of organelleshaping GTPases is limited. Drp1, in particular, is subject to complexposttranslational modification by ubiquitylation, sumoylation, nitrosylation, andphosphorylation PKA. Phosphorylation traps Drp1 in large, slowlyrecycling complexes, allowing mitochondria to fuse into a neuroprotectivereticulum.We previously showed that Bβ2, a neuron-specific and OMM-targeted regulatorysubunit of protein phosphatase 2A (PP2A), sensitizes neurons to various insults bypromoting mitochondrial fission In an effort to identify signal transduction pathways that alter themitochondrial fission/fusion balance, we investigated the effect of elevatingcAMP levels in PC12 pheochromocytoma cells and primary hippocampal neurons.Mitochondria were visualized by transfection with mitochondria-targeted GFP orby live-staining with the fluorescent dye MitoTracker Red. Application of theadenylate cyclase agonist forskolin either alone or in combination with thephosphodiesterase type IV inhibitor rolipram resulted in the conversion ofmostly punctiform or short and stubby mitochondria to highly elongated andinterconnected organelles . MitochoTo implicate PKA in the morphogenetic effects of cAMP, we established clonal PC12cell lines that inducibly express the PKA inhibitory peptide PKI fused to GFPand the outer mitochondrial anchor domain of MAS70p (omPKI). Cells withinducible expression of an OMM-directed form of the protein phosphatase 1 (PP1)modulator inhibitor-2 (omInh2) were analyzed for comparison . Inducti2O2 andstaurosporine GFP (targeted via the N terminus ofcytochrome oxidase VIII). After 7–14 d, mitochondrial shape was assessedby automated morphometry of confocal z-stacks from perfusion-fixed brainsections .CompareWe carried out epistasis experiments in hippocampal neurons to determine whetherPKA-mediated changes in mitochondrial shape and neuronal survival are causallyrelated. Initially, we showed that overexpression of the antiapoptotic Bcl2protein prevented apoptosis induced by inhibiting PKA with omPKI but had noeffect on omPKI-induced mitochondrial fragmentation . SimilarTo distinguish between these possibilities, we interfered with mitochondrialfission by dominant-negative (K38A mutant) Drp1 or Fis1 knockdown. Blocking themitochondrial fission machinery fully restored viability of neurons transfectedwith omPKI, demonstrating that PKA inhibition kills by inducing mitochondrialfission. In contrast, AKAP1 silencing with two shRNAs compromised neuronalviability in a manner that could not be reversed by inhibiting mitochondrialfission . Our datThe data thus far suggest a model in which the cAMP signaling through the outermitochondrial PKA/AKAP1 complex opposes the mitochondria fragmenting activity ofseveral protein phosphatases including PP2A R2>0.99) and was largely independent of thesize of the bleached area, indicating that Drp1 mobility follows reaction-rather than diffusion-limited kinetics PKA/AKAP1 could maintain elongated mitochondria by inhibiting fission orpromoting fusion reactions. The mitochondrial fission enzyme Drp1 was previouslyshown to be phosphorylated by PKA and phospho-mimetic substitution of thetargeted Ser residue causes mitochondrial elongation 1/2); therefore, we expressedDrp1 turnover as the ratio of the two curve-fitting parameters  andanalyzed mitochondrial morphology at basal or forskolin/rolipram-stimulated cAMPlevels. Mutation of SerPKA in Drp1 rendered cells largely refractoryto mitochondrial elongation by cAMP  on the other hand were completely refractory toH89-induced mitochondrial fission  GFP-Drp1 was completely insensitiveto PKA/AKAP1 recruitment, displaying rapid recovery comparable to wild-type Drp1cotransfected with AKAP1ΔPKA , amembrane-permeant, reversible, and Cys-reactive crosslinker. Transfected COScells were crosslinked in vivo, and cell lysates were subjected toultracentrifugation  to assess Drp1 assembly into large,sedimentable complexes. Compared to co-expression of OMM-targeted GFP,OMM-targeted PKA enhanced oligomerization of wild-type but notSerPKA-mutant Drp1 . Aiming for a milder phenotype, we targeted Thr55 inthe Drp1 GTPase domain (cat) without affecting the Km or apparent affinityof dynamin-2 for GTP This hypothesis predicts that GTPase-impaired Drp1 mutants should behavesimilarly to phospho-Sere domain  based onPKA, the T55D mutation alsoincreased the propensity of Drp1 to form sedimentable oligomeric structuresafter intact cell crosslinking . Drp1 T55Dexpression resulted in a dose-dependent increase in mitochondrial length,demonstrating that the mutation impairs Drp1's fission activity . Remarkaslinking .PKA by PKA/AKAP1) leads to a stableaccumulation of Drp1 at mitochondria, likely by interfering with a disassemblystep that is required for mitochondrial fission.Therefore, inhibition of GTP hydrolysis . Wild-typeGFP-Drp1 foci were mostly found to abut mitochondria, seemingly randomlytranslocating along their length and pivoting around the organelle .MitochoMovies were subjected to automated particle tracking analysis PKA-phosphorylatable Drp1, indicating acritical role for the PKA-Drp1 axis.This report establishes a role for outer mitochondrial PKA and, in particular, thePKA/AKAP1 complex in the maintenance of mitochondrial integrity and the protectionfrom neuronal injury. The importance of cAMP/PKA signaling in cell survival is welldocumented However, our results do not rule out the possibility that PKA may cause mitochondrialelongation by promoting mitochondrial fusion events in addition to inhibitingmitochondrial division. For instance, a recent report showed that forskolin canstimulate mitochondrial fusion in a cell-free assay AKAP1 is a large, multifunctional adaptor protein with several splice variants and ahighly conserved N-terminal transmembrane domain that acts as a mitochondrialtargeting sequence A recent study described nuclear aggregation of mitochondria upon overexpressing anN-terminal fragment of AKAP1 containing the PKA binding domain. The authors did not,however, investigate whether this effect was PKA dependent and instead attributedthe phenotype to a lack of RNA binding to the missing C terminus Both the PKA and the PTPD1/Src interaction domains are important for maintenance ofmitochondrial membrane potential by AKAP1 Consistent with an essential role for AKAP1 in neuronal survival, a recent reportdemonstrated that ischemia induces expression of the E3 ubiquitin ligase SevenIn-Absentia Homolog 2 (Siah2), which targets AKAP1 for rapid proteasomal degradationPKAphosphorylation, and mitochondria of cells expressingSerPKAAla-substituted Drp1 were unresponsive to cAMP and PKA/AKAP1. As toa mechanism, Drp1 activation via AKAP1 silencing was associated with acceleratedcycling of the fission enzyme between cytosolic and mitochondrial pools. Conversely,Drp1 inhibition via cAMP or PKA recruitment or overexpression resulted in theaccumulation of stable Drp1 oligomers at mitochondria and in an extension of thelifetime of mitochondrial Drp1 foci. Because SerPKA phosphorylationdecreased the Kcat of GTP hydrolysis and because a mutation thatstabilizes the GTP bound form of Drp1 mimicked the effects of PKA phosphorylation onlocalization and dynamics of the fission enzyme, modulation of Drp1's GTP cycleemerges as a probable mechanism for the mitochondria-stabilizing and neuroprotectiveactions of PKA/AKAP1.We have identified a conserved PKA phosphorylation site in the GTPase effector domainof Drp1 as the principal mediator of PKA/AKAP1-induced mitochondrial remodeling.AKAP1-mediated redistribution of PKA was shown to augment Drp1 SerPKAA previous FRAP study demonstrated that the apoptosis inducer staurosporine causesaccumulation of slowly recycling mitochondrial YFP-Drp1 complexes, which colocalizewith Bax and Bak. Since the arrest of Drp1 cycling occurs after mitochondria havefragmented but before they release cytochrome C, this phenomenon may be related tothe proapoptotic christae remodeling activity of Drp1 PKA promotestranslocation of the fission enzyme to mitochondria, a conclusion largely based onoverexpression of phosphorylation site-mutant Drp1 PKAD-mutant) GFP-Drp1 partitions mostly with the cytosolic fraction(>90%), oligomerizes less readily than wild-type Drp1, and onlyinfrequently forms mitochondrial punctae (unpublished data), which is essentiallyopposite to the phenotype of Drp1 phosphorylated by PKA. Because Asp substitution ofDrp1 SerPKA at most incompletely reproduces the inhibitory effect ofSerPKA phosphorylation on in vitro GTP hydrolysis PKA with an acidicresidue locks Drp1 into a partially inhibited state, arresting the enzyme at adifferent stage of its subcellular translocation cycle.Seemingly at odds with our findings, another study previously suggested thatcalcineurin-mediated dephosphorylation of Drp1 at SerPKA phosphorylation or mutation of theGTPase domain.Enhanced colocalization or cofractionation of Drp1 with mitochondria has previouslybeen interpreted as evidence for Drp1 activation e.g. . Our datIn support of a model in which PKA/AKAP1 fuses mitochondria by accumulating Drp1 ininactive superstructures , recent Outer mitochondrial PKA-induced super-oligomerization of Drp1 could inhibit celldeath by several, non-mutually exclusive mechanisms. For instance, mitochondrialnetworks resulting from unopposed fusion can sustain higher metabolic activity 1 anti-MTCO2 ,rabbit anti-ERK (Santa Cruz), mouse anti-phospho-SerPKA Drp1 CDK Drp1 , rabbitanti-LacZ and mouse IgG2a anti-V5 epitope tag (Invitrogen), mouseanti-neurofilament , andmouse anti-MAP2B (BD Transduction Laboratories). For immunofluorescencestaining, Alexa fluorophore-coupled secondary antibodies were purchased fromInvitrogen. Infrared fluorophore-coupled secondary antibodies for quantitativeimmunoblot analysis were purchased from LI-COR Biosciences .The following antibodies were used: rabbit anti-GFP , mouseIgG32–2391–30 for PKA and MAS70p1–29 for PKI. Forthe V5-tagged AKAP1 constructs, the AKAP1 cDNA was excised from the GFPconstructs with BglII and SalI and ligated into the pcDNA3.1HisV5 vectordigested with BamHI and XhoI. AKAP1 and Fis1 were silenced by H1-promoter-drivenexpression of shRNAs The core domain of rat AKAP1 that is present in all published splice variants(residues 1–524) was isolated by reverse transcriptase PCR and fused tothe N terminus of EGFP. The PKA-binding deficient mutant I310P, L316P wasgenerated by mutagenesis according to the QuikChange protocol (Stratagene).N-terminally GFP tagged PKA, PKI For analysis of Drp1 phosphorylation, COS cells were cotransfected withGFP-tagged AKAP1 and Drp1 at 2∶3 plasmid mass ratios using Lipofectamine2000. After 24 h, cells were stimulated with various concentrations offorskolin/rolipram for 45 min, lysed in SDS sample buffer containing 2 mM EDTAand 1 µM microcystin, and sonicated with a probe tip to shear DNA. ForAKAP1 immunoprecipitation, PC12 cells were transfected with AKAP1 WT- orAKAP1ΔPKA-GFP. After 48 h, AKAP1 was immunoprecipitated with GFP antibodiesand protein A-agarose beads essentially as described COS cells were transfected with triple HA-tagged Drp1 and either omGFP or omPKAexpressing plasmids at 3∶1 mass ratio and cultured for 24 h. After a washwith PBS, cells were incubated with HBSS containing up to 0.5 mMdithiobismaleimidoethane for 5 min at 37°C. The HBSS/DTME was removed and cells were lysed in buffercontaining 60 mM Tris pH 6.8 and 2% SDS. Insoluble debris was removed bycentrifugation  and cleared lysates (300 µl) werelayered onto 1 ml of a 300 mM sucrose cushion and subjected toultracentrifugation . Pellets were dissolved in SDSsample buffer containing 5% β-mercaptoethanol to cleave thecrosslinker and analyzed by SDS-PAGE alongside an aliquot of the lysate prior toultracentrifugation.Hippocampal neurons from E18 rat embryos 2 chamber, Nalge Nunc) were infected with lentivirus ortransfected using LipofectAmine 2000 as above. Hippocampal cultures wereinfected or transfected at 8 to 10 d in vitro and analyzed 3 to 5 d later. Forlive cell imaging 24 to 96 h post-transfection, cells were incubated with 100 nMTMRM for 30 min at 37°C to visualize mitochondria, and images through themidplane of the soma were captured using a Zeiss LSM 510 laser-scanning confocalmicroscope. For analysis of fixed cells, cultures were subjected toimmunofluorescence staining with antibodies to cytochrome oxidase II (HeLa cellsonly) and GFP HeLa cells, PC12 cells, and hippocampal neurons cultured on, respectively,untreated, collagen-, and poly-D-lysine-coated, chambered No. 1 cover glasses(20 mm2/(4π*a)) andcumulative area:perimeter ratio (Σa/Σp). The form factor is reported asan average of all particles in a region-of-interest (ROI), has a minimum valueof 1 (for perfect circles), and captures well the transition from punctiform toelongated, complex shaped, but still isolated mitochondria. The cumulativearea:perimeter ratio is computed as the summed particle area in a ROI divided bythe summed particle perimeter (including the perimeter of enclosed spaces or“holes”). This metric is particularly effective at detecting thetransition from elongated, isolated mitochondria to a reticular network ofinterconnected mitochondria. Colocalization of GFP-Drp1 with mitochondria  of lentivirus (feline immunodeficiency virus), prepared by theUniversity of Iowa Viral Vector Core, were bilaterally injected into thestriatum and hippocampus using a 10 µl Hamilton syringe with a 30°beveled 30 gauge needle. Virus expressing mitochondria-targeted GFP (viaresidues 1–31 of cytochrome oxidase subunit VIII) and AKAP1-GFP weredelivered into the left and right hemisphere, respectively. The followingcoordinates were used with the incisor bar at 3.3 (from bregma): Striatum,AP = 0, ML = ±3.5,DV = −4.5; Hippocampus,AP = −4.0, ML = ±2.0,DV = −3.5. Animals were sacrificed 7–14 d laterand transcardially perfused with 4% paraformaldehyde. 40 µm thickcoronal cryostat sections were processed for indirect immunofluorescence withantibodies to GFP and MAP2B and counterstained for nuclei with TOPRO-3. Ten to22 confocal z-sections of infected neurons, 0.8 µm apart, were capturedand analyzed for mitochondrial shape by digital morphometry as describedabove.All animal procedures were approved and carried out according to theInstitutional Animal Care and Use Committee (IACUC) at the University of Iowa.Adult male Lewis rats were anesthetized with 91/9.1 mg/kg of ketamine/xylazineand placed in a stereotaxic apparatus. Concentrated stocks (∼101/2) and mobile fraction (mFx). Drp1dynamics followed double exponential recovery kinetics withR2 values of generally greater than 0.99.Control experiments, in which bleach recovery of cytosolic and mitochondrialDrp1 was tracked separately by masking the GFP channel with the MitoTrackerchannel, showed that mitochondrial masking does not significantly affect theresults (unpublished data).Hela and PC12 cells were grown on collagen-coated, chambered No. 1 cover glasses, transfected 24 to 48 h, and stained with MitoTracker DeepRed 30 min prior to analysis. Cells with non-mitochondrial GFP-Drp1 aggregates,a sign of overexpression, were excluded from the analysis. Using the 488 nmlaser line of a Zeiss LSM 510 confocal microscope, an approx. 5×5 µmregion of mitochondria-associated GFP-Drp1 was bleached and fluorescencerecovery was tracked by capturing images every 5 s for 5 min. Image stacks wereanalyzed, and recovery curves normalized to pre-bleach intensity and correctedfor acquisition bleach 2(5%) control. Frames were captured at 30 s intervals for 60 to 90 min,contrast-enhanced , and analyzed with the Particle Trackerplugin for ImageJ HeLa cells cotransfected with GFP-Drp1, dsRed2/mito, and either empty vector orPKA catalytic subunit at 15∶4∶1 plasmid ratios were subjected totime lapse imaging using the 63× objective of a motorized Leica AF6000epifluorescence microscope under temperature (37°C) and COCOS cells expressing GFP-Drp1 with or without omPKA were permeabilized in 0.5mg/ml digitonin, 100 mM KCl, 20 mM HEPES pH 7.4, 1 mM EDTA, 1 mM EGTA, 1 mMbenzamidine, 5 µg/ml leupeptin, 1 mM dithiotreitol (DTT), and 1 mMphenylmethylsulfonyl fluoride (PMSF). Cytosolic and heavy membrane fractionswere prepared by centrifugation .t test (two-tailed) forsingle comparisons and by one-way analysis of variance (ANOVA) followed bypairwise Bonferroni post hoc tests for multiple comparisons.Data were analyzed by Student's Figure S1Reference image-based and automated methods yield similar mitochondrialmorphology scores. (A) Confocal images of TMRM-stained mitochondria inhippocampal neurons were used as reference images for quantification ofmitochondrial morphology (increasing length scores from 0 to 4). Comparison of blinded reference image-based length scores (B) and digitalmorphometry (C) applied to confocal images of hippocampal neurons expressingBβ2- or AKAP1-directed or nonsense (NS) shRNAs (means ± s.d. of12–28 neurons). The cumulative area:perimeter ratio (Σa/Σp)can account for mitochondrial clusters and achieves significance valuesapproaching that of the reference-image-based method. (D) Cell-by-cellcorrelation of reference-image-based length scores and cumulativearea:perimeter ratios in neurons expressing Bβ1 or Bβ2 and Bcl2.Small symbols represent values from individual neurons; the large symbolsare population means (± s.d.).(0.38 MB TIF)Click here for additional data file.Figure S2Renilla luciferase, and shRNAs .Knockdown was assessed 3 d later by dual-luciferase assays. AKAP1-directedshRNAs were as effective as a positive control shRNA targeting the luccoding sequence. (C) PC12 cells transfected with the indicated shRNAplasmids were probed for endogenous AKAP1 and ERK1/2 expression (duplicatecultures). At least two AKAP1 splice variants are expressed in PC12 cells,with the ∼120 kD variant predominating. (D) HeLa cells were transfectedwith GFP-Drp1 expression plasmids that include (+) or exclude (−)a H1 promoter-shRNA cassette to silence endogenous Drp1, and GFPimmunoprecipitates were probed with Drp1 antibodies. Endogenous Drp1assembles with (RNAi-resistant) GFP-Drp1 only in the absence ofDrp1-directed shRNA.A point mutation and two shRNAs interfere with AKAP1 function/expression andDrp1 shRNA permits assembly of pure GFP-Drp1 oligomers. (A) Wild-type (WT)and mutant  AKAP1-GFP wereimmunoprecipitated via GFP from transfected COS cells and analyzed forassociation with the PKA regulatory subunit RIIα; the asterisk indicatesimmunoglobulin heavy chain. (B) Hippocampal cultures were cotransfected witha 1∶1∶2 mass ratio of plasmids expressing the indicated targetproteins fused to the N terminus of firefly luciferase (luc),(0.25 MB TIF)Click here for additional data file.Figure S3p values refer to comparisons to control(nonsense shRNA) transfected conditions.AKAP1 regulates mitochondrial shape in dendrites of hippocampal neurons.Hippocampal cultures were transfected with mitochondrial GFP and theindicated constructs at 1∶4 mass ratio at 10 DIV and fixed 3 d laterfor analysis of mitochondrial shape in dendrites. (A) Representativeepifluorescence images and graphs (B) showing mitochondrial form factor(mean ± s.e.m. of 40–50 neurons from a representativeexperiment). (0.26 MB TIF)Click here for additional data file.Figure S4Rescue of rat AKAP1 knockdown-induced mitochondrial fragmentation byexpression of human AKAP1. PC12 cells were cotransfected with mitochondrialGFP, the indicated shRNAs, and either human AKAP1 cDNA or empty vector,fixed after 3 d, and analyzed by epifluorescence microscopy. (A)Representative images  and graphs (B)depicting mitochondrial shape analysis from a representative experiment(mean ± s.e.m. of 300–400 cells per condition).(1.31 MB TIF)Click here for additional data file.Figure S5Neuronal death due to AKAP1 silencing cannot be rescued by inhibitingmitochondrial fission. Hippocampal cultures were cotransfected with plasmidsexpressing AKAP1-directed or control shRNAs and the indicated rescueplasmids and analyzed after 5 d for mitochondrial morphology (A) andapoptosis (B) (means ± s.e.m. of 3–5 experiments). Bcl2, butneither dominant-negative Drp1 nor Fis1 RNAi, attenuates apoptosisassociated with AKAP1 knockdown.(0.14 MB TIF)Click here for additional data file.Figure S6Protonophore-mediated mitochondrial fragmentation does not affect Drp1dynamics. HeLa cells expressing GFP-Drp1 were treated with either vehicle or 10 µM carbonylcyanide p-trifluoromethoxyphenylhydrazone(FCCP) and 2 µM oligomycin  for 1 to 4 h, during which time Drp1 turnover wasmeasured by FRAP. FCCP leads to dramatic mitochondrial fragmentation asquantified by form factor (A) but has no effect on Drp1 recovery .(1.53 MB TIF)Click here for additional data file.Figure S7PKA Drp1 (pDrp1) and total Drp1 on the sameblot using a dual-channel infrared imager. Drp1 phosphorylation wasquantified as the ratio of phospho- to total Drp1 signals normalized to thehighest value. Shown is one experiment representative of two.AKAP1 enhances forskolin-stimulated Drp1 phosphorylation in PC12 cells. PC12cells cotransfected with GFP-Drp1 and either empty vector, AKAP1, or AKAP1ΔPKA were stimulated for 45 min with the indicated concentrations offorskolin in the presence of rolipram (2 µM). Total cell lysates wereprobed for phospho-Ser(0.11 MB TIF)Click here for additional data file.Figure S8PKA. Primary hippocampal neurons cotransfected with wild-typeor SerPKA-mutant GFP-Drp1 and either vector, wild-type, or PKAbinding-deficient (ΔPKA) AKAP1 were analyzed for morphology of dendriticmitochondria after immunofluorescence staining for the OMM-protein TOM20(representative images in A). (B) and (C) show mitochondrial form factor andlength, respectively, from the same set of neurons (means ± s.e.m. of12–24 neurons per condition from two culture dates).Elongation of dendritic mitochondria by AKAP1 requires Drp1Ser(0.36 MB TIF)Click here for additional data file.Video S1AKAP1 slows Drp1 fluorescence recovery. Coexpression with AKAP1 slowsrecovery of GFP-Drp1 in the photobleached square compared to when eitherprotein is mutated.(0.80 MB MPG)Click here for additional data file.Video S2A mutation in the GTPase domain slows Drp1 fluorescence recovery. FRAPkinetics of the T55D mutant are attenuated compared to wild-typeGFP-Drp1.(0.89 MB MPG)Click here for additional data file.Video S3PKA and GTPase mutation extend the lifetime of mitochondrial Drp1 punctae.Coexpression with the PKA catalytic subunit or T55D substitution leads tothe accumulation of large GFP-Drp1 punctae that turn over more slowly thanwild-type Drp1 without PKA.(1.87 MB MPG)Click here for additional data file.


Bulk degradation of cytoplasmic material is mediated by a highly conserved intracellular trafficking pathway termed autophagy. This pathway is characterized by the formation of double-membrane vesicles termed autophagosomes engulfing the substrate and transporting it to the vacuole/lysosome for breakdown and recycling. The Atg1/ULK1 kinase is essential for this process; however, little is known about its targets and the means by which it controls autophagy. Here we have screened for Atg1 kinase substrates using consensus peptide arrays and identified three components of the autophagy machinery. The multimembrane-spanning protein Atg9 is a direct target of this kinase essential for autophagy. Phosphorylated Atg9 is then required for the efficient recruitment of Atg8 and Atg18 to the site of autophagosome formation and subsequent expansion of the isolation membrane, a prerequisite for a functioning autophagy pathway. These findings show that the Atg1 kinase acts early in autophagy by regulating the outgrowth of autophagosomal membranes.  •The Atg1 kinase phosphorylation consensus was identified on peptide arrays•Atg9 is a direct target of the Atg1/ULK1 kinase in vitro and in vivo•Atg9 phosphorylation recruits Atg18 and Atg8 to the PAS•Atg9 phosphorylation is required for isolation membrane expansion/autophagy function Autophagy function is pivotal to cell health. Papinski et al. identify the phosphorylation consensus of the central kinase in this pathway, Atg1. The autophagy-related protein Atg9 is a direct target of Atg1. Atg9 phosphorylation by Atg1 is required for autophagosome formation. This finding sheds light on how Atg1 controls autophagy. Macroautophagy, hereafter referred to as autophagy, is an important cellular mechanism for the bulk degradation of cytoplasmic material. Autophagy is a sequential process beginning with the sequestration of cytoplasmic cargo in an expanding membrane sac known as the phagophore, which forms at the preautophagosomal structure (PAS). The phagophore expands to form a double-membrane autophagosome that engulfs the cargo and subsequently fuses with the lysosome or vacuole where the cargo is degraded. This conserved process serves to ensure cellular homeostasis, and is integral for the cellular response to stress conditions such as nutrient starvation as well as in embryonic development and the defense against several human pathogens. Indeed, defects in autophagy pathways have been associated with numerous human pathologies including infectious diseases, neurodegenerative disorders, and cancer. Despite these fundamental functions, the molecular mechanisms underlying autophagy regulation remain poorly understood.Caenorhabditis elegans and Drosophila melanogaster, and the homologs Unc-51-like kinase 1 (ULK1) and ULK2  tag  quantification. We purified Atg1, the kinase-dead Atg1 D211A mutant, Atg2, and Atg9 from rapamycin-treated wild-type and HTB) tag . These cHTB) tag B (KijansHTB) tag .Eleven autophosphorylation sites on Atg1 and four Atg1-regulated phosphorylation events on Atg2 were detected C and S1EIn addition to S249, S1086 on Atg2 also conforms to the Atg1 consensus F. Thus, atg1Δ cells, showing that these sites are indeed phosphorylated in an Atg1-dependent manner in vivo . Indeed, whereas soluble Atg1 complex purified from yeast readily phosphorylated the C-terminal Atg9757–997 fragment, phosphorylation was strongly decreased upon mutation of all four consensus phosphorylation sites  purified from on sites D.In summary, these findings suggest that Atg9 is a bona fide target of Atg1, which directly phosphorylates Atg9 on several sites in vitro and in vivo.atg9Δ cells. S657, S831, and S948 were analyzed individually, in all possible double-mutant permutations, as well as the triple mutant  or aspartate/glutamate  and analyzed for their effect on the Cvt pathway or autophagy when expressed in  3A, 3D; C. In add 3A, 3D; D. First, 3A, 3D; C, Atg9 c 3A, 3D; A. Variou 3A, 3D; A, indicaatg9Δ cells. Interestingly, in Atg9 nonphosphorylatable mutant cells , the starvation-induced increase in Pho8Δ60 activity amounted to only 10%–30% of that in wild-type cells, indicating that Atg9 phosphorylation is required for bulk autophagy in vivo  and efficient recruitment of this key autophagy factor and Atg8 to the PAS. This in turn is required for PAS assembly capable of Atg8-positive isolation membrane formation and elongation.Consensus motifs of a large number of yeast kinases have previously been analyzed using peptide arrays . HoweverAtg2 and Atg18 are essential for autophagy and the Cvt pathway. Atg18 contains a WD40 propeller and directly binds to Atg2 , and botAtg9 phosphorylation by Atg1 is required both under rich conditions and upon starvation, as the Cvt pathway and autophagy are nonfunctional in Atg9 mutant cells. The serine-to-aspartate mutations in Atg9 seem unable to mimic the phosphorylated state. Although this unfortunately does not allow analysis of Atg9 function during continuous phosphorylation, it is conceivable that cycles of phosphorylation and dephosphorylation are required for progressive autophagy and the Cvt pathway. Further analyses of the precise mechanism, temporal control, and involvement of Atg9 phosphorylation during Atg9 vesicle cycling and PAS formation will be important directions for future studies.Interestingly, although Atg1 kinase activity is required for Atg9 retrograde transport, direct phosphorylation of Atg9 by Atg1 was not required for this event, as fluorescence microscopy analyses revealed no significant alterations in the localization of nonphosphorylatable Atg9 mutants. Our data do not exclude the possibility that both antero- and retrograde trafficking kinetics are altered and the overall movement of Atg9 vesicles is slowed down. However, it seems more likely that Atg1 regulates Atg9 trafficking by other means than direct phosphorylation of Atg9. Thus, the critical Atg1 kinase substrate controlling Atg9 shuttling remains to be identified. Notably, the Atg1 LIR mutant, which is defective in binding to autophagosomes but still localizes to the PAS normally , shows nWith the complex interplay of many autophagy proteins in PAS function and autophagosome formation, future studies are required to unravel the precise interplay and mechanism underlying Atg1 signaling in autophagy and Cvt pathway function. The identification of Atg9 as an important and direct Atg1 kinase target adds another piece to this still largely unassembled puzzle.Yeast strains and plasmids are listed in 600 of 2 and treated with 220 nM rapamycin for 1 hr, harvested by centrifugation, and washed in PBS, 2% glucose. Cells were then resuspended in a pellet volume of lysis buffer  and frozen in droplets in liquid nitrogen. After cell disruption with a freezer mill , the extract was thawed in lysis buffer and cleared by centrifugation. The cleared extract was incubated with 160 μl of IgG-coupled magnetic beads  for 1 hr at 4°C with rotation. The beads were washed six times for 5–10 min in lysis buffer with rotation and cleaved with lysis buffer containing 0.5 mM DTT and TEV protease for 1 hr at 16°C with slow shaking.Yeast cells were grown in 2 l of YPD medium to an ODAtg1 was genomically tagged at the C terminus with histone H3-HA (H3HA), and Atg9 with a C-terminal histone lysine methyltransferase domain (HKMT) was cloned in a CEN plasmid under the control of its endogenous promoter. Both proteins were tested for functionality by analyzing Ape1 processing. The M-Track methylation assay was performed as described . Methyla3D: 0.0025; P5D: 0.0020; P6D: 0.00022; P3A: 0.00033; P5A: 0.00013; P6A: 0.0000091; Pdelta: 0.0000006. All p values were calculated using an unpaired two-tailed Student’s t test. A p value less than 0.05 was considered to be significant. The intensities from the MS1 filtering in Additional methods can be found in the D.P., M.S., W.R., L.W., C.A.B., A.M., I.H., T.P., J.H.L., and C.K. performed the experiments shown in the manuscript. M.K., I.S., S.S.L., and A.B. performed initial experiments. D.P., W.R., L.W., C.A.B., A.M., B.E.T., R.A., G.A., M.P., and C.K. participated in the experimental design. M.P. and C.K. wrote the paper.


By mass spectrometry analysis of mouse Cystic Fibrosis Transmembrane-conductance Regulator (mCFTR) expressed in yeast we have detected 21 phosphopeptides accounting for 22 potential phospho-residues, 12 of which could be unambiguously assigned. Most are conserved in human CFTR (hCFTR) and the majority cluster in the Regulatory Domain, lying within consensus sequences for PKA, as identified in previous mammalian studies. This validates our yeast expression model. A number of phospho-residues were novel and human conserved, notably mouse Ser670, Ser723, Ser737, and Thr1467, that all lie in acidic sequences, compatible with their phosphorylation by protein kinase CK2. Thr1467 is localized in the C-terminal tail, embedded in a functionally important and very acidic sequence (EETEEE) which displays an optimal consensus for protein kinase CK2. Herein, we show that Thr1467, homologous to human Thr1471 is readily phosphorylated by CK2. Indeed a 42 amino acid peptide encompassing the C-terminal segment of human CFTR is readily phosphorylated at Thr1471 with favorable kinetics (Km 1.7 µM) by CK2 holoenzyme, but neither by its isolated catalytic subunit nor by other acidophilic Ser/Thr kinases . Our finding that by treating CFTR expressing BHK cells with the very specific CK2 inhibitor CX4945, newly synthesized wild type CFTR (and even more its Phe508del mutant) accumulates more abundantly than in the absence of CK2 inhibitor, supports the conclusion that phosphorylation of CFTR by CK2 correlates with decreased stability of the protein. Cystic fibrosis (CF) is caused by mutations affecting a polytopic integral membrane protein, termed Cystic Fibrosis Transmembrane-conductance Regulator (CFTR) functioning as a phosphorylation stimulated, ATP-dependent anion channel. Pathogenic mutations of CFTR either impair its anion channel functions or make the protein unstable determining its premature degradation. This latter event is promoted by the commonest mutation of CFTR, a deletion of the amino acid residue phenylalanine 508 (Phe508delCFTR), which is found in 70–90% of CF patients. Phe508delCFTR is only partially functional when it is induced to traffic to the cell membrane In an attempt to disclose molecular events that may underlie deregulation of CFTR functionality and/or stability, attention has been focused on post-translational events, with special reference to phosphorylation. These studies clearly demonstrated that multiple phosphorylation of the regulatory domain of CFTR by PKA and probably also by other basophilic protein kinases such as PKC is essential to confer the full channel functionality to CFTR. The same residues phosphorylated in vitro by PKA Intriguingly however CFTR also includes several potential phospho-acceptor sites that display the consensus for the most pleiotropic acidophilic protein kinase, CK2  is abundantly expressed and where a methodology for the expression of CFTR by a protocol that overcomes proteolysis problems has been recently set up By MS analysis of CFTR expressed in yeast we have now been able to show that besides all the “canonical” PKA sites concentrated in the R domain (whose detection demonstrates the reliability of the “yeast” approach) we additionally observe that some novel phospho-residues previously never found in CFTR from animal cells, are present in mouse CFTR expressed in yeast. One of these is the best predicted site for CK2, Thr1467 that is conserved in human CFTR (Thr1471): this residue is also readily phosphorylated in vitro with favourable kinetics by CK2 holoenzyme once included in a recombinant fragment of human CFTR encompassing its C-terminal segment. This outcome is described in the present report together with pharmacological evidence supporting the view that CFTR phosphorylation by CK2 may correlate with accelerated degradation of the protein.33P]ATP (3000 Ci/mmol) as well as HRP (horseradish peroxidase)-conjugated antibodies were purchased from PerkinElmer. Mouse monoclonal anti-CFTR antibody (10B6.2) that recognizes a specific epitope on NBD1 domain was from CFTR folding consortium (www. cftrfolding.org). Complete protease inhibitor cocktail tablets were from Roche Diagnostic. Okadaic acid was purchased from Alexis Biochemicals. Methotrexate was from Santa Cruz Biotechnology. CX4945 was from SYN|thesis med chem.All chemicals, cell culture media, anti-β tubulin antibody, and phosphatase inhibitor cocktails 2 and 3 were from Sigma-Aldrich. ATP . The reaction was started by the addition of the indicated protein kinases. The reaction mixtures were incubated for 10 minutes at 37°C and stopped by the addition of 2× concentrated Tricine sample buffer  glycerol, 4% (w/v) SDS, 0.0025% (w/v) Coomassie Blue G, 0.0025% (w/v) Phenol Red and 105 mM DTT). Samples were separated on Tricine-SDS-PAGE following the Schägger’s protocol 33P incorporation was evaluated by gels autoradiography on PerkinElmer’s Cyclone Plus Storage Phosphor System. Initial rate data were analyzed with the program Prism .Peptides derived from human CFTR C-terminal region were phosphorylated by incubation in a 20-µl volume containing 50 mM Tris/HCl, pH 7.5, 10 mM MgClStructural model of the last 42 aminoacids of CFTR was built using ab initio Rosetta tool Protein-protein docking analysis was performed using two FFT-based docking software PIPER Molecular dynamics (MD) simulations of the final complex (parameterized with AMBER99) were performed with NAMD 2.8 Saccharomyces cerevisiae was subjected to tryptic digestion followed by TiO2 phosphopeptide enrichment prior to MS analysis as detailed in the Experimental section. From the MS spectra (Xenopus oocytes Full length mouse CFTR (mCFTR) expressed in  spectra  and S2 i spectra  leading  spectra . In a feSeveral phospho-residues identified by our analysis however were not previously reported to be phosphorylated in vivo: one or more are located in the N-terminal segment (Ser45 and/or Ser50) and at the end of the R domain (Ser808 and Ser811), most of which are conserved in hCFTR. Also conserved is Ser670 that lies at the end of the R domain, whose in vitro phosphorylation by both PKA Phospho-Thr1467, close to the C-terminus and sitting in a region which is 100% conserved in hCFTR (Thr1471), attracted our attention for three reasons: i) it conforms to the optimal sequence recognized by CK2, ii) it is adjacent to the PDZ interacting motif which is essential for interaction with AMP-activated protein kinase To corroborate the implication of CK2 as a phosphorylating agent of CFTR Thr1471 a 42 amino acid-long recombinant fragment encompassing the C-terminal segment of hCFTR was generated and tested for its phosphorylation by CK2. As shown in We also determined whether other acidophilic Ser/Thr protein kinases, besides CK2, are able to phosphorylate Thr1471. While protein kinases CK1 and the genuine Golgi apparatus casein kinase (GCK), recently identified with FAM20C protein Given the location of Thr1471 close to the PDZ-interacting motif that favors retention of CFTR within the cell by interacting with Golgi associated CAL  To provide evidence about the implication of CK2 in processes affecting the stability of CFTR and/or of its Phe508del mutant which is the commonest cause of Cystic Fibrosis, advantage has been taken of the most selective cell permeable CK2 inhibitor available, CX4945. In order to evaluate the efficacy of CX4945 on the level of de novo synthesized CFTR, protein synthesis was inhibited by two hours cell treatment with cycloheximide (CHX). Blockade was then removed by washing off CHX and newly synthesized CFTR was quantified after 2, 4, and 8 hrs incubation either in the absence or presence of CX4945. As shown in It can be concluded from the data of Expression of CFTR in yeast was primarily aimed at producing sufficient amount of the protein for structural studies Based on this supposition we wanted to see if CFTR expressed in a foreign environment like yeast, where CK2 (as well as PKA and many other important Ser/Thr protein kinases) are abundant, but where the homeostatic mechanism leading to CFTR degradation is lacking and proteolysis has been prevented by a number of devices, CFTR is phosphorylated, and at which residues, by CK2.Xenopus oocytes The study presented here led to the identification of 22 potential phospho-residues (12 of which were unambiguously assigned) in mouse CFTR expressed in yeast, all of which, with just a few exceptions, are conserved in human CFTR. Seven of these coincide with phospho-residues previously identified in human CFTR expressed in mammalian cells and/or To the best of our knowledge the other phospho-residues detected by us in CFTR were not reported before: one however (Ser670) was previously reported to undergo in vitro phosphorylation by both PKA At variance with the previously identified CFTR phospho-sites, which are almost exclusively located in the R domain and display the PKA consensus, several of the newly identified ones described here are located close to acidic residues which may suggest their targeting by CK2 and some are located outside the R domain: in particular one or two are located in the N-terminal segment, but could not be assigned with good confidence to individual positions see , though Our attention was mainly focused on phospho-Thr1471 (human numbering), since besides being highly conserved in human CFTR and displaying an optimal sequence for CK2 phosphorylation, it is adjacent to the PDZ interacting motif which is essential for interactions with the scaffold protein NHERF-1, anchoring CFTR to the cytoskeleton To get clues about the possible consequences of CFTR phosphorylation by CK2, advantage has been taken of the most selective CK2 inhibitor available to date, CX-4945 By treating CFTR expressing BHK cells with cycloheximide, in order to block protein synthesis and then restoring protein synthesis by washing away the inhibitor we observed that the re-appearance of CFTR reached higher levels if cells were incubated with the CK2 inhibitor CX-4945, as compared to control. This indicates that inhibition of CK2 results in larger accumulation of newly synthesized CFTR, consistent with diminished degradation of the protein. Interestingly such an effect is definitely more pronounced in the case of Phe508delCFTR with a two to three-fold increment of band B upon treatment with the CK2 inhibitor see , suggestAlthough additional work is needed in order to understand the significance of CFTR phosphorylation by CK2, with special reference to the enigmatic role of Thr1471, our data disclose the importance that a number of previously undetected phosphoacceptor sites may have in determining the stability and the functionality of CFTR. Our experiments also highlight the role of the master kinase CK2 in these processes, providing, from an empirical stand point, a new strategy to increase the yield of functional CFTR in CF cells, possibly by combining CK2 manipulation with usage of correctors and/or potentiators.Figure S1The sequence of mouse CFTR with all phosphopeptides identified is reported together with peptide identification confidence and phosphorylation site probability that are indicated with a color code. Table at the end of the file summarizes the data, by listing all identified phosphopeptides with their corresponding phosphorylation sites.(TIF)Click here for additional data file.Figure S2Annotated fragmentation spectra of the phosphopeptides identified in this study.(PDF)Click here for additional data file.Figure S3MS/MS spectrum of phosphopeptide EETEEEVQDTR from Human CFTR.(TIF)Click here for additional data file.Figure S4BHK cells expressing WT, WT/T1471A or Phe508delCFTR were exposed to either cycloheximide  or Brefeldin A . CFTR synthesis was blocked by two hours treatment with CHX as it is shown by the attenuation/disappearance of the ER-resident band B of CFTR. On the contrary the fully mature band C is not affected by CHX treatment due to its slower turnover. Conversely, when the escape from ER is prevented by using Brefeldin A, band B increases [a]. [a] Glozman R, Okiyoneda T, Mulvihill CM, Rini JM, Barriere H, Lukacs GL (2009) N-glycans are direct determinants of CFTR folding and stability in secretory and endocytic membrane traffic. J. Cell Biol. 184, 847–862.(TIF)Click here for additional data file.Table S1Phosphopeptides identified from mouse CFTR. For each peptide the following details are reported: aminoacid sequence, modifications, phosphorylation site/s, pRS score, pRS probability and pRS site probability, q-value, PEP score and number of missed-cleavages.(DOC)Click here for additional data file.Material S1(DOC)Click here for additional data file.


Hippo pathways are ancient signaling systems that contribute to cell growth and proliferation in a wide diversity of eukaryotes, and have emerged as a conserved regulator of organ size control in metazoans. In budding yeast, a Hippo signaling pathway called the Regulation of Ace2 and Morphogenesis (RAM) network promotes polarized cell growth and the final event in the separation of mother and daughter cells. A crucial regulatory input for RAM network control of cell separation is phosphorylation of a conserved hydrophobic motif (HM) site on the NDR/LATS family kinase Cbk1. Here we provide the first direct evidence that the Hippo-like kinase Kic1 in fact phosphorylates the HM site of Cbk1, and show that Kic1 is allosterically activated by Hym1, a highly conserved protein related to mammalian MO25. Using the structure of mammalian MO25 in complex with the Kic1-related pseudokinase STRAD, we identified conserved residues on Kic1 that are required for interaction with Hym1. We find that Kic1 and Hym1 protein levels remain constant throughout the cell cycle but the proteins’ association is regulated, with maximal interaction coinciding with peak Cbk1 HM site phosphorylation. We show that this association is necessary but not sufficient for this phosphorylation, suggesting another level of regulation is required to promote the complex to act upon its substrates. This work presents a previously undiscovered cell cycle regulated interaction between a Hippo kinase and a broadly conserved allosteric activator. Because of the conserved nature of this pathway in higher eukaryotes, this work may also provide insight into the modularity of Hippo signaling pathways. Drosophila melanogaster, the Hippo/MST signaling pathway includes a set of genes responsible for controlling tissue size by regulating cell proliferation and cell death ATP. We incubated reaction mixtures at room temperature for the indicated amount of time and quenched the reactions by addition of 5× SDS-PAGE sample buffer with immediate mixing followed by 10 min of incubation at 85°C. We resolved proteins on a 12% SDS-PAGE gel, and then stained with Coomassie Blue. We evaluated the extent of Cbk1 labeling by using a Storm 860 Phosphorimager (Molecular Dynamics) to quantify the amount of 32P incorporated into Cbk1. We measured pixel intensities by using ImageQuant software (Molecular Dynamics) and compared volume over gel background in regions of interests encompassing the Cbk1 bands with regions of interests encompassing simultaneously acquired spots of known 32P quantities. For Kic1-Hym1 kinase assays, we used equimolar (1 µM) GST-Kic1(1-359)-His All strains generated and used in this study are derived from the BY4741 genetic background. We generated deletion, hemagglutinin (HA)-tagged, and myc-tagged strains by standard gene replacement or tagging . We cultKIC1 and HYM1 coding sequence along with sequences 500 bp upstream and downstream of the start and stop codons from genomic DNA into pRS315 and pRS316 vectors. To generate kic1 alleles, we introduced substitutions by site-directed mutagenesis using Pfu Turbo (Agilent). We constructed the Hym1-Kic1 fusion construct by cloning the Hym1-13myc sequence 5’ to the Kic1-3HA in a pGREG506 vector [ADH1 promoter, resulting in pGREG506-PADH1-HYM1-13myc-KIC1-3HA. We introduced plasmids by standard lithium acetate transformation and cultured cells in synthetic minimal selection medium . We constructed centromeric Kic1 and Hym1 plasmids by cloning full length 6 vector  with an CTS1 and ACT1 primers as previously described [ For QPCRs, we prepared RNA from log-phase cultures by hot acid phenol extraction and performed quantitative RT-PCRs using escribed .GAL-CDC20 synchrony experiments, we grew cells to log phase at 30°C in either YP or synthetic minimal media + 2% galactose, filtered, and resuspended into an equal volume of YP + 2% glucose and continued to grow the cells at 30°C for 2.5 h until M phase arrest (assessed by accumulation of large-budded cells). We filtered the cells again, resuspended them into YP + 2% galactose, grew at 30°C, and removed samples at indicated time points. We harvested cells by centrifugation, fixing a small fraction in 3.7% formaldehyde for microscopy of budding index and then freezing the remainder in liquid nitrogen. For the For Kic1-Hym1 co-immunoprecipitations, we prepared cell lysates by bead beating as previously described , and imm For all Western blot assays, we transferred proteins to Immobilon-FL polyvinylidene difluoride  membrane. We blocked the membranes for 30 min at room temperature in Odyssey blocking buffer (LI-COR). We probed for Kic1-HA with 1:1000 anti-hemagglutinin (HA) mAb  and Hym1-myc with 1:1000 rabbit polyclonal anti-c-Myc (Rockland). We probed for phosphorylated Cbk1 HM site with 1:500 affinity-purified phosphospecific rabbit antibody (anti-pT743) . We incu We grew cells to mid-log phase performed differential interference contrast (DIC) microscopy using an Axiovert 200 M microscope  with a Cascade II-512B camera . We captured images with a 100×/1.45-numerical aperture oil immersion objective using the Openlab software (Improvision).in vitro , which is comparable to the equilibrium binding constants of mammalian MO25 and MST kinases (2-10 µM) [Kic1 was identified as an interacting partner of the helical repeat protein Hym1, which is closely related to mammalian MO25 -30. Strung cbk1∆ ,27,28. T2-10 µM) .in vitro results suggest that, like the mammalian MO25-STRAD complex, Hym1 acts as an allosteric activator to Kic1. While Kic1-Hym1 clearly phosphorylated the Cbk1 HM site, this was only robustly detectable using the anti-pT743 antibody, and overall our findings suggest that the HM site is relatively inefficiently phosphorylated in the context of the Cbk1-Mob2 complex compared to a short peptide of Cbk1. Because Cbk1 is only functional when bound to its co-activator Mob2 , we asseGiven that Hym1 is highly similar to the mammalian MO25 protein and can enhance Kic1 kinase activity, we hypothesized that the two pathways likely have similar biochemical mechanisms. Previous studies have shown that MO25 binds to a Kic1-related pseudokinase STRAD as well as several MST/Hippo kinases ,34. MO25in vivo when we made this mutation endogenously  interaction of proteins following lysis to form complexes not present in the cells and (II) dissociation of complexes present in live cells during isolation. Since the Kic1-Hym1 interaction is likely in the low micromolar range, there is probably significant dissociation during immunoprecipitation that is difficult to quantify. Although co-immunoprecipitation of Kic1-HA with Hym1-myc clearly peaked during cell separation in multiple experiments, we saw a consistently high level of basal Kic1-Hym1 interaction throughout the time courses. To determine if this could be attributed to post-lysis association we prepared lysates from cells expressing either Kic1-3HA or Hym1-13myc separately, mixed them, and then assessed Kic1-HA co-immunoprecipitation with Hym1-myc. We saw a significant level of co-immunoprecipitation, demonstrating that post-lysis association probably contributes to the basal level of interaction between these two proteins in our experiments. Importantly, however, we did not see an increase in post-lysis Kic1-Hym1 association in synchronized cells, further supporting the regulated formation of this complex in vivo following mitotic exit  demonstrating that both proteins were functional. Surprisingly, when we expressed the fusion protein in metaphase/anaphase arrested cells where both endogenous Kic1 and Hym1 were deleted, we found that Cbk1 HM site phosphorylation was not persistent throughout the release -His6 as the kinase and GST-Cbk1(673-756)-His6 as the substrate where the phosphoacceptor site Thr743 was either WT or mutated to a non-phosphorylatable residue (T743E). We detected more phosphorylation of the Cbk1 fragment when the Thr743 phosphoacceptor site was WT. We observed significant phosphorylation of the Cbk1 T743E fragment, likely due to other phosphoacceptor sites present on the fragment.(TIF)Click here for additional data file.Figure S2Analysis of putative Kic1-Hym1 interaction residues.A. We performed 32P-ATP kinase assays with different alleles  of bacterially purified recombinant GST-Kic1(1-359)-His6 as the kinase and GST-Cbk1(673-756)-His6 as the substrate. Where indicated, we added bacterially purified recombinant full-length Hym1-His6. The D57A and WDF-AAA alleles of Kic1 abolished the kinase activation upon addition of Hym1. L90Q and C134F alleles retained Hym1 activation of Kic1 as WT. B. We performed a yeast two hybrid analysis to confirm that the WDF motif and Asp57 on Kic1 is important for Kic1-Hym1 interaction. We cloned Kic1 alleles and Hym1 into pGAD-C1 and pGBD-C1, and we coexpressed these constructs as fusion proteins with the GAL4 activation domain and DNA-binding domain, respectively. We used plasmids expressing the indicated proteins either as prey or bait alone as negative controls and wild type Kic1 and Hym1 as a positive control. We found both the WDF mutant and the D57A allele abolished Kic1-Hym1 interaction.(TIF)Click here for additional data file.


The sequence similarity search of the predicted epitopes of osmotin using Structural Database of Allergenic proteins (SDAP) showed similarity with known allergens from tomato, kiwifruit, bell pepper, apple, mountain cedar and cypress. Mutants M1, M2 and M3 showed up to 72%, 60% and 76% reduction, respectively in IgE binding whereas M123 showed up to 90% reduction with patients' sera. The immunoblot of M123 mutant showed 40% reduction in spot density as compared to osmotin. All mutants showed decreased inhibition potency with M123 exhibiting lowest potency of 32% with osmotin positive pooled patients' sera. The three B- cell epitopes of osmotin predicted by in silico method correlated with the experimental approach. The mutant M123 showed a reduction of 90% in IgE binding. The present method may be employed for prediction of B- cell epitopes of allergenic proteins.The identification of B-cell epitopes is an important step to study the antigen- antibody interactions for diagnosis and therapy. The present study aimed to identify B- cell epitopes of osmotin using bioinformatic tools and further modify these regions to study the allergenic property. B-cell epitopes were predicted based on amino acid physicochemical properties. Three single point mutations M1, M2, and M3 and a multiple point mutant (M123) were selected to disrupt the IgE binding. These mutants were cloned, expressed and proteins purified to homogeneity. The IgE binding of the purified proteins was evaluated by ELISA and ELISA inhibition with patients' sera. Three regions of osmotin M1 (57–70 aa), M2 (72–85 aa) and M3 (147–165 aa) were identified as potential antibody recognition sites using  The prevalence of food allergy is increasing worldwide and as many as 8% of children and 2% of adults are affected in western countries The knowledge of the B-cell epitopes can be applied in diagnosis, therapy or development of effective vaccines for immunotherapy Nicotiana tabacum). It accumulates in cells on osmotic stress adaptation and provides osmotolerance to plants in silico approach. The potential IgE binding residues in these epitopes were mutated to reduce allergenicity of osmotin.Osmotin is a stress-responsive multifunctional protein from tobacco , flexibility (Karplus and Schulz method), accessibility (Emini method) and turns (Pellequer method) EcoR I site and 5′GCTCGAG CTCCTTAGCCACTTCATCACTTCCAG 3′ with Xho I site. The PCR products were digested with EcoRI and XhoI enzymes, subcloned in pET22b+ vector and sequenced to confirm the mutations. Constructs containing the mutations in the epitopes were used for the expression of fully modified recombinant osmotin protein mutants.Osmotin was mutated at three residues to generate three single point mutants M1, M2 and M3. A fourth mutant M123 with all three point mutations was also generated. Mutagenesis was performed using oligonucleotide primers and mutation-specific oligonucleotide primers accommodating each mutation. Internal amino acid substitution in osmotin was performed by a three step PCR reaction. The first two steps of PCR reaction was carried out with an internal mutant primer and one of the flanking primers. Final products of these two steps were purified from agarose gel, mixed and amplified using flanking primers. The fourth mutant with all three point mutations was generated step wise using mutant M2 as template. The mutation 1 was generated using forward primer 5′ATCAATGCGCCACGATTTACTAAAAAG3′ and reverse primer 5′CATTTTAGTAAATCGTGGCGCATTG3′, mutant 2 was generated using forward primer 5′ATGCTGCTGGTAGGTTTACGTGCCAA3′ and reverse primer 5′TTTGGCACGTAAA CCTACCAGCAGCA3′, mutant 3 using forward primer 5′ACGGCTAATATATTCGGCGAATGTCC3′ and reverse primer 5′GGACATTCGCCGAATATATTAGCCGTAC3′ and the flanking primers 5′GAATTGAATTCGATGGGCAACTTGAGATCTTCTTTTG3′with Escherichia coli cells. The cells were grown till 0.6 O.D., induced with 1 mM isopropyl-beta-D-thiogalactopyranoside for 3 h, harvested and the pellet was suspended in binding buffer containing 50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole (pH 8.0), sonicated and centrifuged. The supernatant was incubated for 2 h with 2 ml Ni-NTA equilibrated with binding buffer. The column was washed with buffer containing 20 mM imidazole, 300 mM NaCl and 50 Mm NaH2PO4 (pH 8.0) and bound proteins were eluted using buffer containing 250 mM imidazole, 300 mM NaCl and 50 mM NaH2PO4. The purified mutant proteins were resolved on 12% SDS-PAGE, transferred to nitrocellulose membrane and probed with osmotin positive patients' pooled sera. Densitometry of the immunoblot was performed by Gene Tools software from SynGene .The constructs of M1, M2, M3 and M123 mutant sequences in pET22b+ vector were transformed into BL-21 2PO4/K2HPO4 buffer (pH 7.4). Circular dichroism spectroscopy was performed on a J-815 S spectropolarimeter (Jasco) with constant nitrogen flushing at 25°C. Wavelength was analysed with a step width of 0.2 nm and a band width of 1 nm. The spectral range was 200–240 nm at a scan speed of 50 nm/min. The final spectra were corrected by subtracting the corresponding baseline spectrum obtained under identical conditions. Average of three scans was taken and the mean residue molar ellipticity was calculated.Osmotin protein and its mutant M123 were dialyzed against 10 mM KHThe patients of allergic rhinitis and asthma aged 15–50 years were skin prick tested with various aeroallergens and food extracts to detect sensitization. The guidelines of the American Thoracic Society were followed for diagnosis of asthma 2SO4 and the absorbance was read at 492 nm.Specific IgE binding for the mutants and osmotin protein was determined by ELISA. Briefly the proteins were incubated in carbonate buffer overnight at 4°C in a microtitre plate . After washing with PBS– Tween 20 (0.05%), the nonspecific sites were blocked with 3% defatted milk for 3 h at 37°C. The plate was washed again and incubated with 100 µl diluted (1∶10 v/v) sera of food allergic patients which were positive to osmotin, overnight at 4°C. Normal human sera (n = 4) were used as negative control. The plate was washed and incubated with antihuman IgE-horse radish peroxidase  for 3 h at 37°C. The plate was washed with PBS- Tween 20 and followed by PBS and colour was developed with orthophenylene diamine in citrate – phosphate buffer, pH 5.0. The reaction was stopped after 30 min by adding 5 N HELISA inhibition with the mutants was performed to determine the allergenic potency and relative ability of the mutants for IgE binding. The microtitre plate was coated with purified osmotin protein and blocked with 3% defatted milk. Further it was incubated with pre- incubated mixture of 1∶10 v/v osmotin positive pooled patients' sera (n = 5) and graded amounts of osmotin and each of the purified mutants i.e. 0.1, 1, 10, 100 and 1000 ng as inhibitor. Sera of non-allergic subjects were used as control. The remaining steps were the same as followed for ELISA. Inhibition was presented as percentage decrease in absorbance of sample with inhibitor to that of sample without inhibitor and allergenic potency was defined by establishing the concentration of protein required for 50% inhibition.Statistical analysis of results was done by using GraphPad Prism and GraphPad Instat software . The statistically significant difference was determined using one way ANOVA followed by Bonferroni comparison test between osmotin and the mutant M123 challenged mice groups. The p value <0.05 was considered as significant.in silico approach showed reduction in the antigenic index in DNAstar program (http://fermi.utmb.edu/SDAP/), these epitopes showed similarity with tomato allergen Lyc (NP24), kiwi fruit allergen (Act c 2), cypress allergen (Cup s 3.0102), bell pepper allergen (Cap a 1), apple allergen  and mountain cedar allergen (Jun a 3) with a PD (property distance) value of less than 8.5 , 80 (Gly → Phe) and 151(Asn → Phe) in each epitope were selected for substitution mutation. These epitopes on mutation using  program . In Struthan 8.5 .E. coli and purified as His-tag proteins. The yield obtained of these proteins were 2.5 mg/L, 2 mg/L, 2 mg/L, 3 mg/L of the culture. The protein expression in the culture after induction at 1h, 2 h and 3 h with IPTG was visualized by SDS-PAGE , 22%–60% (M2), 43%–76% (M3) and 50%–90% (M123) as compared to osmotin . All the50. Only M2 showed the inhibition with an EC50 of 500 ng whereas the other mutants did not reach the EC50 value  to known IgE epitopes could contribute to IgE binding and cross-reactivity Linear epitopes are more relevant as compared to conformational epitopes for food allergens as these allergens are encountered by the immune system only after they are subjected to digestion/heating In the present study, three single point mutants  and a triple point mutant, M123 was subcloned and protein purified to homogeneity. These were developed by mutating a single residue in each of the three IgE binding predicted areas. Previous study has shown that single point allergen mutants do not lead to significant changes in protein structure but reduces allergenicity Brassica pollen allergen Bra r 1 showed 74% and 94% inhibition of IgE binding with two patients' sera respectively at the highest inhibitor concentration. The double mutant of Bra r 1 showed 13% and 19% inhibition indicating reduced affinity of the IgE antibodies The in silico and in vitro studies The potential of introduced foreign or engineered proteins in genetically modified crops to cause allergy has been of concern worldwide Specific immunotherapy against allergic disorders requires frequent injections of allergen extract to slowly build up the maintenance dose in silico method that correlated well with the experimental approach. Osmotin mutant M123 showed a reduction of 90% in IgE binding. The present method may be employed for prediction of B- cell epitopes of allergenic proteins.In conclusion, three B- cell epitopes of osmotin were predicted by


In silico transcription factor binding analysis of the HIV-1 subtype B LTR sequences revealed a potential downstream CCAAT enhancer binding protein (C/EBP) binding site. This binding site (+158 to+172), designated DS3, was found to be conserved in 67% of 3,858 unique subtype B LTR sequences analyzed in terms of nucleotide sequence as well as physical location in the LTR. DS3 was found to be well represented in other subtypes as well. Interestingly, DS3 overlaps with a previously identified region that bind members of the nuclear factor of activated T cells (NFAT) family of proteins. NFATc2 exhibited a higher relative affinity for DS3 as compared with members of the C/EBP family (C/EBP α and β). DS3 was able to compete efficiently with the low-affinity upstream C/EBP binding site I with respect to C/EBP binding, suggesting utilization of both NFAT and C/EBP. Moreover, cyclosporine A treatment, which has been shown to prevent dephosphorylation and nuclear translocation of NFAT isoforms, resulted in enhanced C/EBPα binding. The interactions at DS3 were also validated in an integrated HIV-1 LTR in chronically infected U1 cells. A binding knockout of DS3 demonstrated reduced HIV-1 LTR-directed transcription under both basal and interleukin-6-stimulated conditions only in cells of the monocyte-macrophage lineage cells and not in cells of T-cell origin. Thus, the events at DS3 positively regulate the HIV-1 promoter in cells of the monocyte-macrophage lineage.Transcriptional control of the human immunodeficiency virus type 1 (HIV-1) promoter, the long terminal repeat (LTR), is achieved by interactions with cis-acting elements present both upstream and downstream of the start site.  Human immunodeficiency virus type 1 (HIV-1) is transcriptionally regulated by cellular and viral proteins interacting with the cis-regulating elements in the viral promoter, the long terminal repeat (LTR). The HIV-1 LTR is approximately 640 base pairs in length and is divided into three components, the unique 5′ (U5), unique 3′ (U3), and the repeat (R) regions Three CCAAT enhancer binding protein (C/EBP) binding sites have been previously described within the upstream HIV-1 LTR: upstream site 1 (US1), located immediately 5′ of the distal nuclear factor-κB (NF-κB) binding site (−105 to −116); upstream site 2 (US2), located upstream of US1 (−164 to −173); and upstream site 3 (US3), located upstream of US2 (−245 to −253) N-terminally deleted p30 C/EBPα form does not function as a transcriptional activator. C/EBPβ, conversely, has four internal AUG codons that result in 38-, 35-, 21-, and 14-kDa proteins, respectively. C/EBPβ is also referred to as nuclear factor interleukin-6 in human and liver-enriched activating protein, in rodents. A 21-kDa liver inhibitory protein isoform also exists as a proteolytic cleavage product of full-length C/EBPβ C/EBPs are a bZIP (basic leucine zipper) family of transcription factors that includes C/EBPα, C/EBPβ, C/EBPγ, C/EBPδ, C/EBPε, and C/EBPζ (CHOP10 [C/EBP homologous protein 10]/GADD153 [growth arrest and DNA damage inducibility]). C/EBPs bind a consensus DNA sequence (A/G)TTGCG(C/T)AA(C/T) through their basic regions either as homodimers or as heterodimers with other family members via their leucine zipper motifs, as previously reviewed + T-cells 2+) mobilization and to heterotrimeric G protein–coupled receptors results in dephosphorylation and nuclear translocation of NFATs The NFAT family of transcription factors consists of five proteins that are related to the REL-NF-κB family. These include NFAT1 , NFAT2 (NFATc1 or NFATc), NFAT3 (NFATc4), NFAT4 (NFATc3 or NFATx), and NFAT5 , as previously reviewed We identified a novel downstream cis-acting element in the HIV-1 LTR that is utilized by C/EBP and NFAT family members to positively and selectively control transcription in cells of the monocyte-macrophage lineage. These results provide additional evidence pointing to the importance of downstream LTR binding sites with respect to nucleosome positioning within the context of the viral promoter.+ T-cells  were cultured and maintained in Roswell Park Memorial Institute (RPMI)-1640  supplemented with penicillin (100 U/mL), streptomycin (100 µg/mL), sodium bicarbonate (0.15%) (Cellgro), and fetal bovine serum (10%) . HEK 293T cells  were cultured in Dulbecco minimal essential medium (Cellgro) supplemented with penicillin (100 U/mL), streptomycin (100 µg/mL), sodium bicarbonate (0.15%) (Cellgro), and fetal bovine serum (10%) (GemCell). U-937 and U1/HIV-1 cells were stimulated for 12 hours, where indicated, with human interleukin-6 recombinant protein  prior to harvesting. U-937 and U1/HIV-1 cells were also treated with 1 µM Cyclosporine A  for 24 hours followed by overnight treatment with IL-6, where indicated.The human histiocytic lymphoma cell line U-937  is a promonocytic cell line that was used as a model of cells in the monocytic lineage and will be referred to herein as U-937 cells 7) were collected by centrifugation, washed once with ice-cold 1× Dulbecco phosphate-buffered saline (Mediatech), and lysed in ice-cold lysis buffer . After centrifugation (1000×g), the supernatant (cytoplasmic extract) was discarded. The pelleted nuclei were gently resuspended in nuclear extract buffer , shaken vigorously on a rocker for 30 min at 4°C, and subjected to centrifugation for 10 min . The supernatant (nuclear extract) was then dialyzed two times for 45 minutes using minidialysis units  in dialysis buffer . The nuclear extract was recovered from the dialysis units and transferred to an Eppendorf tube and stored at −80°C in small aliquots. The protein concentration was determined by Bradford assay as described by the manufacturer .Small-scale nuclear extracts were prepared from low-passage, exponentially growing cells. Briefly, cells  as previously described The sequences used to generate double-stranded, gel-purified oligonucleotides for EMS analysis as previously described Madison, WI). Each sample was subjected to 95°C for 5 min followed by 40 cycles at 95°C for 30 s 56°C for 30 s, and 72°C for 30 s. The final extension was performed at 72°C for 5 min. The following primers were used to amplify the +91 to +238 region (148 base pairs) of the HIV-1 LTR that encompasses the TFBS (DS3) exclusively: 5′ – GCTTCAAGTAGTGTGTGCCCGT- 3′ and 5′-TGCGTCGAGAGAGCTCCTCTG –3′. The DNA immunoprecipitated with RNA polymerase-II antibody was also amplified using primers for the GAPDH promoter , as a positive control for the assay. Each PCR was run on 2% agarose gel containing ethidium bromide and analyzed using Alphaimager.U1/HIV-1 cells were either stimulated with IL-6 or treated with a combination of cyclosporine A (1 µM) and IL-6 (20 ng/ml) as described earlier. At the time of harvest, the cells were cross-linked with 16% formaldehyde achieving a final concentration of 1%. The cells were incubated with gentle rocking at room temperature for 10 min, transferred to centrifuge tube on ice, collected by centrifugation at 1,100 rpm for 5 min, and washed twice with cold phosphate-buffered saline (PBS). Another wash was performed with PBS containing HALT protease inhibitor cocktail . Subsequent steps of chromatin immunoprecipitation were performed as previously described The anti-C/EBPα , anti-C/EBPβ , anti-NFATc2 , anti-NFATc1 , anti-NFATc1 , anti-NFAT5 (sc-13035×), and control rabbit IgG (sc-2027) were obtained from Santa-cruz Biotechnology, Dallas, Texas. RNA pol II antibody (61082) was obtained from Active Motif, Carlsbad, CA. Mouse β-actin and anti-FLAG monoclonal antibodies were obtained from Sigma-Aldrich, St. Louis, MO.HindIII and BamHI restriction endonuclease sites in the multiple cloning site. The vector was verified by sequencing and expression was confirmed by western immunoblotting with anti-FLAG antibody (Sigma-Aldrich).pGL3-based HIV-1 LAI-LTR luciferase reporter vector was used as a template to generate the DS3 knockout (9C) mutant (involving a G-to-C change at position number 9 in the DS3 binding site) utilizing the QuickChange II site-directed mutagenesis procedure . The terminal FLAG-tagged C/EBPβ expression vector was constructed by cloning human C/EBPβ cDNA  in pFLAG-CMV2 vector  between 5 cells/mL. Transient transfections were performed using Lipofectamine LTX with Plus Reagent as recommended by the manufacturer . Luciferase expression constructs (1 µg) [HIV-1 LAI-LTR-luc or DS3 knockout (9C)-luc] and 50 ng of pRL-TK Renilla luciferase internal control vector were co-transfected in U-937 cells or Jurkat T-cells. For U-937 cells, interleukin-6 (20 ng/mL) stimulation was done 3 hours after transfection. The cells were harvested 24 hours after transfection and assayed using the dual-luciferase assay system as described by the manufacturer . Firefly luminescence was normalized to Renilla luminescence. Each value shown represents the average of duplicate or triplicate transfection reactions and was representative of at least three independent experiments. Error bars indicate the standard error. HEK 293T cells were transfected using Xtreme Gene HP DNA transfection reagent as described by the manufacturer . Cells were transfected with either the vector (pFLAG-CMV2) or C/EBPβ expression vector (pFLAG-CMV2-C/EBPβ). Cells were harvested for nuclear extract preparation 24 hours after transfection and used for EMS analysis.Exponentially growing U-937 cells or Jurkat T-cells were plated into 12-well tissue culture plates on the day of transfection at a concentration of 2×106 cells/well) were seeded in a six-well plate and were either left untreated or stimulated with interleukin-6 (20 ng/mL) overnight before harvesting. The cells were washed with ice-cold phosphate-buffered saline and lysates were made in radioimmunoprecipitation assay buffer . Protein estimation was performed using the Bradford assay and samples were diluted 1∶2 with Laemmli Sample Buffer (Bio-Rad). Protein (30 µg per lane) was loaded and blotted using specified antibodies.U-937 cells  to identify Genbank records with a full-length LTR sequence. As of 10/01/2013 there were a total of 510,217 HIV sequences. To ensure that each sequence was from a different patient, only a single LTR was selected from each referenced paper in the Genbank records, which resulted in a total of 9,712 full-length LTR sequences from all HIV subtypes. HIV-1 LTRs from subtypes A, B, C, and D totaling 7,577 sequences were then analyzed using the MUSCLE alignment tool to align these sequences. TRANSFAC software  was then used to examine predicted TFBSs for known TFs. Utilizing this approach, a predicted binding site for C/EBP factors was identified from+158 to+172 with respect to the start site (+1) in 2600 (67%) of the 3858 subtype B sequences analyzed  when compared with other well-characterized upstream and downstream TFBSs in subtype B LTRs. This was performed by measuring the average percentage of point mutations from ConB for the C/EBP US2, US1, and DS3; NF-κB sites I and II; Sp1 sites I, II, and III; the ATF/CREB site; and AP1-I, -II, and -III sites. For each sequence and each binding site, the best matching location was identified, within 10 nucleotides of the expected position, and the average deviation from ConB was calculated. Then that number was averaged for the entire binding site to give the mean percentage nonconsensus configuration of each site . Based oTo determine whether the predicted DS3 site was functional in terms of its ability to bind C/EBP factors, EMS analyses were performed using U-937 cell nuclear extracts. Because the C/EBP transcription factor family plays a critical role in transcriptional regulation in cells of the monocyte-macrophage lineage compared with many other types of cells targeted by HIV-1, U-937 cells were selected to model this interaction because of their monoblastoid cell heritage and because they have been extensively used in studies focused on HIV-1 gene regulation in monocytic cells. HIV-1 subtype B LTR upstream C/EBP binding site 2 (HIV-1 US2), which has been identified as a relatively high-affinity C/EBP binding site, was radiolabeled and used as a probe and incubated with U-937 nuclear extracts . Because2GGAAAATCCAGTGT. These reports prompted the question as to whether NFAT isoforms also bind DS3, and if so, is the element a composite element with C/EBP or is there a competitive interaction at DS3 with C/EBP factors? To answer these questions, EMSAs were performed by incubating stimulated U-937 extracts with labeled DS3 oligonucleotide and relevant C/EBP and NFAT antibodies. Results show that NFATc2 antibody resulted in abrogation of the complex while other antibodies  did not   did not . This fu did not , whereas did not . Similar did not . These rOverall, these results lead to conclusion that NFATc2 isoforms preferentially occupy the DS3 binding site in the HIV-1 LTR and also that affinity of DS3 was optimized for NFATc2 isoforms. It was also concluded that the interactions at DS3 between NFATc2 and C/EBPs are competitive, as antibody supershift/abrogation was not observed with either of the respective antibodies.NFATc1 through c4 are activated via stimulation of receptors linked to calcium signaling and G protein–coupled receptors (discussed in the introduction). Highly phosphorylated forms of NFATs have been shown to be inactive and stay in the cytoplasm and are only translocated to the nucleus as a result of dephosphorylation mediated by calcineurin phosphatase and other inducible or constitutive kinases. Because the results have suggested that the DS3 binding site was utilized competitively by C/EBP and NFAT isoforms and appeared to have higher relative affinity for NFAT isoforms, it was important to determine whether C/EBP preferentially occupies DS3 in situations where NFAT would be inactive and therefore remain in the cytoplasm. Cyclosporine A–treated U-937 nuclear extracts were used in EMS analyses to assess binding of C/EBP family members to DS3 . As expeThe U1/HIV-1 cells, a cell line derived from U-937 cells chronically infected with HIV-1, is a standard model used to study the recruitment of transcription factors to the LTR and also to understand the transcriptional regulation of an integrated HIV-1 provirus The DS3 element exhibited a higher relative affinity for NFATc2 than that observed with C/EBPα and β, and the binding appeared to be in a mutually exclusive manner. Such interactions have been previously described within the context of the HIV-1 promoter; for example, one AP-2 site has been shown to be embedded within two κB elements and AP-2 and NF-κB were shown to bind to this region in a mutually exclusive manner in Jurkat T-cells In the HIV promoter a cluster of functional transcription factor binding sites downstream of the transcriptional start site has previously been defined, with binding sites including AP-1, NFAT, DBF/IRF, and Sp1 The interactions at TFBS (DS3) were also analyzed using U1/HIV-1 cells that are chronically infected with HIV-1 and are a good model system to examine TF binding at selected regions of the LTR. ChIP experiments did recapitulate the data obtained with gel shift analyses and C/EBPα, C/EBPβ and NFATc2 occupancy was shown in this downstream region . MeasuriH17 cells, CD8+ T cells, and also in dendritic cells and mast cells The studies reported herein also extend the functional knowledge of NFATc2 in HIV-1 regulation in cells of the monocyte-macrophage lineage and also identify an additional element bound by members of the NFAT family. This binding site adds to the LTR sites that are regulated by NFATs Upon integration into the cellular genome, the HIV-1 proviral DNA packaged into chromatin and nucleosomes is positioned precisely such that two nucleosome-free regions are established (−254 to +2 and +156 to +266) N-terminal region prevents chromatin remodeling C/EBPs and NFATs are both known to participate in the process of chromatin remodeling and the ensuing transcriptional activation. Several reports, in fact, have suggested that chromatin remodeling might as well be the primary function of NFAT elements as even high-affinity NFAT-binding sites are comparatively poor transcriptional activators in the absence of the collaborating TFs that normally associate/cooperate with NFATs With regard to the ability of C/EBP factors to regulate the LTR from downstream binding site cues can also be taken from simian immunodeficiency virus studies in which we have previously described downstream elements with which C/EBPs are able to interact and that control the rate of transcription Figure S1IL-6 stimulation of U-937 cells increases levels of C/EBPβ. (A) Western immunoblot of U-937 cell nuclear extracts using C/EBPβ polyclonal antibody. (B) Competitive and supershift/abrogation EMS analyses were performed with 32P-labeled oligonucleotides (indicated below the figures) and incubated with nuclear extract from unstimulated or stimulated  U-937 cells as indicated. IL-6 stimulation resulted in increased binding to C/EBP US2 probe (compare lanes 2 and 5), and this enhanced complex formation was abrogated by addition of C/EBPβ antibody (lane 7).(TIF)Click here for additional data file.Figure S2Reciprocal competitive EMS analysis to show DS3 complex specificity. (A) Competitive EMS analysis was performed in which nuclear extracts from U-937 cells were incubated with labeled NFAT oligonucleotide and cold competition was performed with molar excess of DS3 oligonucleotide. (B) Competitive EMS analysis was performed with nuclear extracts from U-937 cells that were incubated with labeled NFAT oligonucleotide, and cold competition was performed with molar excess of unlabeled C/EBP US2 or C/EBP US1 oligonucleotides. (C) Competitive EMS analysis was performed with nuclear extracts from U-937 cells that were incubated with labeled C/EBP US2 oligonucleotide, and cold competition was performed with molar excess of unlabeled NFAT oligonucleotide.(TIF)Click here for additional data file.Figure S3DS3 binds to NFAT isoforms in Jurkat T cells. Nuclear extracts from Jurkat T cells were incubated with labeled DS3 oligonucleotide and supershift/abrogation analysis was performed with indicated antibodies.(TIF)Click here for additional data file.


Mitogen-activated protein kinases (MAPKs), which are found in all eukaryotes, are signal transducing enzymes playing a central role in diverse biological processes, such as cell proliferation, sexual differentiation, and apoptosis. The MAPK signaling pathway plays a key role in the regulation of gene expression through the phosphorylation of transcription factors. Recent studies have identified several RNA-binding proteins (RBPs) as regulators of MAPK signaling because these RBPs bind to the mRNAs encoding the components of the MAPK pathway and regulate the stability of their transcripts. Moreover, RBPs also serve as targets of MAPKs because MAPK phosphorylate and regulate the ability of RBPs to bind and stabilize target mRNAs, thus controlling various cellular functions. In this review, we present evidence for the significance of the MAPK signaling in the regulation of RBPs and their target mRNAs, which provides additional information about the regulatory mechanism underlying gene expression. We further present evidence for the clinical importance of the posttranscriptional regulation of mRNA stability and its implications for drug discovery. Schizosaccharomyces pombe (S. pombe). S. pombe, with its powerful genetics, is an excellent model system for the study of the mechanisms of signaling pathways in higher eukaryotes. In S. pombe, homologs of many signaling molecules involved in tumorigenesis, such as Ras, Rho, Protein kinase C, and MAPK, as well as drug targets, including TOR (target of Rapamycin) and the phosphatase calcineurin (target of the immunosuppressant drug FK506), are highly conserved. Spc1/Sty1/Phh1 and Pmk1 in S. pombe are homologs of mammalian p38 and ERK, respectively. The purpose of this review is to highlight the significance of several data using the fission yeast model system as well as other studies in higher eukaryotes that uncovered the cross-talk mechanism between MAPKs and RNA-binding proteins (RBPs) in the regulation of posttranscriptional gene expression, biological phenomena, and signal transduction . Fu. Fuα mRN in vivo  32]. . α mRNA α [Notably, phosphorylation by MK2 inhibits TTP activity. Several studies have described the molecular mechanisms underlying this regulation, although this subject is controversial. The phosphorylation of TTP by MK2 facilitates the dissociation of TTP from the ARE motif, thereby allowing the biosynthesis of TNFα . Previouα , 48. Furα , 50. In α  and hnRNα , therebyα . S. pombe [+atf1 mRNA, which encodes a member of the ATF/CRE-binding protein family of transcription factors, including ATF2, in higher eukaryotes [Interestingly, the budding yeast p38 homolog Hog1 was shown to be essential for stabilization of these ARE-containing mRNAs , thus inS. pombe . Csx1 iskaryotes . Moreovekaryotes . Consistin vivo and in vitro, thereby stabilizing Cdc4 mRNA and regulating cytokinesis in fission yeast. Nrd1 shows significant sequence similarity with TIA-1 and the TIA-1-related (TIAR) proteins in higher eukaryotes [cdc4M1M2) within the Cdc4 ORF abrogated the binding between Nrd1 and Cdc4 mRNA, and the cdc4M1M2 mutant cells exhibited cell separation defects, suggesting that the loss of Nrd1 binding affects cytokinesis.Recent paper by Satoh et al. revealed an interesting link between RBP and cell cycle control mediated by modulating myosin mRNA stability . They idkaryotes , 58. It karyotes , 60. NotDD, the phosphorylation mimic version of Nrd1, only weakly binds to Cdc4 mRNA, while Nrd1AA, the unphosphorylatable version of Nrd1, in which T40 and T126 are substituted by alanine residues, strongly binds to Cdc4 mRNA [Interestingly, Nrd1 is also phosphorylated by Pmk1 MAPK at two threonine residues (T40 and T126) and this phosphorylation negatively regulates Nrd1 activity to bind and stabilize Cdc4 mRNA and cytokinesis. Nrd1dc4 mRNA . The delα mRNA stability [α mRNA through its 3′-UTR, resulting in increased Gadd45α protein levels. Gadd45α itself is then required to maintain MK2 activity, which is critical for prolonged Cdc25 inhibition and maintenance of a G2 arrest [α is essential to allow cells to recover from DNA damage, before committing to the next mitotic cell division. These reports on RBP-mediated cell-cycle control through mRNA stability provide further evidence for the phosphoregulation of RBPs through MAPK, thereby revealing an additional level of cell cycle regulation.There are also interesting examples of cell-cycle control by modulation of mRNA stability in higher eukaryotes, especially with regard to DNA damage checkpoints . Reinhartability  and show2 arrest . Thus, tα, one of the most important proinflammatory cytokine containing AREs, prevent arthritis in transgenic mice expressing ARE-lacking stable TNFα transcripts, which produce pathological overexpression of TNFα [Considerable number of genes that code for AU-rich mRNAs, including cytokines, chemokines, growth factors, RBPs, and transcription factors, are commonly involved in both chronic inflammation and cancer . Therefo of TNFα , 64. Thu of TNFα –67.We have presented several examples of functional relationships and cross talk regulatory mechanisms between RBPs and MAPK signaling in fission yeast model system and have drawn some parallels with animal cells. A recent paper by Belle et al. on the global investigation of protein half-lives in budding yeast indicates that the protein products of genes that are coregulated on the transcriptional level also tend to be coregulated on the level of protein turnover . Similar


A quick and easy solution to the absence of adequate documentation during clinical trial conduct is the use of notes to file. Over the years, the use of notes to file has evolved from a last resort solution to a common working practice amongst clinical teams, bordering on misuse and abuse of this tool. This article explores this evolution from the perspective of an independent observer. A decade and a half of conducting global clinical trials in India has taught us many things, especially the importance of documentation in research. The nuances of the extent and the ways to document clinical trial data, however, continue to elude us. One of the most common questions asked by investigators participating in clinical trials is: How much documentation is enough? Their frustration has to do with the fact that no amount of detailed documentation seems to satisfy monitors, auditors and inspectors. There are several instances where, in the absence of an adequate documented explanation, the investigators are asked to generate a note to file (NTF), which contains a detailed explanation. An example would be a case where the site staff has not followed the protocol-specified investigational product dosing schedule for several subjects. In such cases, it is common to see an NTF in the site master file explaining this deviation and the reason for the same.Initially, this practice of using NTFs to document deviant processes began when the site team and the monitoring team could not find an appropriate place to document certain issues. However, nowadays, little or no effort goes into even considering whether issues can be documented elsewhere and NTFs are generated, without batting an eyelid, to document everything ranging from banal logistic issues to those of serious non-compliance.The indication that the excessive use of NTFs is not a desirable practice should come from the fact that no guidelines2 mentionIn general, NTFs are used in the following instances:To document the reason for missing, delayed or erroneous documents in the clinical trial master file or in the site master file.To explain protocol deviations or investigator site practices that are different from the norm or from what is prescribed in the protocol.During the review of trial master files, one often comes across an additional way that NTFs are used to document that certain sections of the files are not applicable. For example, for an ongoing study, the file will contain a section for the clinical study report, which is expected to be blank; however, it is a common but unnecessary practice to place a file note stating that this section is not applicable. It is, however, acceptable and recommended that if contents of a particular section of the file are placed in a separate file, this should be indicated via a file note in that section.One of the most common ways to misuse an NTF is to use it in place of source documents. This happens when the investigator site staff misses out essential information about trial conduct for subjects in their respective source documents or medical records. It can even happen when the documentation in the source document needs further clarification. In such cases, all additional information and clarifications are documented on NTFs instead of entering them in the source documents directly or explaining them in the monitoring visit reports. For example, if the site staff omit to document the pill counts done after every visit for one or more subjects, this is tabulated and entered for all those subjects together in an NTF placed in the site master file retrospectively.memos to file are inadequate to address the falsification (backdating) of study documents”. A more recent example can be seen in the warning letter issue to Johnson and Johnson PRD in 2009,[The misuse of NTFs has been cited in warning letters issued by the US-FDA. In these cases, the inspectors from the US-FDA Department of Scientific Investigations (DSI) have admonished sponsors for documenting important events in a clinical trial simply by generating a “Memo-to-File”. One such example is illustrated in the warning letter issued to Sanofi-Aventis in October 2007, where se in 2009, where anThe need to document long drawn-out explanations in the NTFs stems from our need to assign a reason for why and how a deviation happened and to document it. It is perfectly acceptable that during the course of clinical trial conduct, there are accidental deviations from the protocol. These do not always need to be explained and analyzed via NTFs as long as the site team and the monitoring team realize the error and ensure that it does not get repeated. The more important action item, rather than generating NTFs, should be to ensure training of all parties involved and assessing and communicating the impact of the deviation to the clinical team.An interesting viewpoint for the use of NTFs to document site errors or deviations in the protocol made by the investigator site team surfaced during an investigator site audit. The monitor would insist that the Principal Investigator (PI) must sign all NTFs as a punitive action so that such errors do not occur again and to document that he was taking responsibility for these errors.The realization that the use of NTFs has evolved to abuse hits when we come across working practices and guidance documents supporting the use of NTFs. In many cases, even version dated templates are provided for creating NTFs and a separate section in the site or trial master file is created to file them. There are organizations where NTFs are submitted as attachments to monitoring reports by the site monitors to prove that they have successfully closed out site issues during their monitoring visits. This not only sends the message to the monitors and site staff that NTFs are acceptable but also makes it a preferable and a convenient means to document issues.Now you may wonder why this is of concern and what is the impact of generating numerous NTFs when their only purpose is to clearly document events in a clinical trial. The primary concern is that it is often used as a solution to issues arising at sites. If an investigator site is cited for having too many protocol deviations, it is not uncommon for them to retort that NTFs were generated to explain the deviations. They do not realize that the documented explanation does not take away from the fact that deviations have occurred.Also, when the use of NTFs to document issues at the site is a common practice, there arise situations where the same issue is explained in the source document, in the monitoring visit report, in a letter to the ethics committee and also in a separate NTF. This not only overstates the obvious, but also makes the site staff sound excessively defensive and wastes precious time of all those who are involved in creating, printing and signing the NTF. Alternatively, it can also happen that since these issues are already documented in the NTF, they often get omitted from places where they should be documented like the monitoring visit reports and site correspondence.It is only fair that some alternative solutions are provided while strongly recommending avoiding the use of NTFs. This is not difficult as the information documented on NTFs logically belongs to one or more of following locations:The subject’s source document file.The monitoring or site visit report where that issue was first noted and discussed with the site staff.The correspondence between the site staff and the monitoring team, internal correspondence within the sponsor/ contract research organization (CRO) teams or correspondence with the ethics committee and regulators.In very rare instances, it would be necessary to generate an NTF if the information does not belong to any of the above categories. But it is highly likely that these instances would be exceptions rather than the norm.The institutionalization of the use (or misuse) of NTFs does not reflect well on our ability to understand and interpret the spirit of clinical research guidelines. It indicates that we are increasingly becoming incapable of exercising judgment and playing it safe by documenting almost everything that happens during the conduct of a trial in NTFs rather than where they should logically be documented. It is important that all of us stop and think before generating the next NTF, whether it is necessary to do so or can the documentation of this issue be done more appropriate elsewhere.