During two consecutive cruises to the Eastern Central Arctic in late summer 2012,we observed floating algal aggregates in the melt-water layer below and betweenmelting ice floes of first-year pack ice. The macroscopic (1-15 cm in diameter)aggregates had a mucous consistency and were dominated by typical ice-associatedpennate diatoms embedded within the mucous matrix. Aggregates maintainedbuoyancy and accumulated just above a strong pycnocline that separated meltwaterand seawater layers. We were able, for the first time, to obtain quantitativeabundance and biomass estimates of these aggregates. Although their biomass andproduction on a square metre basis was small compared to ice-algal blooms, thefloating ice-algal aggregates supported high levels of biological activity onthe scale of the individual aggregate. In addition they constituted a foodsource for the ice-associated fauna as revealed by pigments indicative ofzooplankton grazing, high abundance of naked ciliates, and ice amphipodsassociated with them. During the Arctic melt season, these floating aggregateslikely play an important ecological role in an otherwise impoverishednear-surface sea ice environment. Our findings provide important observationsand measurements of a unique aggregate-based habitat during the 2012 record seaice minimum year. The ongoing thinning and loss of Arctic sea ice will lead to changes in the surfaceenergy budget of the Arctic Ocean ,2 and wiMelosira arctica [Melting of summer sea ice results in habitat deterioration for ice-associatedorganisms and the formation of a stratified surface meltwater layer ,10.Ice  arctica , and the arctica .Lance (ICE12)and (2) IceArc expedition ARK-XXVII/3 with RV Polarstern. In orderto elucidate the significance of these aggregates, we sampled and quantified theirabundance, biomass and production using three different approaches: (i) trapping ofaggregates in an ice hole (“inverted sediment trap” approach), (ii) collection byscuba divers, and (iii) under-ice video transects with a remotely operated vehicle(ROV). Combined, these different approaches enabled us to extrapolate our findingsto a square metre scale and assess the significance of the floating ice-algalaggregates for the ice-associated ecosystem during the oligotrophic summermonths.Large numbers of ice-algal aggregates were encountered during two consecutiveresearch cruises to the Eastern Central Arctic Ocean in summer 2012: (1) the Centrefor Ice, Climate and Ecosystems (ICE) cruise with RV The three drift ice stations were situated over the deep Arctic basin and wellnorth of the ice edge . The paca maximum).Profiles of salinity, temperature, and dissolved oxygen from the under-icewater column were obtained on 28, 30 and 31 July and 1 August using ahand-held MicroCat sensor (Seabird Electronics 37-SM) lowered through an icehole. Subsequently, water samples from the meltwater layer underneath thesea ice were pumped through the same ice hole. Additionally video profiles(GoPro Hero2) provided information on the distribution of floatingaggregates in cracks and openings in the sea ice. Three video profiles wereobtained: one on 29 July at 19:30 GMT and two on 31 July at 08:30 -09:00 GMTthrough the ice hole used to collect the aggregates (further informationbelow). On 29 July at 13:00 GMT, an additional video profile was obtainedthrough an ice crevice in close proximity to the ice hole. Biological seaice samples were collected on 28 July and 1 August using an ice corer withan inner diameter of 9 cm . Immediately after collection, ice cores were cut into 10-20 cm thicksections with a stainless steel saw, transferred into polyethylene zip-lockbags, stored dark and melted on board the ship at 4°C. Sea ice samples weremelted directly, not in filtered seawater, because nutrient samples weretaken from the same core sections. Comparison between direct melting andmelting in filtered seawater at 4°C showed no significant differences intaxonomic composition of sea ice assemblages [a),particulate organic carbon (POC) and nitrogen (PON) were filtered onto 25 mmØ GF/F and pre-combusted (500°C for 4 h) GF/F filters, respectively. Between500 and 1000 mL of seawater, and meltwater and between 150 and 500 mL ofmelted sea ice, were sampled for Chl a, POC and PON. Onereplicate sample per depth was collected for each parameter for seawater andmeltwater, while sea ice samples were obtained from triplicate ice corescollected within 10 cm of each other. Fifty mL were sampled per analysis ofammonium. On 26 and 29 July, aggregates were collected for speciesidentification with a coarse-meshed sieve from the side of open melt pondsand through a specially drilled ice hole also used to collect accumulatedaggregates (further details below). Aggregate samples used for iodine, Chla, and transparent exopolymer particle (TEP) analysiswere collected directly underneath the sea ice on 31 July by divers using aslurp gun (modified 3.5 L Trident® suction gun).Throughout the drift ice station from 26 July to 3 August, conductivity,temperature and depth (CTD) profiles were taken daily using a rosette system(Seabird Electronics SBE911) deployed from the side of the ship. Seawatersamples were collected on 26 and 30 July and daily from 1 to 3 August atfive depths  of a diver-collected aggregate with a totalvolume of 38 mL were taken for Chl for 12 h and meas-1) were converted to carbon using a conversion factor of 0.63 [For TEP analysis, two diver-collected aggregates were transferred into 15 mLcentrifuge tubes and immediately frozen at -20°C. After thawing, the sampleswere each suspended in 169 mL of <0.2 µm filtered ISOTON (BeckmannCoulter) and divided into subsamples. Subsamples of 3-5 mL, with 4replicates each, were filtered onto 25 mm polycarbonate filters (Nucleopore0.4 µm pore size) and stained with Alcian Blue. TEP was measured with thecolorimetric method according to Engel . TEP con of 0.63 . In ordeFor species identification and determination of assemblage composition,aggregates were transferred into brown glass bottles, preserved with abuffered aldehyde mixture  assuggested by Tsuji and Yanagita and stor2) below theice between two wooden bars placed through core holes and connected by arope with marks every metre. The method was repeated three times along 30 to35-m profiles covering a mix of bare ice, and various melt ponds. Thespectral energy fluxes between 400 and 700 nm were converted to photonfluxes and integrated to get values for photosynthetically active radiation(PAR).Ramses-ACC-VIS hyperspectral radiometers (TriOS Mess- und Datentechnik GmbH)were used to measure incoming energy fluxes at the ice surface andtransmitted fluxes at the underside of the ice. One instrument was mounted1.5 m above the ice surface, levelled and facing upwards, while a second wasswum by a diver with semi-closed rebreather  as in Hudson et al.. The inv2 hole cutthrough the ice at 12 h intervals from 29 to 1 August to obtain an estimateof aggregate accumulation over time in the surface meltwater layer. Welikely underestimated the amount of aggregates as not all of them would havefloated up into the ice hole. The aggregate material from each samplinginterval was dried at 60°C and the dry weight determined. Triplicatesubsamples of 0.5-2 µg dry weight were analyzed for POC and PON. Triplicatemeasurements had a precision better than 7% for both POC and PON.We collected all aggregates accumulating in a 3.2 mRelative current velocities (relative to the drifting ice floe) were measuredbelow the ice with an ice-tethered acoustic doppler current profiler (Nortek600 kHz Aquadopp) in the vicinity of the ice hole. The shallowest data cellwas located about 1.5 m below the ice. These data (means) were extrapolatedto 0.2 m below the under-ice surface through a log decay formulation.Mean ra, 1-40 mL of aggregate-seawater mixture, 1 L of meltedsea ice, 1 L of seawater and 0.5 L of melt pond water was sampled atstations Ice1 and Ice2 respectively. The ice core was cut into 10 cmsections and each section melted for 24 h in 2 L of 0.2 µm filteredseawater. The samples were filtered on GF/F filters and immediately frozenin liquid nitrogen. Sample storage prior to analysis was at -80°C. Pigmentswere measured with high-performance liquid chromatography as described inTaylor et al. [Aggregates were collected together with ambient seawater using a plasticspoon or bucket. This aggregate-seawater mixture was sub-sampled for POC,PON, pigments and net primary production (NPP) analysis. In order toestimate the dilution factor with ambient seawater, individual aggregates ofknown volume (10-25 ml) were filtered through pre-combusted GF/F filters forPOC and PON analyses as stated above. For pigment analysis including Chlr et al. .For NPP experiments, ice-algal aggregates were collected from the sameaggregate-seawater mixture sampled for POC, PON and pigment analysis;seawater was collected 0–2 m below the ice with a peristaltic pump whilewater from closed melt ponds (without connection to the seawater below) wassampled with a hand pump; ice cores were taken as stated above and dividedin two sections: top and bottom. The aggregate mixture, seawater and meltpond samples were directly transferred into incubation bottles . The ice core sections were meltedin the dark for 24 h at 4°C prior to incubation. No filtered seawater wasadded during melting to avoid addition of nutrients.a concentrations and NPP of the aggregate-seawatermixture were corrected for dilution by a factor calculated from the POCmeasurements performed in the aggregate-seawater mixture and in individualaggregates in order to derive aggregate specific Chl aconcentrations and NPP rates.Chl All samples were collected within 500 m of each other at both ice stations.Water and sea ice samples were taken on the same day while melt pond sampleswere obtained on the subsequent day but always within 24 h of the othersamples.Detection of aggregates and their spatial distribution was monitored with anupward-looking video system  mounted onto aremotely operated vehicle navigated directly under sea ice at stations Ice1 and Ice2 .2 and 3 h 52 min of dive video, while at station Ice2 five divescovered a total area of 1809 m2 and 3 h 51 min of dive video. Due toROV-attitude or wrong detection, 34% and 35% of the data from stations Ice1 fromstation Ice2, respectively, had to be discarded. For details on ROV-divestatistics and up-scaling calculations see Aggregates were automatically identified on the video images using a thresholdalgorithm. Images were extracted each 5 sec from the dive videos. All imageswere registered according to the distance between camera and ice given by theacoustic altimeter value and cropped leaving the central part of the image inorder to remove overlays and edge effects. Images with an ROV tilt of more than10° and those recorded deeper than 5 m were discarded. Only the green channel ofthe RGB image was used for analysis, as it showed the clearest aggregate signal.Pixels with a green value <100  were consideredas algae. Detection was verified manually. To average over the spatiallyunevenly distributed data , allrecords were grouped in 3×3 m cells. Means were calculated for each cell.Analysis showed that different cell size choices did not significantly changecalculated numbers with the exception of total covered area. ROV video materialat station Ice1 consisted of four dives covering a total area of 5184m14C uptake method[-1 of 14C sodiumbicarbonate. At the end of each incubation period, samples were filtered onto0.2 µm nitrocellulose filters and the particulate radioactive carbon uptake wasdetermined by liquid scintillation counting using Filter count (PerkinElmer)scintillation cocktail. The average of the dark values was subtracted from thelight triplicates. Dissolved inorganic carbon (DIC) was estimated for eachsample using the flow injection system [During the IceArc cruise NPP was measured using the e method with minn system  and the -2 s-1 andthe other in the dark at -1.3°C for 24 h (long enough incubation to measure netand not gross primary production). Triplicate measurements for the aggregateshad a precision of 7 and 17 % for stations Ice1 and Ice2, respectively. Fordirect comparison with depth-integrated seawater, melt pond and sea ice NPP, theaggregate NPP was normalized to the dilution-corrected Chl aconcentration (see sample collection) and up-scaled based on the ROV surveys. In situ NPP rates were inferred for eachmetre of water depth and for each 10 cm of sea ice and melt pond depth from theChl a normalized photosynthesis-irradiance curves (P-E curves)(r2 = 0.83-0.96) [-1 for sea ice [-1 forAtlantic-influenced Arctic seawater, based on data from the TransArc expeditionARK-XXVI/3 to the same area during the previous year. Subsequently, these Chla-normalized rates were multiplied by the measured Chla profile and summed up to give the depth-integrated NPPrates. For the water column, NPP was integrated over the euphotic zone (1% lightdepth).Seawater, sea ice and melt pond samples were distributed in 10 cell cultureplastic bottles of 20 mL each. Subsequently, they were incubated for 12 h at-1.3°C under a range of light irradiances (0–420 µmol photons m83-0.96) , asa fu sea ice  and 0.1 http://doi.pangaea.de/10.1594/PANGAEA.803221and http://doi.pangaea.de/10.1594/PANGAEA.803115)while aggregate NPP, TEP and species composition, “inverted sediment trap”calculations and the oxygen profiles are archived in the Marine data-base of theNorwegian Polar Institute and publicly available under http://data.npolar.no/dataset/67eede8a-fe8f-11e2-ba11-005056ad0004.Sea ice observations and station data from the IceArc cruise are freely availablein PANGAEA, the Data Publisher for Earth & Environmental Science towed above the ice by helicopter [-2 s-1, with astandard deviation and range of 152 µmol m-2 s-1 and288-857 µmol m-2 s-1, respectively. Melting was evidencedby actual thinning of the sea ice measured during the occupation of the driftice station [The investigated ice floe consisted of FYI and covered a total area of 0.5kmlicopter and a 23licopter . The aveNaviculapelagica, Hantzschia weyprechtii,Entomoneis paludosa and Cylindrothecaclosterium , the under-ice water column (0.22 ±0.11 µgL-1) and melt ponds that had not melted through (0.05 µgL-1). Assuming a thickness of 0.5 m for the meltwater layer (asquare meter would thus correspond to 500 liter), that the majority ofaggregates floated within this layer and were similar in volume to the onemeasured (38 mL), the Chl a concentration of aggregates in arandom liter of seawater corrected for their square metre abundances  molar POC:PONratio of the aggregates was 9.7 ±0.7. Corrected for mean relative under-icecurrent velocities calculated for each sampling interval, we estimated anaggregate standing stock of 0.74 mg C m-2 and abundance of 0.24aggregates m-2 over a 499 m2 catchment area.During the six sampling intervals between 29 July and 1 August, the ice floedrifted 64 km with a largely southward component and a mean velocity of 0.22±0.06 m s-1 . Mean unOC value. The cumereafter . The agg-1 were measured during the peak aggregate accumulationon 31 July. Oxygen profiles measured through the same ice hole showed a decreasein oxygen concentrations in the upper 3 m of the under-ice water column betweenthe two sampling occasions on 30 and 31 July 2012  andPOC:PON (9.3) ratios further suggested the occurrence of senescent algae in theaggregates  and Ice2 (1.33 mg C m-2) (-2) fallsin between these two estimates  . The meastimates . Areal astimates .Howeverstimates .a concentrations were low both in sea ice and theunderlying water column. As we did not observe the formation of the floatingice-algal aggregates described herein, the conditions conducive to theirformation had already been established prior to our investigation period.Interestingly, all previous observations [The FYI encountered during this study was in a late stage of melt as indicated bya low modal ice thickness, high PAR transmittance, well-developed melt ponds anda freshwater layer below the ice . Chla crvations -21 were rvations ,43. The rvations .Melosira arctica [M. arctica havebeen summarized under sub-ice assemblages [M. arctica areattached to the underside of the ice, but grow in the underlying water column.The floating ice-algal aggregates we observed originated from the interstitialassemblage dominated by pennate diatoms that grow in the bottom of sea ice andare embedded in a mucous matrix, reminiscent of a biofilm. This continuous,inter-connected community likely already sloughed off from the bottom of the seaice during the initial stages of melting as the darker ice-algal patches on theunderside of sea ice accelerate bottom ablation. Extensive flushing of sea icethrough melt pond drainage and higher light availability under thinning ice[The species composition clearly distinguished our aggregates from the metre-longfilaments or strand-like aggregates formed by the centric sea ice diatom arctica ,42,45-47 arctica .The iceemblages  and furtemblages  as plankning ice,50-52 liExtracellular polymeric substances (EPS) have been proposed as a binding agent ofaggregates  and an aThe distribution of ice-algal aggregates underneath sea ice was very patchy asevidenced by the large temporal variability in accumulated aggregate biomassinside our ice hole, and during diver observations and spatial ROV surveys. Theskewed size-frequency distribution illustrates that the majority of ROV-detectedaggregates were smaller <15 cm and likely represent individual aggregates.This is supported by the ICE12 drift station observations. However, occasionaldetections of patches >15 cm represent accumulations of individual aggregatesthat concentrate in under-ice domes, open melt ponds or leads as observed bydivers and when surface sampling during ICE12. The lower aggregate abundanceestimated with the “inverted sediment trap” approach, as compared to the ROVsurveys, indicates that aggregates are easily transported below the ice beforethey settle in domes or crevices. This was also observed by divers, sinceexhaled air or fin movements rapidly dislodged and often dismantled the floatingaggregates. Our “inverted sediment trap” estimate of aggregate abundance istherefore conservative because not every passing aggregate got trapped.Nevertheless, it lies in the range reported from an earlier study based on divetransects . Changes-2 are small when compared todepth-integrated ice-algal biomass and primary production rates reported for theArctic, which range from 1-340 mg C m-2 in the former and <1-463mg C m-2 d-1 in the latter [Apherusaglacialis, are able to swim in the boundary layer below the ice andadapted to exploit patchy food sources [Melosira arctica dominated aggregates [Aggregate NPP and standing stocks me latter . However sources . Further sources . In case sources ,64 and agregates ,47. The gregates  similar a concentrations andNPP several orders of magnitude higher than in the surrounding water column andsea ice. High POC:PON and POC: Chl a ratios inside theaggregates during ICE12 and at station Ice2 were likely mediated by preferentialbacterial degradation of labile organic matter, in particular PON, and couldexplain oxygen consumption and elevated ammonium concentrations in surfacewaters where aggregates accumulated. The occurrence of the Chla degradation products phaeophorbide a andpyrophaeophorbide a at station Ice2 indicated increased grazing[a indicates a high fraction ofsenescent algae [The algal aggregates supported high levels of biological activity on the scale ofindividual aggregates as revealed by Chl  grazing. Older ant algae  within tnt algae  within tChaetoceros socialis and Melosira arctica,and the epiphytic diatom Synedropsis hyperborea [The aggregates studied herein are reminiscent in shape, colour, dominance ofpennate diatoms and association with the meltwater layer of the aggregatesreported from the Norwegian Fram Expedition in 1894  and theperborea , represeAlthough the biomass associated with the floating ice-algal aggregates was lowcompared to ice-algal blooms, they sustained high rates of biological activity atthe scale of the individual aggregate and provided a concentrated food source forthe ice-associated fauna during the oligotrophic Arctic summer months. This type ofaggregate-based habitat is likely to be fairly unique because it constitutes anextension of the sea ice community into the under-ice water column during the Arcticsummer melt season. The potential significance of the ice-algal aggregates forsurface consumption, energy transfer to the benthos and seeding of the next springbloom remains an interesting topic for future studies, and is an urgent reminder toimprove our understanding of the rapidly changing Arctic sea ice ecosystem.Table S1Results of aggregate extraction from ROV video transects at stationsIce1 (five dives) and Ice2 (four dives).(DOCX)Click here for additional data file.Table S2Up-scaling of aggregate biomass.(DOCX)Click here for additional data file.Table S3a normalized aggregate net primaryproduction (NPP).Up-scaling of Chl (DOCX)Click here for additional data file.Table S4Composition of ice-algal aggregates.(DOCX)Click here for additional data file.Figure S1Iodide concentrations measured in aggregate filtrate and ambientsurface seawater during cruise ICE12, July-August 2012. .(TIF)Click here for additional data file.Figure S2Relationship between mean relative under-ice current velocities(exemplified here for 3.5 m below the ice) and accumulated POC at eachsampling interval between 29 July and 1 August, 2012.(TIF)Click here for additional data file.Figure S3Oxygen profiles from the upper 5.5 m of the under-ice water columnmeasured with a MicroCat oxygen sensor on 30 and 31 July, 2012.(TIF)Click here for additional data file.Figure S4Pigment composition of ice-algal aggregates.Marker pigments and chlorophyll and its degradation products atstations Ice1  and Ice2 . Chl c 3 =chlorophyll c 3, Chl c1+2 = chlorophyllc1+2, 19-hex = 19-hexanoyloxyfucoxanthin, Fuco =fucoxanthin, Prasino = prasinoxanthin, Chl b = chlorophyllb, Phorbid a = phaeophorbide a,PyroPhorbid = pyrophaeophorbide a, Chl a =chlorophyll a, Phythin a = phaeophythina.(TIF)Click here for additional data file.


Sensitive and specific biomarkers of protein kinase inhibition can be leveraged to accelerate drug development studies in oncology by associating early molecular responses with target inhibition. In this study, we utilized unbiased shotgun phosphotyrosine (pY) proteomics to discover novel biomarkers of response to dasatinib, a small molecule Src-selective inhibitor, in preclinical models of colorectal cancer (CRC). We performed unbiased mass spectrometry shotgun pY proteomics to reveal the pY proteome of cultured HCT-116 colonic carcinoma cells, and then extended this analysis to HCT-116 xenograft tumors to identify pY biomarkers of dasatinib-responsiveness in vivo. Major dasatinib-responsive pY sites in xenograft tumors included sites on delta-type protein kinase C (PKCδ), CUB-domain-containing protein 1 (CDCP1), Type-II SH2-domain-containing inositol 5-phosphatase (SHIP2), and receptor protein-tyrosine phosphatase alpha (RPTPα). The pY313 site PKCδ was further supported as a relevant biomarker of dasatinib-mediated Src inhibition in HCT-116 xenografts by immunohistochemistry and immunoblotting with a phosphospecific antibody. Reduction of PKCδ pY313 was further correlated with dasatinib-mediated inhibition of Src and diminished growth as spheroids of a panel of human CRC cell lines. These studies reveal PKCδ pY313 as a promising readout of Src inhibition in CRC and potentially other solid tumors and may reflect responsiveness to dasatinib in a subset of colorectal cancers. Tyrosine phosphorylation is a key signaling mechanism regulating central aspects of mammalian cell behavior including proliferation, motility, metabolism, and differentiation . ProteinTargeted therapy with tyrosine kinase inhibitors (TKIs) is an ever-expanding modality that enables personalized cancer therapy ,4. LandmIn colorectal carcinoma (CRC), a large majority of cases display elevated activity of Src-family nonreceptor tyrosine kinases ,12, whicIn evaluating targeted TKIs in clinical oncology, there is a need to identify relevant biomarkers that can be used to guide dose selection in preclinical development and to monitor anti-tumor activity in clinical trials. Biomarkers may also be of value in predicting whether a patient is likely to benefit from a particular treatment. Several studies have utilized varied approaches in an attempt to identify such markers -26. RatiThe objectives of the present study were to use shotgun pY proteomics to obtain a global view of tyrosine phosphorylation in the well-known HCT-116 human colon adenocarcinoma cell line, and to extend the analysis to HCT-116 xenograft tumors treated with dasatinib to identify dasatinib-responsive pY biomarkers. We identified pY sites on signaling proteins including PKCδ CDCP1, and RPTPα as major dasatinib-responsive sites in HCT-116 xenograft tumors that may be useful as predictive biomarkers of SRC inhibition. Finally, using spheroid cultures established from a number of human CRC cell lines, we observed a correlation between datatinib-mediated inhibition of proliferation and reduction of PKCδ pY313. Our results reveal PKCδ pY313 as a candidate biomarker for predicting response to dasatinib in CRC.2, 37°C atmosphere. Growth medium consisted of Dulbecco’s Modified Eagle’s Medium  for all cell lines except Colo205 which was grown in RPMI (Cellgro). Growth media was supplemented with 10% fetal bovine serum , 1% antimycotic antibiotics (Mediatech), and 100 μM non-essential amino acids (Gibco-Invitrogen). All cells were passaged using 0.25% EDTA-trypsin (Gibco). HCT-116 (ATCC CCL-247), Caco-2 (ATCC HTB37), Colo205 (ATCC CCL-222), DKO-1, DLD-1 (ATCC CCL-221) were obtained from ATCC and Lim1215 cells  were obtDasatinib (BMS-354825) was kindly provided by Richard Smykla from Bristol-Myers Squibb Oncology . Dasatinib was dissolved in dimethyl sulfoxide (DMSO) in a 10mM stock solution. HCT-116, Caco-2, Colo205, DKO-1, and DLD-1 cells were treated with escalating dasatinib concentrations  for 24 h. At the end of treatment, culture medium was removed and cells were harvested for analysis.6 cells. Palpable tumors were detected within 2 to 4 weeks. Dasatinib was reconstituted in DMSO. Immediately prior to treatment, nine parts sterile saline was added to the drug solution. Tumor-bearing mice (0.5-1 cm longest dimension) were administered 100 μL dasatinib (60 mg/kg) or DMSO/saline vehicle by oral gavage. Animals were given either a single dose or treated daily for 5 consecutive days. For each treatment regimen, cohorts of animals were sacrificed and tumors were excised 4 h and 24 h following the final drug administration.All studies were approved by the Vanderbilt University Institutional Animal Care and Use Committee and all efforts were made to minimize animal suffering. HCT-116 xenografts were generated in athymic nude mice (Harlan Sprague-Dawley) following subcutaneous injection of 2-4×10g for 15 min) to clear insoluble material. Cleared tumor lysates containing approximately 40 mg protein (as determined by BCA assay) were used in the analysis. Tryptic peptides were generated by treating lysates overnight at 25°C with trypsin-TPCK (60:1 cellular protein:trypsin).  Tryptic peptides enriched in phosphotyrosine were prepared from HCT-116 monolayer cultures  and from HCT-116 xenograft tumors. Cell lysis, protein digestion, and immunoaffinity enrichment for pY-containing tryptic peptides was achieved using the PhosphoScan Kit  according to the manufacturer's protocol. For cultured cells, each round of analysis was performed using 60 mg total cellular protein (obtained from eight 150 mm dishes). Protein concentration was determined using BCA (Thermo Scientific), with bovine serum albumin  as a standard. Tumor lysates were prepared by homogenization on ice to disperse the frozen tissue (approximately 400 mg tumor wet weight in 9 mL in PhosphoScan Lysis Buffer), followed by brief sonication and centrifugation  equipped with an Eksigent NanoLC-AS1 Autosampler, Eksigent NanoLC-1D plus high performance liquid chromatography (HPLC) pump (Eksigent), nano-electrospray source, and Xcalibur instrument control software. Peptides were separated on a fused silica capillary column (100 μm × 18 cm) packed with reversed phase resin , and coupled with an in-line, fritted (generated with liquid silicate Kasil) trapping column (100 μm × 4 cm) also packed with C18 resin  . BiphasiTo improve the detection of phosphopeptides, an additional data-dependent algorithm in the Xcalibur software was enabled in which the presence of a predominant phosphate neutral loss  would trigger the acquisition of an MS/MS/MS [ Methods were similar to database searching strategies previously described , with miin vivo phosphorylation sites, version 1.5 (http://www.phosphosite.org), maintained by Cell Signaling Technology, Inc.  False-discovery rate was then estimated from peptide matches to the reverse sequences multiplied by two and divided by the total number of peptide hits. Peptides retrieved from database search algorithms with 5% false-discovery rate were further filtered by three additional steps. First, the score threshold was set depending on peptide category. For charge 1 peptides, peptides were chosen with xcorr greater than or equal to 1, RSp less than or equal to 5, and Sp greater than or equal to 350; for charge 2 peptides, threshold was set at xcorr 1.8, RSp 5, and Sp 350; for charge 3 peptides, threshold was set at xcorr 2.5, RSp 5, and Sp 350. Secondly, only peptides with at least a tyrosine and a phosphorylation were retained. Finally, of the remaining peptide hits, only pTyr-containing peptides with at least one of three additional criteria were retained: (1) at least two matching spectra had xcorr values above high threshold , (2) the pTyr site was independently identified from two or more distinct peptides, or (3) the pTyr site had been independently identified elsewhere and included in the PhosphoSite online resource of https://chorusproject.org/).  With a Chorus user account, any subset of these raw data can be downloaded or viewed online. Alternatively, these data can be downloaded directly as a 2.8 GB file without registering at: https://chorusproject.org/anonymous/download/experiment/-2997969850554343767).All original mass spectrometry data from the xenograph tumor experiments has been made publically available through the Chorus online mass spectrometry data resource (TM Plus-ECL (PerkinElmer) substrate was used for detection on a Xenogen IVIS 200. Densitometry was conducted using Living Image 3.2 software . Cell samples were collected after 24 h of dasatinib exposure. Xenograft tumor samples were collected 4 h or 24 h following the final administration of dasatinib. Tumors were snap frozen in liquid nitrogen, then homogenized and diluted to 1 µg/µl in lysis buffer. Between 20 and 40 µg of total protein from each sample was loaded into 7.5-12% SDS PAGE gels and resolved by electrophoresis prior to transferring to PVDF membranes (PerkinElmer). Membranes were blocked overnight at 4°C in tris-buffered saline 0.1% Tween-20 (TBST) containing 5% w/v non fat dry milk powder. Subsequently, membranes were immunoblotted with antibodies to p-Src Y416 , total Src , p-PKCδ Y313 , total PKC , p-RPTP Y789 , total RPTP (Upstate #07-472), β-actin , and β-tubulin . Probing occurred for 1 hr at room temperature with antibodies diluted as suggested by suppliers in TBST with 3% bovine serum albumin (BSA) followed by incubation for 1 hr at room temperature with the species-appropriate horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoReserach) diluted 1:5000 in TBST containing 3% BSA. Western Lightning Serial microscopic images of cell spheroids from 0, 24, 48 and 72 h post dasatinib treatment were imported into the ImageJ software package (NIH). Images were automatically converted to binary and holes in the binary image were closed using the Fill Holes function in ImageJ. The Wand Selection tool was used to segment all connected pixels from the spheroid, and the number of pixels in this area was recorded. Total area for each spheroid was normalized to the 24 h time point.202 to eliminate endogenous peroxidase activity. The sections were subsequently blocked with a serum-free protein blocking reagent for 20 minutes. Serial sections were incubated overnight with a 1:100 dilution of p-PKCδ pY313  antibody or p-SHIP2 Y986/987 . Detection of primary antibodies utilized Envision + Systems (Dako) with a horseradish peroxidase labeled polymer (30 min incubation) followed by addition of DAB substrate (5 min incubation). Stained samples were imaged at 40x magnification and analyzed for expression of histological markers.  Immediately following sacrifice, tumor samples of excised tumor were fixed in 10% formalin for 24 h and transferred to 70% ethanol. Samples were then processed for paraffin embedding and sectioned prior to immunostaining. Following sectioning, tissue samples were deparaffinized, rehydrated, and antigen retrieval step was conducted using a citrate buffer (pH 6.0) solution applied for 20 minutes at 105 °C followed by 10 minutes at room temperature. The samples were then treated with 3% Hin vitro [ We previously conducted a comprehensive analysis of the phosphotyrosine proteome of Src- transformed mouse fibroblasts in vitro . The purInitially, we sought to characterize the pY proteome of cultured HCT-116 cells, in the absence of dasatinib treatment. From 15 independent LC-MS/MS runs , 249 distinct pY peptides were identified. These peptides represent 162 distinct pY sites on 116 different proteins. Criteria for peptide retention included high-threshold xcorr value and identification in > 1 biological replicate.  Of the protein kinase pY sites, we identified 24 sites on 14 different tyrosine kinases and 15 sites on 14 different CMGC kinases. CMGC kinases  were prominent among the most frequently identified HCT-116 sites . CDK1 pY Having obtained the pY profile of HCT-116 cells in culture, the pY proteomics strategy was then employed to identify HCT-116 pY sites that are prominent in xenograft tumors and that have potential to serve as biomarkers for dasatinib responsiveness. Subcutaneous HCT-116 xenograft tumors were established in athymic nude mice and pY-enriched tryptic peptides were prepared from each of 3 tumors from both vehicle-treated mice and from mice treated 4 hours earlier with 60 mg/kg dasatinib. Each peptide preparation was analyzed through three replicate LC-MS/MS runs to obtain the tumor pY profiles. From this analysis, 71 pY sites, representing 58 different proteins, were recognized as being prominent in the xenograft tumors as well as being detected in the cultured cells . Similar CDCP1 pY707 , PKCδ pY313 , and PKCδ pY334  were among the most frequently identified sites in untreated xenograft tumors. Dasatinib treatment greatly reduced the identification frequency of all three sites , Table 4 Of the most prominent dasatinib-responsive pY sites identified from the phosphoproteomic analysis of HCT-116 xenograft tumors , Table 4To evaluate the effect of dasatinib treatment duration on the durability of Src inhibition, HCT-116 tumors treated with 5 daily doses of dasatinib were assessed by western blot analysis to determine the response of identified residues under steady-state drug administration. Similar to tumors treated with a single pharmacological dose, these tumors exhibited coordinate reductions in Src pY416, RPTPα pY798, and PKCδ pY313 observed 4 h following the final administration of dasatinib . HoweverIn addition to immunoblotting, PKCδ pY313 was evaluated in formalin fixed, paraffin-embedded tumor tissues. HCT-116 xenografts treated with dasatinib (60 mg/kg) or vehicle were evaluated 4 h and 24 h after a single pharmacological dose. IHC analysis of PKCδ pY313 showed robust membranous and cytosolic staining in vehicle treated mice . Analysiin vitro including HCT-116, Caco-2, Colo205, DKO-1, DLD-1, and Lim1215. In all cell lines evaluated except Lim1215, dasatinib exposure reduced Src pY416 in a concentration-dependent manner , further validating this phosphoproteomic approach [ HCT-116 colonic carcinoma cells grow rapidly in monolayer culture as adherent, closely packed polygonal cells and readily give rise to epithelioid tumors when injected subcutaneously into nude mice . TumorigRAS gene . To asseRAS gene . ExtensiRAS gene . Utiliziapproach . Many idapproach  rather tapproach . Given t Notable among the other most frequently identified HCT-116 cell pY sites is pY707 on CUB-domain-containing protein 1 (CDCP1). This site has the distinction of being the most frequently identified site not previously found in our MEF analysis . CDCP1,  Further indication of the capacity of pY proteomics to reveal distinct signaling activities in HCT-116 cells are pY sites readily detected on tyrosine kinases EGFR (pY1197), macrophage-stimulating protein receptor (MST1/RON (pY1238)), and the JAK-family member TYK2 (pY292). These tyrosine kinase sites were prominent in the HCT-116 cell profile with 9, 15, and 13 spectral IDs, respectively, but none were detected in the previous MEF analysis . An intrin vitro findings, many sites were identified on active signaling proteins, including many protein kinases. Notably, CDCP1 pY707 and two distinct sites on PKCδ (pY313 and pY334) were again identified. The ready identification of these three sites suggests high activity of the Src/CDCP1/PKCδ signaling complex in the HCT-116 tumors, indicating that observed pY hits on PKCδ may indeed serve as surrogates of Src activity and inhibition. Phosphoproteomics of HCT-116 xenograft tumors returned results simliar to those seen in cultured HCT-116 cells. Differential analysis of these samples revealed 71 distinct pY sites conserved from cultured cells to xenograft tumors. Similar to Additional dasatinib-responsive sites found in xenograft tumors included RPTPα Y798 and Annexin A2 pY30. RPTPα Y798 resides in the cytoplasmic tail near the C-terminus, is a Src target , and hasin vivo [Western blot confirmation of PKCδ pY313 and RPTPα Y798 as potential biomarkers illustrated that the effect of dasatinib on these sites was highly transient. The lack of durability in Src inhibition is consistent with the 3-5 h half-life reported for dasatinib in vivo . AssessmOur results also suggest that the Src autophosphorylation site pY416 and the pY313 site on PKCδ are correlated in CRC cells that are responsive to dasatinib. This was observed in 4 human CRC cell line models. Importantly, though Src pY416 was inhibited at elevated dasatinib concentrations in Lim1215 cells, PKCδ pY313 did not correlate with p-Src in this model and the lack of an effect on PKCδ pY313 predicted a lack of response. These results suggest that measuring Src pY416 levels alone may be insufficient to predict patient responses to dasatinib and that incorporating PKCδ pY313 may be a complementary measure of treatment response.in vitro and xenograft tumor screens and were subsequently validated in xenograft tumors using phosphospecific antibodies. These studies suggest that a subset of these potential markers, RPTPα pY798 and PKCδ pY313 in particular, represent promising biomarkers of response to dasatinib and may represent sensitive measurable downstream surrogates of Src inhibition and overall response in CRC. In summary, we have utilized a shotgun pY proteomics approach to identify candidate pY biomarkers of dasatinib response in HCT-116 cells and xenograft tumors. Several dasatinib-responsive pY sites representing important signaling proteins were identified in both Table S1Phosphotyrosine peptides from HCT-116 cultured cells.(XLS)Click here for additional data file.Table S2Phosphotyrosine peptides from HCT-116 tumors: untreated vs. dasastinibReferences.(XLS)Click here for additional data file.


Phosphorylation has been shown to have a significant impact on expanded huntingtin-mediated cellular toxicity. Several phosphorylation sites have been identified on the huntingtin (Htt) protein. To find new potential therapeutic targets for Huntington's Disease (HD), we used mass spectrometry to identify novel phosphorylation sites on N-terminal Htt, expressed in HEK293 cells. Using site-directed mutagenesis we introduced alterations of phosphorylation sites in a N586 Htt construct containing 82 polyglutamine repeats. The effects of these alterations on expanded Htt toxicity were evaluated in primary neurons using a nuclear condensation assay and a direct time-lapse imaging of neuronal death. As a result of these studies, we identified several novel phosphorylation sites, validated several known sites, and discovered one phospho-null alteration, S116A, that had a protective effect against expanded polyglutamine-mediated cellular toxicity. The results suggest that S116 is a potential therapeutic target, and indicate that our screening method is useful for identifying candidate phosphorylation sites. Huntingtin gene on chromosome 4 coding for a polyglutamine repeat expansion in the Huntingtin protein (Htt) Huntington's disease (HD) is a fatal progressive neurodegenerative disorder involving movement, cognitive and emotional symptoms, with no current neuroprotective therapy The pathogenesis of HD is still incompletely understood, but is believed to arise predominantly via a genetic gain of toxic function due to the CAG repeat expansion Htt is a very large protein with many protein interactions, and likely with many normal functions in the cell in vitro and in vivoThe N-terminal 17 amino acids of Htt, being immediately adjacent to the polyglutamine repeat, appear to be especially important for Htt pathogenesis As the region of the protein near the N-terminal polyglutamine repeat may be especially important for pathogenesis, we sought to determine additional sites of phosphorylation of Htt within this region. We used immunoprecipitation and mass spectrometry to identify novel phosphorylation sites within an N-terminal region fragment ending shortly before the known caspase cleavage sites. We found a number of candidate sites, and screened them for modulation of toxicity. In addition to the confirmation of known phosphorylation sites, we find that that alteration of the serine at position 116 has striking modulatory effects on Htt cell toxicity.Goat polyclonal 909 antibody, prepared against the N-terminal Htt exon-1 fragment was described previously For primary neurons, CD1 mice were purchased from Jackson Laboratory. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Johns Hopkins Animal Care and Use Committee (Protocol MO09M411).2 at 37°C. Cells were split at a 1/10 ratio when reaching 95% confluency. For Western blot experiments, cells were plated in 6 well plates (Corning) and for mass spectrometry experiment cells were plated in 100 mm dishes (Corning). Primary mouse cortical neurons were prepared as described previously 6 cell/cm2 on poly-D-lysine coated 24 well plates (Corning). Cells were kept in Neurobasal medium supplemented with B27 and 2 mM GlutaMAX (GIBCO) in 5% CO2 at 37°C until the day of the experiment. All cells were transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol.Human embryonic kidney (HEK) 293FT cells were from Invitrogen. Cells were kept in DMEM  supplemented with 10% FBS, 100 units/ml penicillin/streptomycin. (GIBCO), and 100 µg/ml Geneticin in 5% COAll Htt constructs used represent N-terminal fragments. They are referred to as N followed by number of amino acids present (e.g. N586). Htt expression constructs N511-52Q, N586-82Q, and N586-22Q were previously described HEK 293FT cells were transfected with htt N511-52Q construct. 24 h after transfection, cells were lysed in M-PER buffer (Pierce) with protease inhibitors (Sigma) and with or without phosphatase inhibitors . The lysates were diluted 1∶1 with phosphate-buffered saline (PBS) and NaCl was added to a final concentration of 150 mM. FLAG-Htt fusion proteins were immuprecipitated from 20 mg total protein using anti-FLAG M2 affinity gel (Sigma) according to the manufacturer's protocol, followed by elution with 100 µg/mL of FLAG peptide. Eluted proteins were analyzed by Western blot with a FLAG antibody . Samples were loaded on NuPAGE 4–12% BisTris polyacrylamide gel, and proteins were visualized with silver stain .For mass spectrometric analysis Htt containing bands were manually cut out for in-gel digestion. Gel pieces were destained with 30 mM potassium ferricyanide and 100 mM sodium thiosulfate (50∶50 vol/vol), rinsed with water 3 times, incubated in 20 mM ammonium biocarbonate for 10 min, and dehydrated with acetonitrile. The process was done three times, and gel pieces were then dried in a SpeedVac. For in-gel digestion, gel pieces were incubated overnight at 37°C with 10 ng/l trypsin (Roche) in 20 mM bicarbonate. Peptides were extracted two times with 50% acetonitrile and 2% formic acid. Extracts were pooled and evaporated to dryness.Peptides were analyzed using QSTAR Pulsar (Applied Biosystems-MDS Sciex) interfaced with an Eksigent nano-LC system. Peptides were resuspended in 0.2% formic acid (10 µl). The solution was then separated on a 360×75 µm reverse-phase column of 10 cm of C18 beads  and a 10-µm emitter tip (New Objective). The gradient for high pressure liquid chromatography was 5–40% B for 25 min  at a 300 µL/min flow rate. Survey scans were acquired from m/z 350–1200 with up to three precursors selected for MS/MS using a dynamic exclusion of 30 s. Rolling collision energy was used to promote fragmentation. The electrospray voltage was 900 V and MS/MS spectra of ion 416.7 m/z were acquired for 3 min. The MS/MS spectra were searched against NCLInr database, using Mascot Daemon as an interface and our in-house Mascot server.For Western blotting analysis, HEK 293FT cells were lysed 48 h after transfection in M-PER buffer (Pierce) with a protease inhibitors cocktail (Sigma). Protein concentrations were determined using BCA method (Bio-Rad). Proteins were separated on NuPAGE 4–12% BisTris polyacrylamide gels (Invitrogen) and transferred to nitrocellulose membranes (Bio-Rad). After 1 h blocking , membranes were probed with primary antibodies, washed, and incubated with peroxidase-conjugated secondary antibodies (Amersham). The signal was detected using chemi-luminescence . Protein bands were quantified using Molecular Imager Gel Doc XR System and Quantity One software (Bio-Rad).To measure the survival of a small population of transfected primary neurons in culture, we established an analysis based on the nuclear condensation observed during cell death. This assay measures the Hoechst staining intensity of transfected neurons. Neurons were co-transfected at DIV5 with the Htt N586-82Q phospho-mutations and eGFP (10∶1 ratio) using Lipofectamine 2000 (Invitrogen) according to the manufacturer's recommendations. After 48 h of expression, cells were fixed with 4% paraformaldehyde for 30 mn and stained with MAB5492 (Htt1-82) antibody according to the laboratory protocol. Nuclei were stained with Hoechst 33258 . Image acquisition was done using the Axiovision imaging software on an Axiovert 100 inverted microscope (Carl Zeiss) using the automated Mozaix function to cover the integral surface of the wells. Analysis and quantification were performed using the Volocity software (Perkin-Elmer). In order to measure only the intensity of the nuclei of transfected cells, first specific signal of transfected Htt was isolated. Nuclei were subsequently detected in these specific regions corresponding to the transfected cells and their average intensity measured. The percentage of surviving cells was calculated as the percentage of cells for which the average intensity of nuclear staining does not exceed 200% of the average intensity of healthy untransfected nuclei. Results are shown as a percentage of survival of transfected cells, and each independent experiment represent the average of 4 wells per condition.2 were maintained in the chamber throughout the experiment. Morphology of every neuron was quantified blindly using Axiovision software. Cells were given a value for every frame of the experiment: 100 when healthy and 0 when dead. Cells were considered dead when most of the neurites were detached from the soma and fragmented, or when the soma became totally round. Results are expressed as mean ± sem. n = 200 cells analyzed in 5 independent experiments.Primary neurons were co-transfected using Lipofectamine 2000 in the same conditions as described above. 38 h post-transfection, plates were transferred into the incubation chamber of an Axiovert 100 inverted microscope (Carl Zeiss) and healthy neurons expressing GFP were randomly selected and followed for 10 h with a picture taken every 10 min. 37°C and 5% COCells were fixed directly in the cell culture dishes with PBS containing 4% paraformaldehyde for 30 min. After washing three times with PBS, cells were permeabilized with PBS containing 0.2% Triton X-100 for 10 minutes, then treated with blocking buffer containing 0.2% Triton X-100 and 10% goat serum for 2 h. MAB5492 antibody (1∶1500) was incubated overnight at 4°C in PBS containing 10% goat serum. Cells were rinsed in PBS and incubated for 2 h with anti-mouse CY3-conjugated antibody . Nuclei were stained with Hoechst 33342 and, after washing twice in PBS, cells were mounted onto slides using Vectashield. Confocal microscopy was performed using a Zeiss Axiovert 200 inverted microscope with 510-Meta confocal module and 63× objective.In order to identify novel phosphorylation sites in a region of Htt using mass spectrometry, we chose a fragment of 511 amino acids for study. It is slightly smaller than potential fragments generated by cleavage at the several known caspase sites, and thus is unlikely to be cleaved further by caspases in the cell during the experiment. The construct used contained an N-terminal Flag tag  and a glHEK293 cells were transfected with the Htt N511-52Q construct  and eithIn order to screen for the potential functional relevance of the detected phosphorylation sites in Htt, we performed site directed mutagenesis to alter all of the sites. in vivoFor our primary functional assay we chose expanded Htt cell toxicity, since this is a highly relevant cell phenotype for this neurodegenerative disease The results of the screen for Htt toxicity are shown in In order to confirm the effect of the alterations of phosphorylation sites in expanded Htt on its toxicity and to demonstrate directly that these alterations affected Htt-mediated neuronal cell death in culture, we used a time lapse video microscopy assay. This method is comparable to a time lapse analysis method used in the Finkbeiner laboratory In this study we have used mass spectrometry to identify potential phosphorylation sites within the N-terminal region of Htt. We have identified novel potential sites, and have confirmed previously known S421 phosphorylation site. Using a nuclear condensation cell toxicity assay in primary neurons, we set up a screen to evaluate the effects of alterations of phoshorylation sites in polyQ-expanded Htt on its toxicity. Alanine replacement at one site, serine 116, caused a significant decrease of Htt-mediated cell toxicity. We confirmed this effect using a cell death assay based on time lapse video microscopy to directly track cell death.Strengths of this study include the identification of several novel candidate phosphorylation sites, focusing on the N-terminus of Htt near the polyQ repeat, a region of the protein likely to be relevant for HD pathogenesis. We were able to determine a functional effect of an alteration of at least one of these sites.in vitro, and relied on over-expression in non-neuronal cell lines. We did not completely cover the N-terminal 511 amino acids of Htt, though we did achieve 73.14% coverage of the region . A number of the phosphorylation sites we found did not have high ion scores, however our goal was to be inclusive rather than exclusive in pursuing candidate sites, and then to use functional assays to identify sites with potential physiologic relevance.There were several limitations of the study. The discovery experiments were all carried out We have included the phosphorylation sites within the N-terminal 17 amino acid region in the functional studies because of the previous reports of protective effects of Htt phosphorylation within this region. Although, because of the unfavorable configuration of tryptic cleavage sites in this region, we did not identify Htt phosphorylation sites within its N-terminus. Similar to previous studies, we found that phospho-mimetic alteration at the S13/16 sites ameliorated toxicity.We also studied the previously reported phosphorylation at S421 and confirmed the neuroprotection Neuronal cell death is highly relevant to pathogenesis of HD, and cell loss in postmortem HD striatum best reflects premortem motor impairment and functional disability in vitro short-duration system, and will need in vivo confirmation. In our experiments, we saw protection with the serine to alanine alteration, but no change with the serine to aspartate alteration. This could indicate that the stoichiometry of phosphorylation at this site is relatively high or possibly that the aspartate substitution does not well mimic the effects of phospho-serine at this site.The S116A alteration showed significant and reproducible protection in the nuclear condensation assay. This was clearly supported by the data from the time-lapse microscopy data, which showed substantial decrease of Htt-mediated cell death. It should be kept in mind that cell culture experiments with over-expressed protein involve an In this study we have attempted to find phosphorylation sites with functional relevance that could be involved in disease pathogenesis. We found a striking effect of alteration of the S116 site on mutant Htt cellular toxicity. This raises the possibility that phosphorylation of S116 could be involved in HD pathogenesis. This would be reminiscent of other neurodegenerative diseases in which phosphorylation is known to modulate cellular toxicity of the relevant disease protein. For instance, in the case of SCA1, alteration of serine 776 in a transgenic mouse model substantially ameliorated the phenotype Htt phosphorylation has been suggested to have potential therapeutic relevance. Small molecule modulation of phosphorylation within the first 17 amino acids alters Htt sub-cellular localization and Htt cleavage and toxicity in vivo. However, if phosphorylation of Htt at serine 116 can be shown to be relevant in vivo, then we would propose that phosphorylation of S116 has an important modulatory role in HD pathogenesis. Because changing the serine to alanine is protective, inhibiting the kinase or kinases that phosphorylate this site should also be protective. Thus this phosphorylation event could be a candidate therapeutic target for HD.The candidate phosphorylation sites identified here still need to be confirmed Table S1Primer sequences for Htt-N586-82Q site directed mutagenesis.(DOCX)Click here for additional data file.


The RhoA/ROCK pathway contributes to diabetic cardiomyopathy in part by promoting the sustained activation of PKCβ2 but the details of their interaction are unclear. The purpose of this study was to investigate if over-activation of ROCK in the diabetic heart leads to direct phosphorylation and activation of PKCβ2, and to determine if their interaction affects PDK-1/Akt signaling.in vitro. siRNA silencing was used to confirm role of ROCK2 in PKCβ2 phosphorylation in vascular smooth muscle cells cultured in high glucose. Furthermore, the effect of ROCK inhibition on GLUT4 translocation was determined in isolated cardiomyocytes by confocal microscopy.Regulation by ROCK of PKCβ2 and related kinases was investigated by Western blotting and co-immunoprecipitation in whole hearts and isolated cardiomyocytes from 12 to 14-week diabetic rats. Direct ROCK2 phosphorylation of PKCβ2 was examined in vitro. ROCK2 siRNA in vascular smooth muscle cells or inhibition of ROCK in diabetic hearts reduced PKCβ2 T641 phosphorylation, and this was associated with attenuation of PKCβ2 activity. PKCβ2 also formed a complex with PDK-1 and its target AKT, and ROCK inhibition resulted in upregulation of the phosphorylation of PDK-1 and AKT, and increased translocation of glucose transporter 4 (GLUT4) to the plasma membrane in diabetic hearts.Expression of ROCK2 and expression and phosphorylation of PKCβ2 were increased in diabetic hearts. A physical interaction between the two kinases was demonstrated by reciprocal immunoprecipitation, while ROCK2 directly phosphorylated PKCβ2 at T641 This study demonstrates that over-activation of ROCK2 contributes to diabetic cardiomyopathy by multiple mechanisms, including direct phosphorylation and activation of PKCβ2 and interference with the PDK-1-mediated phosphorylation and activation of AKT and translocation of GLUT4. This suggests that ROCK2 is a critical node in the development of diabetic cardiomyopathy and may be an effective target to improve cardiac function in diabetes. Diabetic cardiomyopathy is a common complication of diabetes mellitus. The affected hearts initially show diastolic dysfunction, progressing to systolic dysfunction and to the eventual development of left ventricular dilatation and failure At the cellular and molecular levels, hearts from both human patients and animal models of type 1 and type 2 diabetes show changes in energy metabolism, calcium homeostasis, signaling pathways and contractile protein composition Protein kinase C β2 (PKCβ2) is a member of the conventional subfamily of PKC isozymes The canonical pathway for maturation of conventional PKCs into a catalytically competent form is the sequential phosphorylation of three residues: first, a threonine residue in the activation loop (T500 in PKCβ2) by phosphoinositide-dependent protein kinase-1 (PDK-1), followed by residues in the turn motif (T641) and the hydrophobic motif in the C-terminal region (S660). Although there is evidence that the latter two arise from autophosphorylation, more recently a role for mTORC2 in the phosphorylation of the turn motif in conventional PKCs and in PKCε was demonstrated The purpose of the present study was to investigate the mechanism of interaction between ROCK and PKCβ2 leading to altered phosphorylation of the latter kinase in diabetic hearts. We found evidence that ROCK2 directly interacts with and phosphorylates PKCβ2 at the T641 site, leading to its activation. Interestingly, inhibition of ROCK-induced PKCβ2 T641 phosphorylation and activity was also associated with increased phosphorylation of PDK-1 and Akt and consequently translocation of GLUT4 to the cell membrane.All animal study procedures complied with the guidelines of the Canadian Council on Animal Care and all protocols were approved by the Animal Care Committee of the University of British Columbia.Male Wistar rats were treated with streptozotocin  or its citrate buffer vehicle  as described Isolated working hearts were perfused with normal buffer or buffer containing 1 µM H-1152 for 30 min as described in our previous study 2+-tolerant cardiomyocytes were isolated from control and diabetic hearts as described previously 2+-free Tyrode’s solution , followed by Tyrode’s solution containing 0.05 mM Ca2+, 0.8 mg/ml type II collagenase  and 0.1% BSA. The ventricles were removed, minced, and the resulting cell suspension filtered and centrifuged briefly at 60×g. The cell pellet was washed 3 times in Tyrodes solution containing increasing amounts of Ca2+ . Viability, determined by assessing the percentage of cells that excluded trypan blue dye, ranged from 80% to 90%. The cells were then plated on laminin-coated dishes and cultured in M-199 media for the period of the experiment.CaWestern blotting was performed as described previously Vascular smooth muscle cells were isolated from the aorta of male Wistar rats by sequential digestion with collagenase I and II and elastase 5 vascular smooth muscle cells were seeded in each well of a 6-well culture plate, incubated overnight and transfected the following day. For each transfection, 20 pmols of ROCK2 or scrambled siRNA diluted in 100 µl siRNA transfection medium was mixed with 6 µl of siRNA transfection reagent diluted in 100 µl of siRNA transfection medium at room temperature for 45 minutes to form transfection complexes. The mixture was diluted with 0.8 ml of siRNA transfection medium and was added directly to the cells that had been pre-washed once with 2 ml of siRNA transfection medium. The cells were then incubated for 6 hours. Transfected cells were recovered by adding 1 ml of growth medium with 10% fetal calf serum. After 24 hours, the medium was changed to one containing low glucose (5 mM) or high glucose (20 mM) without serum for another 48 hours. Finally, the medium was removed by aspiration and the transfected cells were washed twice with PBS. SDS gel loading buffer (200 µl) was added to lyse the cells and obtain protein for SDS PAGE and Western blotting.All siRNA and transfection reagents were obtained from Santa Cruz Biotechnology and were used according to the manufacturer’s instructions. Briefly, 2.0×102. At the end of the incubation period, SDS gel loading buffer (5 µl) was added and the samples were boiled for 5 min. Western blotting was performed with anti-phospho PKCβ Τ500, anti-phospho PKCβ2 Τ641 and anti-phospho PKC S660 antibodies .PKCβ2 was dephosphorylated with lambda protein phosphatase and then incubated with or without active ROCK2  in a molar ratio of 1 to 30 in a total volume of 50 µl at 37°C for 45 min. The reaction mixture contained 20 mM tris pH 7.4, 400 µM ATP and 3 mM MgClTotal proteins extracted from untreated or ROCK inhibitor-perfused diabetic hearts were immunoprecipitated in RIPA buffer with anti-PKCβ2 antibody coupled to Dynal beads according to the manufacturers’ instruction. The eluted protein was separated by SDS PAGE, followed either by staining with Coomassie Brilliant Blue R250 to demonstrate total PKCβ2 binding proteins or by Western blotting with phospho-(Ser) PKC substrate antibody  to identify ROCK-dependent PKCβ2 phosphorylation of proteins.Cardiomyocytes were cultured in M-199 medium on glass bottom culture dishes pre-coated with laminin and allowed to attach for one hour. The medium was then exchanged for fresh M-199 and incubation continued in the presence or absence of ROCK inhibitors for another one hour at 37°C. The cells were washed with ice-cold PBS twice and fixed with 3.7% paraformaldehyde for 20 min. They were permeabilized and blocked with 0.1% NP40 plus 2% BSA in PBS for 45 min at room temperature, then treated with anti-GLUT4 and anti-VAMP2 antibodies  at 1∶200 dilution in the same buffer at 4°C overnight. Following three 10 min washes with PBS containing 0.1% NP40 plus 2% BSA, the cells were incubated with a secondary antibody conjugated to Alexa Fluor® 488 or Alexa Fluor® 555 at 1∶1000 for one hour at room temperature. Following another three 10-min washes, the cells were mounted with Prolong Gold Antifade reagents and observed with a Zeiss LSM700 laser scanning microscope.2+-dependent phosphatase and its total activity was determined as described in 2 at 30°C for 30 min with 5 µg of inactive AKT-1 (Invitrogen) and 50 µg of heart protein extract. Following the incubation, levels of the AKT phosphorylation were analyzed by Western blotting with anti-AKT S473 antibody .PHLPP is an MnAll values are expressed as the mean ± SEM; n denotes the number of animals in each group. Statistical analysis was performed by one-way ANOVA followed by the Newman-Keuls test for multiple comparisons, using GraphPad Prism (GraphPad Software). For all results the level of significance was set at P<0.05.We have previously shown that ROCK activity is increased in hearts from diabetic rats, and that its acute inhibition improves contractile function In order to confirm this unexpected observation we investigated the effects of H-1152 and those of a second, chemically distinct, ROCK inhibitor, Y-27632, on the phosphorylation of PKCβ2 in cardiomyocytes isolated from control and diabetic hearts . As was Because of similarities between the different PKC isoforms in the sequences of the activation loop, hydrophobic motif and turn motif, anti-phospho PKCβ2 antibodies may cross-react with the same sites in other isoforms. In order to rule out a contribution of other isoforms to the changes in phosphorylation detected, we also determined phosphorylation of the T500, T641 and S660 residues in PKCβ2 immunoprecipitated from diabetic hearts . The resTaken together, these data suggest that ROCK regulates the phosphorylation of PKCβ2 T641 in diabetic hearts, but that the phosphorylation of the T500 and S660 residues is controlled by a different mechanism, which is examined further below.Since the phosphorylation of T641 is an absolute requirement for PKCβ2 activity In order to determine whether there was a physical association between ROCK2 and PKCβ2 in the heart, we conducted reciprocal immunoprecipitation with anti-ROCK2 and anti-PKCβ2 antibodies. The results show that ROCK2 was immunoprecipitated from both control and diabetic hearts by anti-PKCβ2 antibody  and simiin vitro phosphorylation reaction in the presence of ATP. The results showed that levels of PKCβ2 T641 and S660 phosphorylation were increased by approximately 2 and 2.5 fold respectively in the presence compared to the absence of ROCK2. However, no phosphorylation of the T500 residue could be detected  have been shown to dephosphorylate PKCβ2 and other PKCs AKT is an immediate downstream target of PDK-1, so we determined whether phosphorylation of this kinase was altered in diabetic compared to control hearts. In untreated diabetic hearts, T308 phosphorylation in the AKT activation loop, which is the PDK1 site, was unchanged  while phBoth PDK-1 and Akt co-immunoprecipitated with the anti-PKCβ2 antibody, suggesting that these proteins form a signaling complex with PKCβ2 in cardiomyocytes . Total PSince AKT regulates the translocation of GLUT4 to the membrane, we investigated whether the levels of GLUT4 in the membrane fraction were affected by ROCK inhibition. As reported previously Both acute and chronic inhibition of the RhoA-ROCK pathway improve the contractile function of the diabetic heart ROCK1 and ROCK2 have a molecular weight of approximately 160 kDa and share up to 65% overall homology with 92% homology in the kinase domain in vitro. Finally, the selective silencing of ROCK2 profoundly attenuated PKCβ2 phosphorylation at the T641 site in vascular smooth muscle cells. On the other hand, a role of mTORC2 in this process seems unlikely, since the increased phosphorylation of PKCβ2 in the diabetic heart was not accompanied by a corresponding increase in the phosphorylation of PKCα at the turn motif, while the phosphorylation of AKT at the S473 site, a well-established mTORC2 target used to assess mTORC2 activity The phosphorylation of PKCβ2 at each of the three sites essential for its full activation was uniformly increased in diabetic hearts, although through distinct mechanisms. Previous studies have suggested that mTORC2 is important in the phosphorylation of the turn motif, the site most critical for kinase activity, in conventional PKCs including PKCβ2 PHLPP1 and PHLPP2 are the phosphatases that are believed to control the intracellular levels of PKCβ2 through dephosphorylation of T641 and S660, resulting in degradation of the kinase in vitro phosphorylation assay, although phosphorylation of S660 was increased, likely through autophosphorylation. PDK-1 has been demonstrated to be the kinase responsible for the activation loop phosphorylation in PKCβ2 as well as other PKC isoforms ROCK2 had no effect on the phosphorylation of PKCβ2 at T500 site in the Akt is an important signaling hub that regulates cell metabolism, proliferation and survival ROCK2 siRNA was much more effective than the pharmacological inhibition of ROCK in reducing PKCβ2 T641 phosphorylation in vascular smooth muscle cells incubated in high glucose. Furthermore, ROCK2 silencing also reduced PKCβ2 T641 phosphorylation in normal glucose, although pharmacological inhibition of ROCK had no detectable effect on this parameter. These results may be explained by the observation that proteins treated with small molecule inhibitors may still act as a scaffold for protein-protein interactions that are disrupted by siRNA treatment Inhibition of ROCK leads to greatly reduce PKCβ2 activity as showed the diminution of phosphorylation in 55 and 45 kDa protein bands. Further studies are underway to identify the ROCK2-dependent PKCβ2 targets and determine whether they are new targets contributing to dysfunction in diabetic heart. Importantly, it will be of significance to determine whether ROCK2, PKCβ2, PDK-1 and AKT also form a signaling complex as a mechanism to regulate GLUT4 translocation in adipose tissues and skeletal muscle in type 2 diabetes, since it is well-established that the translocation of GLUT4 to the membrane is the rate-limiting step in insulin-induced glucose sensitivity through activation of AKT in human patients, yet the detailed mechanisms remain elusive In summary, the current study shows ROCK2 acting as a kinase for PKCβ2 T641 phosphorylation and activation in diabetic hearts. Inhibition of ROCK is associated with activation of PDK-1 and its downstream target AKT, leading to increased translocation of GLUT4 to the membrane. Overall, this study establishes that ROCK2 as a critical node in the development of diabetic cardiomyopathy and an important target for its treatment, as it’s inhibition will simultaneously improve multiple signaling pathways in the diabetic heart.


YB-1 mRNA in rabbit organs and several cell lines. As found, in the majority of studied eukaryotic cells a considerable proportion of YB-1 mRNA was stored in free mRNPs, i.e., was poorly translated. Also, we demonstrated that YB-1 synthesis depended on conditions that determined the rate of cell division. Specific suppression of YB-1 synthesis resulted from inhibition of the mTOR signaling pathway with inhibitor PP242, but not rapamycin. Experiments on reporter constructs showed that dependence of YB-1 mRNA translation on activity of the mTOR signaling pathway was dictated by 5′ untranslated regions of this mRNA, irrelatively of the TOP-like sequences at the beginning of 5′ UTR.YB-1 is a eukaryotic protein with numerous intra- and extracellular functions based on its ability to interact with RNA, DNA, and many proteins. In spite of achievements in studying its functions, regulation of YB-1 synthesis in the cell remains poorly understood. In the current study Western and Northern blotting were used to determine the amounts of YB-1 and  YB-1 belongs to proteins with a cold shock domain and performs many functions in the cell . Finally, we have shown that inhibition of the mTOR kinase results in specific negative regulation of both endogenous YB-1 synthesis and translation of reporter constructs containing YB-1 mRNA 5′ UTR.Here, using Western and Northern blotting, we detected amounts of YB-1 and YB-1 mRNA translation, we estimated the amounts of YB-1 and YB-1 mRNA in several eukaryotic cell cultures and various rabbit organs. The amount of YB-1 was determined using Western blot with antibodies against its C-terminal peptide, while Northern blot was used to detect YB-1 mRNA. As seen from YB-1 mRNA shows no considerable variability either, ranging from 0.4 to 0.6 ng per 10 µg of the total RNA  and heart . In other rabbit organs it varies from 0.1 to 0.2 ng per 15 µg of the total RNA. It should be noted that in spite of a low YB-1 mRNA content in rabbit lungs and spleen, the YB-1 amount in these organs was almost as large as in testis, which suggested a high level of YB-1 mRNA translation there or a high YB-1 stability.As to YB-1 and to organ . Rabbit YB-1 mRNA, we determined its content in polysomal- and free mRNP fractions from lysates of various eukaryotic cell lines and rabbit organs. As a rule, the presence of mRNA in a polysomal fraction points to its efficient translation. In contrast, predominant localization of mRNA in a free mRNP fraction is indicative of the absence or low level of translation of this mRNA. The lysate samples were centrifuged through 50% sucrose to give free mRNPs in the supernatant, and polysomal mRNPs in the pellet. Isolation of the total RNA from these fractions was followed by detection of YB-1 mRNA (or GAPDH mRNA as a control) by Northern blot. Applicability of this technique was verified by addition of up to 30 mM EDTA to the lysate prior to centrifugation  more than 90% of YB-1 mRNA is sedimented in the form of free mRNPs. Thus, in the majority of the studied cell lines, YB-1 mRNA proved to be a weak template.It follows from YB-1 mRNA distribution in lysates of rabbit organs showed a lower translational level of YB-1 mRNA as compared to GAPDH mRNA used as a control . To confirm conclusions drawn from the experiments on cell cultures, we studied the rate of YB-1 synthesis in the cell. For this purpose, we proposed a technique implying metabolic cell labeling with -methionine). It appeared that in 3T3, CHO and Vero cells with a high level of YB-1 mRNA in the free mRNP fraction YB-1 was nevertheless synthesized  which underlies the so-called density-dependent inhibition of cell proliferation. We sought to see how an increased cell density  affected YB-1 synthesis. For this purpose 3T3 cells were cultivated up to a confluence of 40–50%, 80–90% and 100%, and additionally incubated with [sity see .35S]-methionine), and then YB-1 synthesis was analyzed  on YB-1 synthesis and its amount in the cell. After 2-day starvation 3T3 cells  and HEK2YB-1 mRNA falls into the set of mRNAs whose translation is strongly dependent on activation of the mTOR signaling pathway. To answer it, we incubated HeLa and HEK293 cells with PP242, a specific mTOR inhibitor, for 2–3 h  suppressing phosphorylation of Akt and downstream substrates  inhibitbstrates  accompanbstrates , producebstrates . TogetheYB-1 mRNA. According to the NCBI database, YB-1 mRNA 5′UTR may comprise from 118 to 171 nt. We used 2 variants of this 5′UTR: one of the minimal length – from rabbit mRNA ) and the other of the maximal length – from human mRNA ). These or luc mRNA with control 5′- and 3′ UTRs . We sought to verify this suggestion using reporter mRNA coding for luciferase (luc) and possessing 5′UTR and/or 3′ UTR from  3′ UTRs  were useluc mRNAs with rabbit YB-1 mRNA 5′ and 3′ UTRs. This result is perfectly consistent with results obtained for endogenous YB-1 synthesis  provide conditions for a high level of YB-1 mRNA translation.Here we studied the amounts of YB-1 and YB-1 mRNA and YB-1 in rabbit organ lysates showed that they are variable and often display no correlation. In this situation, one can suggest several modes of post transcriptional regulation of YB-1 synthesis. Specifically, a high level of YB-1 mRNA translation in the lungs explains a large amount of YB-1 along with a low content of YB-1 mRNA in this organ. A high level of YB-1 mRNA in the testis lysate might suggest a large amount of the protein. However, it is not so, suggesting that the bulk of YB-1 mRNA may be stored in the masked form for a future use. In the brain, the level of YB-1 mRNA translation is also high, but the YB-1 amount is fairly low. Possibly, this is underlain by an enhanced YB-1 degradation in this organ.On the other hand, analysis of the amounts of The developed here technique of YB-1 synthesis assessment allowed us to pioneer in finding the peculiarities of synthesis of endogenous YB-1 in the cell. Specifically, we report the dependence of YB-1 synthesis on the rate of cell division. As shown, the density-dependent inhibition of cell proliferation makes the rate of YB-1 synthesis much lower.Serum starvation that also retards cell division results in a lower amount of YB-1 in the cell. As to YB-1 synthesis, it decreases to a larger extent than the total protein synthesis. The release from the starvation-induced arrest of cell division is accompanied by a rapid and essential increase of the YB-1 synthesis rate followed by renewal of the YB-1 amount in the cell.The role of YB-1 in cell proliferation has been widely reported YB-1 mRNA 5′ UTR. Besides, YB-1 insensitivity to rapamycin  shows that it is phosphorylation of 4E-BP, and hence, its eIF4E-binding activity that regulates translation of YB-1. Of interest, the Erk1/2 inhibitor U0126 had no effect on YB-1 synthesis. It is known that ERK activation causes, in particular, phosphorylation of eIF4E by kinase Mnk1, and hence, an increase in translation of some eIF4E-dependent templates. The fact that YB-1 synthesis remained unaffected by ERK inhibition  suggests that the level of YB-1 mRNA translation depends on the eIF4E/phospho-4E-BP ratio rather than on the amount and phosphorylation of eIF4E.We have shown that the mTOR signaling pathway is one of the signaling cascades responsible for YB-1 synthesis regulation. Inhibition of the mTOR kinase by PP242 and the upstream kinase PI3K (by wortmannin) results in specific negative regulation of both endogenous YB-1 synthesis and translation of reporter constructs containing YB-1 mRNA was supported by the results reported recently YB-1 mRNA was detected in the pool of mRNAs whose translation was suppressed with inhibited mTOR. These authors believed that inhibition of YB-1 mRNA was induced by PRTE  or TOP-like elements of YB-1 mRNA 5′ UTR. But this element was positioned at the beginning of the 171 nt-long 5′ UTR of human YB-1 mRNA, while our reporter construct contained a 5′UTR of rabbit mRNA which is shorter and lacks PRTE. However, luc mRNA with such a truncated 5′ UTR displayed sensitivity to inhibition of the mTOR signaling pathway. Presumably, the sequence responsible for sensitivity to mTOR inhibition within the 5′ UTR, is closer to the coding region than PRTE. It is still to be refined what elements of the primary or secondary structure of YB-1 mRNA 5′UTR are responsible for mTOR-dependent regulation of YB-1 mRNA translation.The role of mTOR in regulation of translation of 2 and passaged by standard methods.3T3 and HEK293 cells  were kindly provided by Dr Elena Nadezhdina (Institute of protein research RAS) and were cultivated in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal calf serum, 2 mM glutamine, 100 U/ml penicillin, and 100 µg streptomycin (PanEco). HeLa cells were cultivated in DMEM/F12 supplemented with 10% fetal calf serum, 2 mM glutamine, 100 U/ml penicillin and 100 µg streptomycin (PanEco). The cells were incubated at 37°C in a humidified atmosphere containing 5% COIn some experiments cells were treated with PP242 , or rapamycin  or wortmannin  or U0126  for 2–3 h.Samples for Western blot analysis were obtained as follows. The rabbit tissue samples were obtained from the Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, where before use in experiment rabbits were kept under standard conditions of the barrier zone in compliance with Program of care and use of animals, and all manipulations with them were performed in compliance with ethical standards in animal research approved by the Institute’s Commission on Biological Safety and Ethics established on October 3, 2011 (# 173/k).g for 10 min to obtain cleared samples. The cultured cells were washed with phosphate buffered saline, lysed in SDS-electrophoresis sample buffer or buffer containing 20 mM HEPES-KOH (pH 7.6), 100 KCl, 5 mM MgCl2, 2 mM DTT, 0.25% Nonidet P-40, 0.2% SDS and protease inhibitors cocktail (Roche). The lysate was centrifuged at 5,000 g for 10 min to obtain cleared samples.Rabbit tissues  were homogenized using a Dounce homogenizer in SDS-electrophoresis sample buffer. The lysate was centrifuged at 5,000 S473 , phospho-p70S6KT389 , phospho-rpS6S240/244 , phospho-4EBP1T37/46 , 4EBP1 . phospho-eIF4ES209 , phospho-eIF2αS51 , phospho-p44/42 MAPK (Erk1/2)Thr202/Tyr204 , rpL7 (Abcam)).Proteins were separated by SDS-PAGE and transferred onto a nitrocellulose membrane. The membrane was blocked for 1 h at room temperature with non-fat 5% milk in TBS  and incubated overnight at 4°C in TBS-T  supplemented with BSA (5%) and appropriate antibodies , polyclonal mouse antibody against α-tubulin (Sigma-Aldrich), polyclonal rabbit antibody against phospho-AKTImmunocomplexes were detected using an ECL Prime kit  according to the manufacturer’s recommendations.260) was separated by electrophoresis on a denaturing 1.5% agarose gel containing 2.2 M formaldehyde and 1x MOPS buffer . RNA was transferred onto a nylon membrane  and crosslinked using a transilluminator-cross-linker (Vilber-Lourmat) at 0.15 J/cm2. Membrane-bound RNA was hybridized to a 1055 nt fragment of YB-1 cDNA probe  or a 466 nt fragment of GAPDH cDNA probe  labeled with [32P]dATP  using a DecaLabel DNA labeling kit (Fermentas) in hybridization buffer  at 65°C for 12–16 h. The membrane was washed twice with 2x SSC, 0.1% SDS for 5 min at room temperature, twice with 0.2x SSC, 0.1% SDS for 5 min at RT, twice with 0.2x SSC, 0.1% SDS for 15 min at 42°C, and twice with 0.1x SSC, 0.1% SDS for 15 min at 68°C, and analyzed by autoradiography. If needed, the relative radioactivity of the bands was determined using a Packard Cyclone Storage Phosphor System .Total RNA from cells or polysomal and post-polysomal fractions of lysates was extracted by the TRIzol method. Total RNA from rabbit tissues was extracted by homogenizing with TRIzol using a Dounce homogenizer. 10–15 µg of total RNA from cells or tissues . Rabbit tissues were homogenized using a Dounce homogenizer in the same buffer. Extracts were transferred into 1.5 ml tubes and incubated on ice for 10 min with occasional mixing. The nuclei and debris were removed by centrifugation at 12,000 g for 10 min in a microcentrifuge. Supernatants were recovered, and 200 µl aliquots were layered onto 50 µl of 50% sucrose cushion composed of extraction buffer lacking Triton X-100 and pelleted at 90,000 rpm for 13 min in a TLA-100 rotor (Beckman) at 4°C . RNA fr35S]-methionine labeling, the cells were cultivated in DMEM lacking L-methionine but containing 0.1 mCi/ml of L-[35S]-methionine  for 1–2 h. The cells were washed with phosphate buffer saline, scraped and lysed with buffer containing 20 mM HEPES-KOH, pH 7.6, 100 KCl, 5 mM MgCl2, 2 mM DTT, 0.25% Nonidet P-40, 0.2% SDS and protease inhibitors cocktail. Cell debris was removed by centrifugation at 10,000 g for 15 min, and extracts were then directly utilized in IP experiments. For IP, cell extracts were incubated with appropriate antibodies  or rat pre-immune antibodies, 100 µg each) immobilized on protein G-Sepharose beads  for 2 h at 4°C. After extensive washing with PBS, proteins were eluted with acid-urea sample buffer , and analyzed by acid-urea 10% polyacrylamide gel electrophoresis and autoradiography. The relative amount of radioactivity in the bands was determined using a Packard Cyclone Storage Phosphor System .For -labeled GAPDH cDNA. Approximately 65% of GAPDH mRNA was detected in polysomal pellet. In the presence of 30 mM EDTA, the entire GAPDH mRNA was found in supernatant. Hence, in the absence of EDTA the entire GAPDH mRNA detected in the polysomal fraction was bound to mono- and polysomes. 18S rRNA was absent too from the polysomal fraction after EDTA treatment. This means that all particles under 40S were retained in supernatant.Analysis of mRNA distribution between polysomal and free mRNP fractions in the cell. HEK293 cells were scraped and lysed. Nuclei and mitochondria were removed by centrifugation, and cytosolic extracts without (lanes 1 and 2) or with (lanes 3 and 4) 30 mM EDTA were then spun through a 50% sucrose cushion at 100,000 rpm in a TLA-100 centrifuge (Beckman) for 13 min to pellet polysomes. Total RNA from postpolysomal supernatant and polysomal fractions (resuspended pellets) were extracted with TRIzol, subjected to agarose gel electrophoresis and Northern blot hybridization to [(TIF)Click here for additional data file.Figure S2Results of peptide mass fingerprint analysis of the protein cut out from the gel after anti-YB- 1 Ab IP and acid-urea PAGE.(TIF)Click here for additional data file.Figure S335S]-Met) incorporation on cell confluence. NIH3T3 cells of various confluence were [35S]-methionine-labeled, harvested and lysed. Cell lysates were analyzed by PAGE and autoradiography Relative radioactivity of the bands was determined using a Packard Cyclone Storage Phosphor System Dependence of radioactive label ([(TIF)Click here for additional data file.Figure S4A simplified scheme of the mTOR signaling pathway. The basic mTOR substrates are 4E-BP (eukaryotic initiation factor 4E binding protein) and S6 kinase (p70S6K). Phosphorylation of 4E-BP results in its lower affinity for eIF4E, which makes the latter accessible for translation initiation. Activated S6 kinase phosphorylates a number of substrates (eukaryotic translation initiation factor 4B (eIF4B), ribosomal protein S6, programmed cell death 4 (PDCD4) - a tumor suppressor that binds to eIF4A, eukaryotic translation elongation factor 2 kinase (eEF2K) etc.), thereby contributing to activation of both initiation and elongation of translation. Inhibitors of mTOR kinase are rapamycin and PP242, the former predominantly inhibiting phosphorylation of p70S6K. The major mTOR activating pathway is the PI3K/Akt kinase cascade. Its inhibition with wortmannin affects, among others, mTOR kinase. Inhibition of Erk kinase with U0126 causes inhibition of Mnk-1, and hence, suppression of eIF4E phosphorylation, thereby decreasing translation of some eIF4E-sensitive mRNAs (here used as a control).(TIF)Click here for additional data file.Protocol S1Plasmid construction.(DOCX)Click here for additional data file.