PKCβ-mediated phosphorylation of serine 36 in p66 has been implicated as a key regulatory step preceding mitochondrial translocation, ROS production, and cell death, and PKCβ thus may provide a target for therapeutic intervention. We performed a reassessment of PKCβ regulation of the oxidoreductase activity of p66. Although our experiments did not substantiate Ser36 phosphorylation by PKCβ, they instead provided evidence for Ser139 and Ser213 as PKCβ phosphorylation sites regulating the pro-oxidant and pro-apoptotic function of p66. Mutation of another predicted PKCβ phosphorylation site also located in the phosphotyrosine binding domain, threonine 206, had no phenotype. Intriguingly, p66 with Thr206 and Ser213 mutated to glutamic acid showed a gain-of-function phenotype with significantly increased ROS production and cell death induction. Taken together, these data argue for a complex mechanism of PKCβ-dependent regulation of p66 activation involving Ser139 and a motif surrounding Ser213.Dysfunctional mitochondria contribute to the development of many diseases and pathological conditions through the excessive production of reactive oxygen species (ROS), and, where studied, ablation of p66Shc (p66) was beneficial. p66 translocates to the mitochondria and oxidizes cytochrome  In particular, complexes I and III are involved in this process  by PKA modulated ATP generation and ROS production , benefit –7, most  process . Work pu process –12. Phosoduction . Loweredoduction , whereasoduction –17. PKCϵduction –, 19. Eviduction –. Interfe2O2 through the oxidation of cytochrome c , a potent PKC activator, was used as a positive control. We observed cell membrane translocation of all isoforms tested  in the case of pro-oxidant treatment in HEK293 cells (A) as well as in mouse embryonic fibroblasts (MEFs) (data not shown). Next we analyzed PKCβ-deficient immortalized MEFs and measured ROS levels 30 min after treatment with the pro-oxidants H2O2 or t-BHP , data not shown) fluorescence, which was comparable with the response observed in cells lacking p66 (B). Expression of PKCβ in PKCβ−/− MEFs restored basal and pro-oxidant-induced ROS levels (data not shown). Conditional knockdown of PKCβ in WT MEF 3T3 cells using siRNA further confirmed its role in stress-induced ROS production (C). siRNA knockdown of PKCβ was confirmed with quantitative real-time PCR (D). Furthermore, PKCβ−/− cells showed increased resistance to cell death induced by pro-oxidants, as reported for p66−/− cells .To define the requirement of PKCβ for ROS production, we first confirmed that the experimental conditions studied here result in the activation of PKC, monitored by cell membrane translocation of PKC, as shown before . 12-O-te93 cells A as wellor t-BHP . PKCβ-deking p66 B. Expresoduction C. siRNA time PCR D. Furthe/− cells   or t-BHP (data not shown), but we were unable to reduce or prevent Ser36 phosphorylation by treatment with two different PKC inhibitors, Gö6850 (data not shown) and Gö6976 (F). Inhibition of PKCβ with Gö6976 was confirmed because it reduced cell membrane translocation of PKCβ upon TPA treatment (G) as well as after pro-oxidant treatment both in HEK293 cells and in MEFs (data not shown). PKCβ-deficient MEFs showed slightly increased Ser36 phosphorylation but, like their WT counterpart, responded with further increased phosphorylation to H2O2 treatment (F). However, because PKCβ inhibition, as well as PKCβ knockdown and knockout, was very effective in lowering mitochondrial ROS levels in response to pro-oxidant treatment without decreasing Ser36 phosphorylation, we hypothesized that Ser36 might not be the critical PKCβ-regulated site on p66.Given the importance of Serith H2O2 F or t-BHd Gö6976 F. Inhibireatment G as wellreatment F. Howeve139, Thr206, and Ser213 (206 and Ser213) or adjacent to (Ser139) the phosphotyrosine binding (PTB) domain of p66 (1SHC) and x-ray diffraction (PDB code 4XWX), and visual inspection suggests that these three sites are located on the surface of the protein (A) and thus freely accessible for the kinases. We confirmed the predicted PKCβ phosphorylation sites by using in vitro kinase assays with recombinant PKCβ and peptides harboring any one of these three predicted sites. We could show phosphorylation of peptides containing Ser139, Thr206, or Ser213, but we were not able to detect substantial Ser36 phosphorylation (B). These results are in agreement with our previous observation where we identified JNK1/2 as p66 Ser36 kinases  or stained with Coomassie. GST-Shc was excised from the gel and digested either with trypsin or Lys C to yield peptides containing Ser139 and Thr206/Ser213, respectively, detectable by MS. As shown in Fig. 3B, incubation with PKCβ resulted in a time-dependent increase in the number of p66 Ser139-phosphorylated peptides alone or in combination with JNK but not with JNK alone. p66 Ser36 phosphorylation was observed with JNK alone or in combination with PKCβ but not with PKCβ alone . However, Thr206- and Ser213-phosphorylated peptides were only detected when both kinases were used together (B–D). The percentage of Thr206/Ser213-containing phosphopeptides detected was significantly lower than that of Ser139-phosphorylated peptides.Next we confirmed these three sites in an 66 Ser36 A or stai36 phosphorylation upon pro-oxidant treatment was not affected by the PKC inhibitor Gö6976 (A), which efficiently decreased Ser139 phosphorylation that is detected by MS (B and C). However, in contrast to the in vitro experiments (B–D), no Thr206/Ser213 phosphorylation was detected.In our cellular assays, increased p66 Serr Gö6976 A, which ed by MS , B and Ceriments , B–D, no139 in p66, equivalent to Ser29 in p52Shc, has already been reported to be phosphorylated by the PKCα, ϵ, and δ isoforms and is essential for the interaction of ShcA with the protein tyrosine phosphatase protein tyrosine phosphatase PEST (PTP-PEST) . As shown in B and C, the alanine exchange caused significantly reduced ROS production upon stress. We were also able to show that reduced ROS production in p66S139A reconstituted MEFs correlated with decreased cell death (D).SerTP-PEST) . Howeverblotting A. As sholl death D.−/− MEFs. Equal protein expression was confirmed by immunoblotting (A). Two different phenotypes were observed (B). Although mutating Thr206 did not affect the ability of p66 to produce ROS, maintaining the integrity of Ser213 was essential for the pro-oxidant function of p66 both under basal growth conditions and in response to H2O2 treatment (B). Additional mutations tested included the individual exchange of these residues to glutamic acid. However, these mutants did not behave differently from the alanine mutants for all parameters studied here (data not shown). Exchanging Thr206 and Ser213 to alanine resulted in a protein that did not differ from S213A (B). In contrast, replacing both residues with glutamic acid created a protein that was hyperactive with regard to ROS production (B). This, to our knowledge, is the first demonstration of a constitutively active form of p66. To assure that mutant p66 specifically affected mitochondrial ROS production, we treated cells with low concentrations of the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Such mild uncoupling of mitochondria is known to decrease mitochondrial ROS production  similar to p66-deficient cells, in contrast to WT MEFs showing a viability of about 50% (A). We could observe the same resistance of cells expressing p66 mutated in Ser213 after treatment with hydrogen peroxide (H2O2), although less pronounced (data not shown). As observed for ROS production, mutation of Thr206 had no effect on the ability of the protein to induce cell death (A), whereas expression of p66T206E/S213E, as expected from its ROS phenotype, greatly enhanced cell death. To assure that apoptosis in response to t-BHP was caused by excessive ROS production, we treated cells with the anti-oxidant N-acetylcysteine (NAC). Pretreatment of cells with NAC rescued them from death after pro-oxidant treatment (B).Next we tested whether the ROS-deficient phenotype of cells expressing p66 mutated in Serbout 50% A. We coull death A, whereareatment B.2+ levels. ROS damage nucleic acids, lipids, or proteins, alter the function of signaling proteins, and eventually cause cell death . These findings confirm the PKCβ dependence of the pro-oxidant function of p66 but strongly argue for the existence of an additional kinase responsible for Ser36 phosphorylation. The sequence surrounding Ser36 predicts phosphorylation by proline-directed kinases , whereas a peptide containing Ser36 only displayed background phosphorylation. Similarly, using p66 phosphorylated in vitro or immunoprecipitated from HEK293, we detected phosphorylation of Ser139 but not of Ser36 (36 was readily detected in in vitro kinase assays with recombinant JNK1 (A). To verify phosphorylation of Thr206 and/or Ser213 in cellular p66, a variety of MS experiments were performed, including collision-induced dissociation, higher-energy collisional dissociation, electron transfer dissociation (ETD), wide-band activation, and three-stage mass spectrometry (MS3). Moreover, immobilized metal affinity chromatography (IMAC) and TiO2 enrichment of phosphopeptides and digestion of proteins with two different enzymes (trypsin and lysozyme C) was carried out. In no case was phosphorylation of Thr206 and Ser213 found, which lets us speculate that the amount of p66 phosphorylated on Thr206 and Ser213 is below the detection limit. This assumption is corroborated by the demonstration that, in the in vitro kinase assay, we were able to detect only a small quantity of Thr(P)206/Ser(P)213 peptide (1.1%) compared with peptides phosphorylated on Ser139 (18%). If we predict the same ratio for our cellular assays, where, on average, we found 1.6% of the peptides to be phosphorylated on Ser139, detection of Thr(P)206/Ser(P)213 peptide will be impossible with the approach used currently.Phosphorylation of Sern of p66 . Howeverrylation  and 4A.  kinases , and MAPases  indicates that only Ser36 conforms to a possible Pin1 binding side. Nevertheless, we have started to address a possible role of the PKCβ phosphorylation sites identified here in Pin1 binding and mitochondrial import of p66. We can confirm the expected reduction in Pin1 binding for p66 mutated in S36A but failed to detect altered binding for the p66 Ser139 or p66T206A/S213A mutants, suggesting that these novel sites are not directly involved in Pin1 binding and, thus, Pin1-dependent mitochondrial translocation of p66 (data not shown). However, the whole situation may be much more complex because Pin1-mediated prolyl isomerization is a critical step in JNK activation . It may therefore very well be that it is the import of de novo phosphorylated protein, which may be a minor fraction, that is critical for the mitochondrial function of p66.We thus cannot exclude that these novel PKCβ phosphorylation sites are somehow involved in mitochondrial import. However, one fact that complicates the analysis of mitochondrial translocation is the observation we consistently made in various cell systems that mitochondrial p66 is present in substantial quantities already in unstimulated cells  and that the mitochondrial translocation following stimulation is not immediately obvious, as In summary, our experiments provide evidence for a complex mechanism regulating the activation of p66 under stress. Given the importance of p66 in the development of various disease conditions, targeting the key kinases (PKCβ and JNK1/2) involved in triggering the pro-oxidant and pro-apoptotic function of p66 may become a realistic therapeutic option.−/− , and streptomycin (100 μg/ml)  at 37 °C, 5% CO2. Stable cell lines were generated after transfection of MEF 3T3 p66−/− cells with pBABEpuro p66 WT and mutant expression constructs using Lipofectamine 2000 (Invitrogen) and puromycin (Invitrogen) selection (0.4 μg/ml) for 2 weeks. HEK293 cells were transfected with 1.5 μg of pECFP-PKCα, β, or θ (laboratory of Gottfried Baier), and fluorescence was detected after 48 h by confocal microscopy as described previously  .For the inhibition of PKC, Gö6976 and Gö6850 were obtained from Calbiochem . It has been shown previously that Gö6976 inhibits PKCα and β1 but not the Ca> δ > ζ) . t-BHP, Primers for mutagenesis of p66 were obtained from Eurofins Genomics . Site-directed mutagenesis was performed using the QuikChange II site-directed mutagenesis kit from Stratagene . Amino acid exchange was confirmed by DNA sequencing at the Department of Genetic Epidemiology at the Medical University Innsbruck or at Microsynth AG (Switzerland).Following electrophoresis, the gel was soaked in fixing buffer consisting of 40% (v/v) ethanol and 10% (v/v) acetic acid for 1 h. After fixation, the gel was washed for 1 h with deionized water and incubated for 2 h in Coomassie stain (Sigma) with continuous agitation. The gel was destained with deionized water.in vitro kinase assay, protein bands were excised from Coomassie-stained gel and digested with trypsin from porcine pancreas (Sigma-Aldrich) or Lys C (Sigma-Aldrich) as described previously  target 1e6. The selected isotope patterns were fragmented by higher-energy collisional dissociation with normalized collision energy of 28 at a resolution of 15,000 with a maximum injection time of 120 ms and automatic gain control target 5e5.After the eviously . TrypticData analysis was performed using Proteome Discoverer 1.4.1.14 (Thermo Scientific) with the search engine Sequest. The raw files were searched against a database (545 entries) containing the most common contaminants and p66. Precursor and fragment mass tolerance was set to 10 ppm and 0.02 Da, respectively, and up to two missed cleavages were allowed. Carbamidomethylation of cysteine, oxidation of methionine, and phosphorylation of serine, threonine, and tyrosine were set as variable modifications. The peak area of the phosphorylated and non-phosphorylated peptides was calculated by summing up the peak areas or the corresponding precursor ions.in vitro kinase assay, except the particle size of the column material, which was 5-μm reverse-phase C18. The LTQ Velos mass spectrometer was operating in data-dependent mode selecting the top four most abundant isotope patterns with a charge >1 from the survey scan with an isolation window of 2.0 m/z. Survey full-scan MS spectra were acquired from 300–1800 m/z at an enhanced scan rate. Two-stage mass spectrometry (MS2) and MS3  scans were fragmented by collision-induced dissociation with a normalized collision energy of 35. The settings for data analysis were the same as with the in vitro kinase assay, except for fragment mass tolerance, which was set to 0.8 Da. The amount of phosphorylated and non-phosphorylated peptide was calculated by the peak heights of the extracted ion chromatogramsImmunoprecipitated proteins were digested on beads with trypsin and analyzed using an UltiMate 3000 nano-HPLC system coupled via an electrospray ionization interface to an LTQ Velos mass spectrometer (Thermo Scientific). HPLC conditions were the same as with the m MitoTracker Red CM-H2XROS from Molecular Probes (Invitrogen) in serum-free DMEM. For this purpose, 30,000–120,000 cells/chamber were seeded in 8-well chambered Lab-Tek coverglasses . Staining was performed for 30 min at 37 °C. Images were taken using an Olympus IX-70 inverted microscope with an Olympus ×40 water  and an Olympus U-RFL-T mercury vapor lamp. Image acquisition was performed with a Kappa ACC1 camera and Kappa ImageBase software . For MitoTracker Red CM-H2XROS, a 568-nm filter was used with a light expose of 1.6 s. Gray values were measured using Scion Image software  for Windows. For every experimental condition, gray values from 100 cells were averaged. Alternatively, cellular ROS levels were determined by staining with DCF-diacetate (DCF-DA) (Sigma). Cells were loaded with 7 μm DCF-DA and incubated for 15 min at 37 °C before FACS measurement. Analysis was done using CellQuest software for FACScalibur (BD Biosciences).Mitochondrial ROS were imaged by fluorescence microscopy after staining the cells with 100 n2 real-time SYBR Green-fluorescein PCR master mix (Bio-Rad) according to the protocol provided on an iQ5 multicolor real-time PCR detection system from Bio-Rad. Using 100 nm PKCβ siRNA, on average 86% knockdown was achieved and was used for further experiments. The RT2 qPCR Primer Assay for Mouse PRKCB1 was ordered from Qiagen. The expression was normalized to the housekeeping gene RSP-29. Data analysis was performed using the 2−ΔΔCt method  and transfected using Lipofectamine 2000 (Invitrogen) according to the protocol of the manufacturer. RNA isolation was performed using Qiagen RNeasy mini kit , and cDNA synthesis was done using the Fermentas RevertAid first strand cDNA synthesis kit . PKCβ expression was determined by real-time PCR using RTt method . MeltingProtein lysates were prepared, separated, and analyzed as described previously . The fol32Pi from [γ-32P]ATP. The following peptides were used: Ser36, NH2-PEELPSPSASSL-COOH; Ser139, NH2-WTRHGSFVNKPT-COOH; Thr206, NH2-GAKGATRRRKPC-COOH; and Ser213, NH2-RRKPCSRPLSSI-COOH. NH2-QKRPSQRSKYL-COOH was used as a positive control. The level of radioactivity bound to phosphocellulose filters was counted using a Wallac MicroBeta 1450 (PerkinElmer Life Sciences). A detailed in vitro kinase assay protocol for phosphorylation of recombinant p66 by recombinant PKCβ has been described previously  and propidium iodide  or by counting stained (dead) and unstained (living) cells with a Neubauer counting chamber after incubation with trypan blue (Sigma-Aldrich). A detailed protocol has been described previously .t test or ANOVA. Significance values were designated as follows: *, p < 0.05; **, p < 0.005; ***, p < 0.0005.All data are presented as mean ± S.D. Statistical analysis was done using GraphPad Prism 5  using M. H. and S. K. designed, conducted, and analyzed experiments and prepared first drafts of the manuscript and figures. M. H. and T. F. performed experiments. F. F. and G. B. conducted PKC kinase assays and provided reagents. L. K. and H. L. performed MS studies. G. B. provided expert advice on PKC signaling. J. G. and A. D. generated mutant p66 constructs and provided technical support for many procedures. M. L. and M. G. provided important tools. J. T. provided overall coordination and supervision of the study and wrote the manuscript together with S. K.


However, its limited availability and poorly known basic chemistry hamper its use. Based on the analogy with iodine, labelling is performed via astatobenzoate conjugates, but in vivo deastatination occurs, particularly when the conjugates are internalized in cells. Actually, the chemical or biological mechanism responsible for deastatination is unknown. In this work, we show that the C−At “organometalloid” bond can be cleaved by oxidative dehalogenation induced by oxidants such as permanganates, peroxides or hydroxyl radicals. Quantum mechanical calculations demonstrate that astatobenzoates are more sensitive to oxidation than iodobenzoates, and the oxidative deastatination rate is estimated to be about 6 × 106 faster at 37 °C than the oxidative deiodination one. Therefore, we attribute the “internal” deastatination mechanism to oxidative dehalogenation in biological compartments, in particular lysosomes. While this name was proposed from a physical perspective (i.e. the absence of any stable isotope), one might tend to consider that it is also suitable from a chemical point of view, since most bonds involving this atom are less stable than the ones involving its closest analogue, iodine. This affects the potential application of 211At to medicine. Nevertheless, 211At is considered as one of the most promising radionuclides for targeted alpha therapy, due to its favourable physical properties 3.Halogens are usually named according to ancient Greek words denoting one of their characteristics. The heaviest of these elements befell the name astatine, as a reference to its instability6. However, labelling with astatobenzoate conjugates suffers from in vivo dehalogenation, which diminishes the tumour uptake and leads to the release of free astatine and its accumulation in stomach and thyroid. Even if stomach and thyroid uptake can be mitigated9, a more stable labelling is needed for systemic administration8. It is particularly interesting to note that when mAbs are labelled with astatobenzoates, deastatination is limited14, while when antibody fragments are used, considerable dehalogenation occurs15. Unfortunately, the slow mAbs pharmacokinetics are not well-suited to be combined with the 211At half-life time16, and astatine-labelled antibodies have been so far limited to locoregional treatments. Moreover, this behaviour is remarkable, as it echoes the one of proteins, iodinated through direct labelling or using the Bolton-Hunter reagent17. In these cases the dehalogenation mechanism has been elucidated: the 2-iodophenol moiety of the catabolites released through carrier metabolization undergoes dehalogenation catalysed by deiodinases. Thus, the deastatination mechanism is most probably initiated through internalization into cells and lysosomal degradation. To overcome deiodination, reagents such as the N-succinimidyliodobenzoate (SIB) have been synthesized by electrophilic radioiodination of N-succinimidylaryltrialkylstannane derivatives19. Indeed, these reagents lack the phenolic hydroxyl group required in the catalytic mechanism of mammal deiodinases21.Clinical trials using either monoclonal antibodies (mAbs) or antibody fragments labelled by astatobenzoate conjugates afforded encouraging results against both recurrent brain tumours and recurrent ovarian cancersN-succinimidylastatobenzoate (SAB), has been developed10. However, the aforementioned studies of astatobenzoate-labelled proteins showed that such labelling with 211At is unstable, leading to dehalogenation, contrary to the iodine case. Hence, it is clear that carrier catabolism favours astatobenzoate deastatination via mechanisms that remain unknown. It should also be mentioned that besides proteins, the injection of small organic compounds such as astatobenzoate-labelled biotin derivatives22 or simply 3-astatobenzoate itself11 leads to a radioactivity biodistribution similar to that of astatide, and very dissimilar to the iodobenzoate one, denoting a fast deastatination. Alternatively, labelling with astatodecaborates instead of astatobenzoate conjugates was envisaged, leading to stable labelling even with small molecules and very encouraging preclinical results25. However, this approach seems hindered by high uptake in kidneys and liver26. Note that the study of such compounds is beyond the scope of the present work, which aims at revealing the mechanism(s) responsible for astatobenzoate dehalogenation.Therefore, an analogous astatination reagent, the 27, and (ii) the relative weakness of the C−At bonds compared to the C−I ones. One may argue that since astatine is absent from the biosphere 29, no At-specific enzyme that catalyses C−At bond breakages is likely to exist. However, some proteins such as the sodium-iodide symporter recognize both astatide and iodide31, demonstrating that the presence of iodine-processing enzymes may also affect astatine compounds. Nevertheless, C−At bond cleavages induced by promiscuous enzymes are unlikely to happen since the analogous C−I bonds are not cleaved. The second and most often quoted justification, that the C−At bonds are weaker than the corresponding C−I ones, although true32, is not sufficient to explain why astatobenzoate-labelled proteins are seemingly stable in blood and not when internalized inside living cells.Two explanations have been proposed so far to justify the astatobenzoate dehalogenation, (i) the action of unidentified enzymes that would catalyse the C−At bond cleavage (similarly to what happens to radioiodinated proteins by direct labelling)in vivo deastatination mechanism(s) should satisfy the following criteria, (i) the analogous C−I bonds must remain stable under conditions that are sufficient for allowing C−At bond cleavages, (ii) these conditions could not be met in blood, but rather in other biological compartments such as the ones the carrier and its catabolites enter during the catabolism process, and (iii) the C−At bond breakage, should occur in the absence of any enzyme. This work aims at providing a satisfactory explanation that fully meets these criteria. One of the most striking differences between I and At lies in the astatine metalloid properties34: its Pourbaix (E−pH) diagram displays cationic species36, contrarily to the iodine one37, and the ionization potential of its free atom28 is lower than the one of iodine by more than 1 eV. Therefore, one may hypothesize that astatinated compounds are more sensitive to oxidation than iodinated ones, which seems particularly relevant since carrier catabolism (which favours deastatination) exposes astatobenzoate moieties to critical changes in redox conditions. Indeed, internalization in cells will lead labelled carriers into lysosomes, where myriads of strong oxidants such as the so-called reactive oxygen species (ROS), a family of compounds including peroxides and oxygen radicals, are present.In order to explain the biodistribution observations from the literature, the sought 125I-iodobenzoate-labelled antibody fragment38, showing the presence of iodobenzoate and of its lysine and glycine conjugates as main catabolites, free iodine being absent. One could thus assume that the astatobenzoate catabolites would be similar to the iodinated ones. Oxidants such as permanganates, peroxides or hydroxyl radicals have been tested. Also, we show that the Fenton reaction, which happens in vivo in lysosomes, leads to deastatination within seconds. Since astatine is only produced in minute quantities, no spectroscopic tool can be used to probe the nature of its chemical species. Therefore, we also present relativistic density functional theory (DFT) calculations to elucidate or at least get insight on the “microscopic” mechanism that eventually leads to deastatination. It is indeed of great interest to combine quantum calculations and experiments to obtain information on astatine species at the molecular scale41. Finally, we conclude by attributing the deastatination mechanism to oxidative dehalogenation.Here, we report the stability of an astatobenzoate conjugate in the presence of various oxidants to assess if its oxidation is possible, and if it eventually leads to deastatination. Indeed, an extensive metabolic study has been made on a 1a) as a model compound for performing stability studies in presence of oxidants such as permanganates  in the presence of 2 mM NaMnO4 (not shown). Figure tert-butyl hydroperoxide, referred to as “TBHP” and peroxodisulfate) are also able to induce deastatination at the same pH value. This is particularly interesting since the most notorious oxidants occurring in vivo are the ROS, among which peroxides can be found. Also, one should note that oxidative dehalogenation induced by TBHP occurs at physiological pH, i.e. 7.4, with similar kinetics as observed at pH = 4.7.Figure benzoate  in the pin vivo ROS, namely hydrogen peroxide (H2O2), does not promote any noticeable deastatination , they undergo an extremely fast deastatination: within the few tens of seconds needed for the HPLC injection, most of the astatobenzoate moieties were dehalogenated, and the majority of the activity was found in a peak having a retention time that appears to match the one of an oxygen adduct of astatine (in particular an At (III) species).The most common eroxide HO2, does i.e. trivalent iron instead of divalent) coupled with hydrogen peroxide are also known to produce hydroxyl radicals, but at a much slower rate45. These conditions are often referred to as “Fenton-like”. The dehalogenation kinetics of ethyl 3-astatobenzoate (1a) incubated under Fenton-like conditions is displayed on Fig. 2O2 and 1% H2O2 plus 10−4 M of Fe3+ ions are displayed in Fig. 47. They also indicate that the released astatine species should be an oxygen adduct, but they do not indicate how and why oxidation leads to deastatination. One should note that this part of the study has been performed at pH = 3 to favour the efficiency of the Fenton reaction in order to probe the effect of the presence of hydroxyl radicals (which are produced in lysosomes) on the dehalogenation mechanism(s). Furthermore, one should note that no discernible deiodination of ethyl 3-iodobenzoate (1b) is observed after 12 h incubation under Fenton-like conditions. In order to gain more insight on the oxidative dehalogenation mechanism(s) and on the differences between the C−At and C−I bonds of interest, a quantum mechanical study was performed.Catalytic amounts of ferric iron , and test calculations confirmed that this dissociation limit is found to be favoured by more than a hundred kcal.mol−1 compared to ionic limits at the considered level of theory.As no spectroscopic information can be obtained at ultratrace concentrations, it is necessary to consider quantum mechanical calculations to get any “microscopic” information on the deastatination mechanism(s). It is of particular importance to treat spin-orbit coupling (SOC), since this relativistic interaction has a strong influence on the geometries and properties of At compounds−1, respectively)49 and the computed ones  was obtained. Furthermore, first ionisation potentials (IP1s) were computed as a model descriptor for the oxidation propensity of halobenzoates. We obtained an IP1 value of 195.5 kcal.mol−1 for iodobenzene, which fits well with the experimental value of 201.3 ± 0.7 kcal.mol−1 50. All these results provide a firm ground to the used level of theory prior to starting the study of the oxidation of halobenzoate conjugates. Note that for the interested reader, additional calculations to illustrate the importance of SOC on the computed quantities are reported in Tables A good agreement between the experimental dissociation energies of astatobenzene and iodobenzene  − qk(N − 1) form, where k is an atom, qk(N) is the electron population of the k atom in the neutral system (N electrons) and qk(N − 1) is the electron population of the k atom in the ionized system (N − 1 electrons). Note that the electron populations are obtained in the present work from natural population analyses. We found that the condensed-to-atom Fukui index, f− 51, is not only maximum for At in 1a, and for I in 1b, but is also at least four times larger for At or I, respectively, than for any other atom of the system. The corresponding f− values are 0.7 and 0.5, meaning that the removed electron is more than 50% localized on the halogen moiety in each case.To probe the relative feasibility of the oxidation of 1b presents a higher IP1  than the one computed for astatobenzoate 1a . The important difference, 10.4 kcal.mol−1, makes 1a actually significantly easier to oxidize than its iodine counterpart. Indeed, the previous difference is greater than the one between iodobenzene and bromobenzene, and even the one between iodobenzene and chlorobenzene 50. Thus, our calculations support the fact that astatobenzoates should be more prone to oxidation than their iodinated counterparts, since the first conceptual step of any potential oxidation, i.e. the withdrawal of one electron, is more favourable in the X = At case. Therefore, since we do not aim at fully elucidating the oxidation mechanism(s), we continue by directly studying the consequences of oxidation on the C−X bonds of interest.It appears that the iodobenzoate compound 2b species is known to exist, it seems reasonable to study the dissociation energies of the 2a and 2b compounds , one can first observe that the C−At and C−I bond energies are almost unchanged. Much more enthralling is the comparison between halobenzoates and their oxidized counterparts, as the bond dissociation energies drop in 2a and 2b by more than a third compared to 1a and 1b, respectively, e.g. diminishing the C−At bond energy from 44.6 to 28.2 kcal.mol−1. While these energy decreases are of course noticeable, their significance may be further assessed with transition state theory in these undoubtedly kinetically controlled systems. As test calculations showed no energy barrier on the potential energy surfaces corresponding to the homolytic dissociations of the C−X bonds in 2a and 2b, as well as in 1a and 1b, these bond breakages are kinetically controlled by the corresponding bond dissociation energies. According to the Eyring equation52, the ratio between the bond breakages rates in 2a and 2b is affected by the following temperature-dependent factor:d(2b) and Ed(2a) are the bond dissociation energies of 2b and 2a, respectively, R is the universal gas constant and T is the temperature. Hence, the 9.6 kcal.mol−1 difference between Ed(2b) and Ed(2a) leads to a dissociation rate in 2a larger by a factor of roughly 6 × 106 at 37 °C (human body temperature) than in 2b, which could explain the iodobenzoate relative stabilities towards oxidizing conditions compared to the astatobenzoate ones. Indeed, the iodosobenzoates will not only be produced much less efficiently than their astatinated counterparts, but also the astatosobenzoates are more prone to homolytic dehalogenation, unlike the iodosobenzoates for which the halogen will be more efficiently reduced back while proteins are oxidized53. On the other hand, the 16.4 kcal.mol−1 difference between Ed(1a) and Ed(2a) leads at 37 °C to an impressive relative increase of the dissociation rate for 2a, by a factor of roughly 4 × 1011 with respect to 1a. This difference is compatible with stable astatobenzoates in blood, given its antioxidant protections54, and efficient dehalogenation of astatosobenzoates after the oxidation of astatine to its +II oxidation state.By comparing the results obtained for in vivo mechanism by which these therapeutically relevant compounds are catabolized. We propose that the in vivo C−At bond cleavage occurs through oxidative dehalogenation of the astatobenzoate moiety. This would explain the stability of the labelled carriers in blood, where the conjugates are protected from oxidative dehalogenation, notably by the thiolates of the human serum albumin (≈43 g.L−1) in its mercaptalbumin form, and by the strong antioxidants properties of erythrocytes54. Also, no strong oxidant is known to be present in blood. On the other hand, after internalisation in cells, the halobenzoates moieties would no longer be shielded from encountering strong oxidants.We report the first experiments of astatobenzoate dehalogenations, and shed light on the probable 46 and more oxidizing than other subcellular organelles56. However, the overall oxidation level is not the only parameter at play. Indeed, strong oxidants and reductants coexist in the lysosomes microdomains57. Similarly, the pH value experiences strong local variations, notably in the strict vicinity of proton pumps. Thus, when catabolized, the astatobenzoate conjugates will be exposed to strong oxidants, typically under acidic conditions in lysosomes. We have shown that the most common in vivo ROS, i.e. hydrogen peroxide (H2O2), does not intrinsically promote any noticeable deastatination. When coupled with ferrous (Fenton conditions) or even of ferric (“Fenton-like” conditions) ions, it promotes a fast cleavage of the C−At bond. It is thus clear that astatobenzoates undergo an extremely fast deastatination in the presence of hydroxyl radicals, ROS known to exist in lysosomes as products of the Fenton reaction. Indeed, due to the degradation of iron-containing macromolecules, many lysosomes are rich in redox-active iron compounds, which results in Fenton-type reactions in these organelles47. The ubiquity of lysosomes in mammalian cells is also consistent with the observed deastatination after cell internalisation regardless of the nature of the cells.We propose reactions with ROS in lysosomes as the most likely path to oxidation. Indeed, lysosomes are the organelles that are responsible for protein degradation, and will be met in the first step of the carrier catabolism. They are known to be acidic 53, akin to the conversion of 1b into 2b. Moreover, it has also been shown, within the halobenzene series, that, the heavier the halogen, the easier it is for CYPs to oxidize it ref. in vivo, it could not be abundant, as it is reduced while it oxidizes proteins53, and that no important dehalogenation of the iodosobenzene has been reported. It is therefore possible that following carrier metabolization, the astatobenzoates conjugates are released in the blood circulation, then captured by the liver, and later undergo oxidation catalysed by CYPs, which ultimately yields to deastatination.Alternatively, another possible oxidation path for halobenzoates is worth mentioning. Indeed, P-450 cytochromes (CYPs) catalyse the oxidation of iodobenzene into iodosobenzenein vivo conditions: they are stable under conditions that are sufficient to provoke deastatination.The oxidative dehalogenation hypothesis nicely meets the criteria we proposed: deastatination by oxidation has been proven to be easily doable in the absence of any enzyme; it explains why the astatobenzoate conjugate stabilities must be very different in blood, where they are protected from oxidation, and within lysosomes where they are exposed to Fenton conditions . It is also consistent with the observed stability of the corresponding iodobenzoates under the same in vivo mechanism, we also aimed at giving an insight of the oxidative deastatination process at the molecular level, especially in regard to iodinated analogues of astatobenzoates. We hypothesized that the difference in in vivo stability between iodobenzoate- and astatobenzoate-labelled proteins with respect to dehalogenation is due to (i) the different sensitivities of the At and I atoms toward oxidation and (ii) the difference in the C−X bond strengths in the oxidized compounds. A plausible scenario is oxidative dehalogenation in which the At atom is oxidized to its +III oxidation state, which weakens enough the C−At bond and eventually leads to its breakage. Our DFT calculations show that it is much easier to start oxidizing astatobenzoates than their iodinated counterparts . We also show that this oxidation results in a vast decrease of the C−X bond dissociation energies, illustrated by a drop of the C−At bond energy in the astatobenzoate from 44.6 to 28.2 kcal.mol−1. Finally, to link this quantity to a more in vivo relevant one, we provide a rough estimate of the kinetic enhancement of the homolytic cleavage rate, showing that the reaction should be accelerated by a factor of about 4 × 1011 (a difference greater than the one between decades and milliseconds).Besides experimental evidences and a proposed 211At-labelling agents, particularly in stimulating new ideas concerning their design and screening. Indeed, just as knowledge of the deiodinase involvement was a necessary step prior to designing SIB, our research demonstrates which types of mechanism should be inhibited for obtaining stable in vivo labelling with 211At. Also, to speed up the quest for new compounds, as the in vivo experiments are costly, labour intensive, and time-consuming, we suggest that prior in silico screenings based on relativistic DFT calculations must be undertaken given both the accuracy and the cost-effective favour of this approach.Finally, we deem that our results could be of interest to the conception of innovative 1a was done following the previously described methods for the synthesis of SAB60. Briefly, to 10 µL of acetic acid were added 25 µL of 2 mg/mL of N-chlorosuccinimide in methanol and 1,1 mg of ethyl 3-(tri-n-butylstannyl) benzoate (1c) in 25 µL of methanol in an HPLC vial. Then 50 µL of 211At in chloroform were added (roughly corresponding to 5–10 MBq of activity). After 20 min incubation, 1a was purified by HPLC using a Dionex Ultimate3000 HPLC device with an Interchrom C18 column piloted by the Chromeleon 6.80 software (ThermoFisher Scientific Inc.). It was coupled with a dual-flow cell gamma detection system42 using a γ-ray detector (raytest GABI Star) piloted by the Gina software (raytest Isotopenmeßgeräte GmbH).The synthesis of 1a (≃2–4 MBq of activity) were incubated in various media at 20 °C directly in the HPLC apparatus (same as described above), and 50 µL samples were injected onto column for analysis. The HPLC sequence was the following: 20 s of 100% acetonitrile (ACN) at 1.05 mL.min−1, a 5 s gradient decrease from 100 to 0% ACN, 65 s of 0% ACN, a 90 s gradient from 0 to 60% ACN, a 720 s gradient from 60 to 72% ACN during which the flow is increased from 1.05 mL.min−1 to 1.3 mL.min−1 during 90 s after the first 6o s of the gradient, a 60 s gradient from 72 to 100% ACN, 300 s of 100% ACN, a 60 s gradient from 100 to 0% ACN during which the flow is decreased from 1.3 mL.min−1 to 1.05 mL.min−1 and 150 s of 0% CAN for a total run duration of 24.5 min. Two subsequent injections of 50 µL of Na2S2O3 (50 mM) and of NaMnO4 (2 mM) were run in a “short run mode” between two kinetic points to wash out any potential residual activity. The short run program consisted of 1 min of 100% H2O, 1 min of 100% H2O to 100% of CH3CN (gradient) and 2 min of 100% CH3CN. The sums of the counts in the “intact” astatobenzoate peak were corrected by the intrinsic decay of 211At .1.4 mL samples of c) DFT methods61 relying on relativistic effective core potentials  and implemented in the NWChem64 and Turbomole65 program packages were used. The hybrid B3LYP exchange-correlation functional66 was selected, according to results of a recent benchmark study led on At compounds48. For treating the 25 valence electrons on both heavy atoms, we have selected triple zeta basis sets supplemented with 2c extensions, referred to as aug-cc-pVTZ-PP-2c67. The aug-cc-pVTZ basis sets69 have been used for the remaining atoms .All the calculations have been performed in gas phase. The two-component (2Supporting Information


AMP-activated protein kinase (AMPK) is a metabolic stress-sensing enzyme responsible for maintaining cellular energy homeostasis. Activation of AMPK by salicylate and the thienopyridone A-769662 is critically dependent on phosphorylation of Ser108 in the β1 regulatory subunit. Here, we show a possible role for Ser108 phosphorylation in cell cycle regulation and promotion of pro-survival pathways in response to energy stress. We identify the autophagy initiator Unc-51-like kinase 1 (ULK1) as a β1-Ser108 kinase in cells. Cellular β1-Ser108 phosphorylation by ULK1 was dependent on AMPK β-subunit myristoylation, metabolic stress associated with elevated AMP/ATP ratio, and the intrinsic energy sensing capacity of AMPK; features consistent with an AMP-induced myristoyl switch mechanism. We further demonstrate cellular AMPK signaling independent of activation loop Thr172 phosphorylation, providing potential insight into physiological roles for Ser108 phosphorylation. These findings uncover new mechanisms by which AMPK could potentially maintain cellular energy homeostasis independently of Thr172 phosphorylation. AMPK is involved in sensing of metabolic stress. The authors show that the autophagy initiator ULK1 phosphorylates β1-Ser108 on the regulatory β1-subunit, sensitizing AMPK to allosteric drugs, and activates signaling pathways that appear independent of Thr172 phosphorylation in the kinase activation loop. AMPK senses increased cellular AMP/ATP ratio during periods of energy stress  and protects the cell from these events by switching off energy-consuming anabolic pathways and switching on catabolic pathways to restore ATP levels. Multiple physiological processes are regulated by AMPK including autophagy, appetite control, mitochondrial biogenesis and cell growth, and proliferation. Consequently, extensive efforts have been made to develop AMPK-activating drugs for potential therapeutic use in treating metabolic diseases  and also cancer and inflammatory diseases.The evolutionarily conserved AMP-activated protein kinase (AMPK) is a key regulator of cellular and whole-body energy homeostasis that controls multiple branches of metabolism to redress energy imbalances caused by physiological and pathological processes4. γ-subunits possess three allosteric adenylate nucleotide-binding sites that bind ATP, ADP and AMP interchangeably, enabling AMPK to sense fluctuations in cellular energy state7.The AMPK αβγ heterotrimer comprises an α-catalytic subunit and regulatory β- and γ-subunits. Multiple isoforms of each subunit exist in mammals  and isoform-specific variations in tissue distribution, regulation, and function have been demonstrated. Both β-isoforms contain a carbohydrate-binding module (CBM) and are myristoylated at position Gly2, a modification that targets AMPK to intracellular membranes and is important for temporospatial regulation of AMPK signaling10), or small compounds binding at the ADaM  site (phosphorylation of β-Ser108 (pSer108) in the β-CBM13). An exception is synergistic activation of unphosphorylated AMPK when γ- and ADaM sites are occupied simultaneously13. The ADaM site, a largely hydrophobic cavity formed between the α-kinase domain small lobe and β-subunit CBM, was identified in crystal structures of AMPK/drug complexes14. These structures revealed that the phosphate group of pSer108 forms electrostatic interactions with α2-kinase domain residues Thr21, Lys31, and Lys33, thereby stabilizing the ADaM site and explaining the role of pSer108 in mediating drug sensitization. The precise mechanism by which ADaM site drugs activate AMPK is not fully understood. pSer108 is absolutely required for AMPK activation by A-76966213, salicylate15 and MT47-10016, and increases by 40-fold potency of the high affinity drug 99112. Using purified enzyme we further demonstrated that activation loop phosphorylation is dispensable for AMPK stimulation by A-76966214. The well-defined character of the AMPK drug site, and its regulation through reversible phosphorylation, has led to speculation that synthetic activators  and salicylate are mimicking an endogenous metabolite(s) that would be capable of sustaining AMPK signaling in the absence of pThr17217.In most instances, ligand-induced allosteric regulation of AMPK is governed by distinct phosphorylation events that either sensitize AMPK to nucleotides/drugs binding at γ-subunit sites (phosphorylation of Thr172 (pThr172) in the α-subunit activation loop2+/calmodulin-dependent protein kinase kinase 2) have been identified as in vivo Thr172 kinases18. Despite some conflicting evidence, current models of AMPK regulation by adenine nucleotides describe a tripartite mechanism in which ATP exchange for AMP and ADP at γ-sites (i) promotes Thr172 phosphorylation, (ii) suppresses pThr172 dephosphorylation, and (iii) (for AMP) allosterically activates Thr172-phosphorylated AMPK6. Hierarchical phosphorylation events in the α-subunit Ser/Thr rich ST-loop (human α1(472–525)) have also been reported to negatively regulate pThr172, either by suppressing Thr172 phosphorylation , or by promoting pThr172 dephosphorylation (α-Thr479 phosphorylation by GSK319). Activity of the autophagy initiator Unc-51-like kinase (ULK), itself an AMPK substrate, is associated with reduced pThr172 via an uncharacterized negative feedback loop20.Thr172 phosphorylation is considered a marker of AMPK activity; identification of upstream kinases, and the mechanisms underpinning pThr172 regulation, have been the subject of intense investigation over several decades. LKB1 and CaMKK2 (Ca21. Closer examination using kinase inactive (KI) AMPK expressed in COS-7 cells revealed that Ser108 is a cis-autophosphorylation site (dependent on intramolecular Thr172 phosphorylation) that is dephosphorylated following removal of the AMPK-activating stimulus. Thus, as for Thr172, Ser108 is largely unphosphorylated under basal conditions13. Identification of alternate upstream kinases for Ser108 would provide advances in two important areas: firstly, characterization of novel Ser108 kinases implicates their therapeutic modulation as a strategy to increase potency of AMPK-targeting drugs; secondly, feed-forward regulation by Ser108 kinases might denote the cellular processes under which concentrations of natural AMPK ligands become elevated. Here, we demonstrate that AMPK β1-Ser108 is a substrate for ULK1 under conditions associated with elevated AMP. We also provide unambiguous demonstration of AMPK signaling independently of Thr172 phosphorylation. These findings underpin a ULK-mediated “ligand switch” model of AMPK allosteric control, in which the adenylate charge-sensing role of β1-AMPK is replaced by an ability to detect perturbations in endogenous metabolite(s) acting at the ADaM site.In contrast, regulation and function of β-Ser108 phosphorylation have been largely unexplored. Ser108 is highly conserved in eukaryotes and was identified as an autophosphorylation site in rat liver AMPK preparations22 (β1/2-dKO). As expected, β1/2-dKO iMEFs were devoid of detectable AMPK α- or β-subunit expression and AMPK signaling as evidenced by lack of phosphorylation of the AMPK substrate ACC-Ser79  cell line derived from mouse embryos harboring genetic deletion of both β1 and β2 isoformsr79 Fig. . Lentivills Fig. . Incubatant Fig. . A-76966ant Fig. . These rβ1/2-dKO iMEFs, transduced with either β1 mutant S108A or S108E  treatment, β1 S108A and S108E transduced iMEF lysates were compared to trace out the changes in proteome and phosphoproteome β1γ1 in HEK293T cells. This complex is a suitable substrate for Thr172-phosphorylating kinases LKB1 and CaMKK2, but does not undergo Ser108 cis-autophosphorylation   or complexed to interaction partners FIP200 and Atg13 (kcat/Km 1.86 × 10−3/s/μM) , compared to ULK1  (S108tide) Fig.  against –114 (S10ly) Fig. . We perfμM) Fig. . ULK2 deLK1 Fig. . We examLK2 Fig. . MS/MS aLK2 Fig. .Fig. 4UL13, or individual A-769662 β1γ1 construct allowed us to investigate the effect of ULK phosphorylation on ligand-mediated regulation of AMPK without the need for prior and extensive phosphatase treatment. We found that ULK pre-treatment sensitized purified AMPK α1(C176S)β1γ1 to activation by A-769662 or salicylate β1γ1 in bacteria produced AMPK that was devoid of pSer108 O2- Fig.  and phenO2- Fig.  induced min Fig. ; this isulk1/2-dKO)29 . We found that H2O2 and phenformin treatments of HEK293T cells that induced Ser108 phosphorylation   Fig. . Therefo. 3 Fig. . ULK1 calls Fig. . These d31. We examined the requirement for β-myristoylation in directing Ser108 phosphorylation during cellular metabolic stress. We found that KI-α1β1γ1 containing a myristoylation-deficient β1-G2A mutant (non-myr) was insensitive to phenformin- or glucose starvation-induced phosphorylation of Ser108 in HEK293T cells , phenformin and A-769662, either alone or in combination, failed to induce Thr172 phosphorylation as expected. Phenformin, but not A-769662, induced a small increase in pACC from an undetectable basal level. Phenformin/A-769662 co-incubation resulted in a further 5.8-fold increase in pACC compared to phenformin alone, producing >30% the pACC signal in WT α2-expressing cells treated with phenformin , independently of Thr172 phosphorylation Fig. . Salicyl13, appears a highly intrinsic activation mechanism. Additionally, independent studies have described a disconnect between AMPK signaling (using elevated pACC as an index) and apparent AMPK activation (no increase in pThr172), most commonly in examinations of the role of skeletal muscle AMPK during exercise42, but also in response to reactive oxygen species (HeLa cells)43 and berberine (LAMTOR1-KO MEFs)44. We now demonstrate that significant cellular AMPK signaling can be triggered independently of pThr172  β1-Ser108 and contacting α-residues are highly conserved among eukaryotes; (ii) the β1-ADaM site is transient and regulated; (iii) both ULK1 phosphorylation of Ser108, and activation of AMPK by small molecule drugs, share a common specificity for the β1-isoform; (iv) synergistic activation of unphosphorylated AMPK, orchestrated across all three AMPK subunits and topographically distant β1-ADaM and γ-sites172 Fig. , althoug46. Other downstream targets are associated with regulation of cell cycle arrest , p53-mediated cell cycle arrest  and cell division . Although functional roles for the majority of sites detected in our analysis are unknown, our findings with PAK2 in particular point to a role for β1-Ser108 phosphorylation in promoting energy stress-induced pro-survival pathways over cell death pathways. This model is consistent with other autophagy-inducing roles for AMPK, and Ser108-phosphorylation as a direct mechanism to activate AMPK by the autophagy initiator ULK1. Future studies will delineate the cellular signaling mechanisms specific to β1-Ser108 phosphorylation, and bridge the gap between AMPK and the other identified downstream substrates.Applying quantitative phosphoproteomics to investigate a physiological role for Ser108 phosphorylation, we identified significant differences in the phosphorylation profiles of several cell cycle-associated proteins in response to the phosphomimetic mutant β1-S108E Fig. . Our appcis-autophosphorylation of Ser108 in the α2(T172A) mutant is unlikely since activity of the unphosphorylated complex can only be achieved synergistically; in the AMP/drug-bound state Ser108 is presumably sequestered to the ADaM site and away from the AMPK active site. The close correlation between pACC, pSer108 and ULK-pSer555 profiles across all conditions provides support for a positive feedback mechanism between ULK and AMPK  AMPK provides further insight into ULK regulation of β1-AMPK signaling Fig. . An inteMPK Fig. . ConsistMPK Fig. . Our modtrans-phosphorylation of Ser108 in cells were confounded by strong background autophosphorylation signals in HEK293T endogenous AMPK, and high basal levels of pSer108 in immortalized MEFs  under which Ser108 is phosphorylated independently of AMPK activity  are considered unfavorable for ULK substrates. However, Ser108 is located at the end of a β-hairpin loop structure12 and C-terminal residues may not participate in substrate recognition by ULK1. β1-Ser108 does not appear to be a favorable substrate for ULK2  , or activate ULK independently of changes in adenylate charge (mTOR inhibitors)  in targeting AMPK to phagophore membranes. A similar mechanism has been proposed to control AXIN-mediated AMPK recruitment to the late endosome/lysosome membrane for Thr172 phosphorylation and AMPK activation by LKB130. An unexpected finding was myristoyl-dependent suppression of Ser108 phosphorylation by ULK1 in cell-free assays  Fig.  are assors) Fig. . The AMPrs) Fig. , or β-surs) Fig. , abrogatays Fig. , indicatIn summary, we have identified an additional layer of communication between AMPK and ULK1, two major regulators of cellular energy homeostasis. ULK1 phosphorylation of Ser108, a post-translational modification important for AMPK drug action, may yield strategic opportunity to increase potency of AMPK-targeting therapeutics. Differential phosphorylation of AMPK at distinct cellular organelles/membranes raises the intriguing possibility of localized AMPK ligand sensitization. Myristoyl-switching is a consistent driving force for both mechanisms; whether secondary targeting signals are required to partition distinct AMPK complexes between organelles represents an area of great interest for the future.DNA oligos were from Sigma , generated by crossing homozygous β1 and β2 null mice54. WT and AMPK β1/2 double knockout (β1/2-dKO) MEFs were immortalized by Fugene HD-mediated transfection with an SV40 large-T antigen expression construct. AMPK α1/2 double knockout (α1/2-dKO) and ULK1/2 double knockout (ulk1/2-dKO) iMEFs were described previously29.COS7 and HEK293T cell lines were purchased from American Type Culture Collection. All cell lines were maintained in Dulbecco’s modified Essential medium (DMEM) containing 10% fetal bovine serum and antibiotics  at 37 °C with 5% COXhoI/EcoRI (AMPK FLAG-α1) or XhoI/HindIII (AMPK FLAG-α2) restriction sites. Lentivirus expression constructs LeGO-iG2, second generation viral packaging vector psPax2 and ecotropic envelope vector pHCMV-EcoEnv were gifts from Carl Walkley . cDNAs for human AMPK β1 (WT and mutants), α1 (WT and kinase inactive D141A mutant), and α2 (WT and T172A mutant) were generated with C-terminal FLAG-tag and cloned into LeGO-iG2 using BamHI/NotI (β1) or EcoRI/NotI (α1 and α2) restriction sites.All mutants were generated using QuikChange site-directed mutagenesis kits (Stratagene). All constructs were sequence verified. Mammalian cell expression constructs were gifts from Reuben Shaw (pcDNA3 mouse FLAG-ULK1 (Addgene #27636) and pcDNA3 mouse FLAG-ULK2 (Addgene #27637)), Noboru Mizushima (pME18s-3xHA-human FIP200 (Addgene #24303)) and Do-Hyung Kim (pRK5 human myc-Atg13 (Addgene #31965)). SV40 large-T antigen expression construct pBSSVD2005 was a gift from David Ron (Addgene plasmid #21826). Complementary DNA (cDNAs) for human AMPK α1 or α2 were generated with N-terminal FLAG-tag and cloned into pcDNA3 using 6-α fusions; WT or mutants as indicated) was expressed in E. coli strain Rosetta (DE3) and purified using nickel-Sepharose and size exclusion chromatography as described previously13. Heterotrimeric human AMPK  was expressed in HEK293T cells as described previously3. Briefly, HEK293T cells at 40% confluency were triply transfected with expression constructs for AMPK α-, β-, and γ-subunits, using transfection reagent FuGENE HD according to the manufacturer’s protocols. Cells were harvested 48 h post-transfection in ice cold lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 50 mM NaF, 1 mM sodium pyrophosphate, 1 mM EDTA, 1 mM EGTA, 1% (v/v) Triton X-100, protease inhibitors). Lysates were clarified by centrifugation at 14,000 rpm for 5 min and flash frozen in liquid N2 until processing. AMPK was purified on glutathione Sepharose 4B and eluted in 50 mM Tris-HCl, (pH 7.4), 100 mM NaCl, 10% glycerol supplemented with 20 mM glutathione. ULK1/2 and ULK1/FIP200/Atg13 complex was expressed by FuGENE HD transient transfection of HEK293T cells. Lysates were harvested after 48 h, clarified by centrifugation and ULK purified on anti-FLAG M2 affinity gel. ULK was eluted in 50 mM Tris-HCl (pH 7.4), 100 mM NaCl, 10% glycerol, 0.01% Tween-20 supplemented with 1 mg/ml FLAG peptide. CaMKK2 isoform 1 was produced in Sf21 insect cells as a C-terminal FLAG fusion as described previously3. Briefly, Sf21 cells were infected at a multiplicity of infection of 10 and harvested 72 h post-infection. Cell lysates were prepared as for recombinant AMPK, and protein was purified on anti-FLAG M2 affinity gel. CaMKK2 was eluted in 50 mM Tris-HCl (pH 7.4), 100 mM NaCl, 10% glycerol, 0.01% Tween-20 supplemented with 1 mg/ml FLAG peptide.Heterotrimeric human AMPK  were mixed together with 2 M CaCl2 solution . The DNA/CaCl2 solution was added drop-wise, with vortexing, into 500 μl of 2xHEPES-buffered saline, pH 7.06. After 20 min incubation at RT, the mixture was transferred drop-wise onto HEK293T cell culture and incubated. Within 16 h of transfection, cell were washed with phosphate-buffered saline (PBS) and replaced with 6 ml fresh media. The lentivirus-containing supernatant was harvested after 48 and 72 h post-transfection and stored at −80 °C.Ecotropic lentivirus was generated by transient transfection of HEK293T cells using calcium phosphate. Briefly, 1 day prior to transfection, 2–2.5 × 105 cells per well in a 6-well plate were seeded in 2 ml media. 200 μl lentivirus supernatant in the presence of 8 μg/ml polybrene in 2 ml media was spun for 98 min at 25 °C, 1100×g (Heraeus Megafuge 2.0 R). Lentivirus-containing media was replaced with an equal volume of fresh media after 24 h. Seventy-two hourspost-transduction, iMEFs were incubated with fresh media for 1 h and treated as indicated. Cells were harvested by washing with ice-cold PBS, followed by rapid lysis in situ using 100 μl ice-cold lysis buffer (50 mM Tris.HCl (pH 7.4), 150 mM NaCl, 50 mM NaF, 1 mM sodium pyrophosphate, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 1% (v/v) Triton X-100 and protease inhibitors), and cellular debris was removed by centrifugation.iMEFs were transduced in 6-well plates by spinoculation. Briefly, 1 day prior to transduction, 0.5 × 10FLAG-tagged AMPK α1 or α2, and acetyl-CoA carboxylase (ACC), were isolated from iMEF cell lysates using anti-FLAG M2 affinity gel or Streptavidin Sepharose high performance, respectively. Immobilized ACC was washed extensively with buffer A and eluted in 2×sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) loading sample buffer for immunoblotting.32P]ATP and 5 mM MgCl2 in a 25 μl reaction volume at 30 °C. Reactions were terminated after 10 min by spotting 15 μl onto P81 phosphocellulose paper (Whatman) and washing in 1% phosphoric acid. Radioactivity was quantified by scintillation counting.ULK and AMPK activities were determined by phosphorylation of a synthetic peptide  with three additional C-terminal Arg residues to promote binding to P81 phosphocellulose paper) using 200 μM [γ-33P]ATP, in a 25 μl reaction volume at 30 °C.Kinase screening (IKPT service) was performed by Kinexus, Canada. S108tide (100 μM) was screened against a panel of 284 selected Ser/Thr kinases with single replicate. Most assays were performed for 15 min duration, 50 μM , [ADP], and [ATP] (Equation Adenine nucleotides from HEK293T perchlorate extracts were measured by LC-MS on an ABISCIEX 5500 QTRAP mass spectrometert-test was used for all comparisons unless stated.The data are presented as mean values ± s.e.m. of at least three independent experiments. The unpaired two-tailed Student’s 59 partner repository with the data set identifier PXD006365. The authors declare that all other data supporting the findings of this study are available within the paper and its Supplementary Information files, or are available from the authors upon reasonable request.The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDESupplementary InformationPeer Review File


However, these variables display a puzzling asynchrony. Well-dated records of Southern Ocean temperature will provide crucial information because the Southern Ocean is likely key in regulating CO2 variations. Here, we perform multiple isotopic analyses on an Antarctic ice core and estimate temperature variations at this site and in the oceanic moisture source over the past 720,000 years, which extend the longest records by 300,000 years. Antarctic temperature is affected by large variations in local insolation that are induced by obliquity. At the obliquity periodicity, the Antarctic and ocean temperatures lag annual mean insolation. Further, the magnitude of the phase lag is minimal during low eccentricity periods, suggesting that secular changes in the global carbon cycle and the ocean circulation modulate the phase relationship among temperatures, CO2 and insolation in the obliquity frequency band.The δD temperature proxy in Antarctic ice cores varies in parallel with CO 2 through glacial cycles. Here, the authors show that a 720,000-year Antarctic temperature record is affected by variations in obliquity-induced local insolation that are associated with phase modulation of eccentricity cycle.The Antarctic temperature record displays a puzzling asynchrony with changes in CO The values of a temperature proxy, the hydrogen isotopic composition (δD), in the Antarctic EDC ice core2 have varied in parallel with CO2 concentrations over the past 800 thousand years 3. However, δD apparently leads CO2 variations. For example, during the last termination (TI), the start of Antarctic warming has been estimated to be synchronous with CO2 increase4 or to lead CO2 increases by 800 ± 600 years5 on the East Antarctic Plateau. The lead is ca. 2000 years at a West Antarctic site6. Over the past 420 kyr, the Vostok ice core shows that the Antarctic δD temperatures lead the CO2 variations by 1.3 ± 1.0 kyr7. During the lukewarm interglacials (430–650 kyr BP), Antarctic δD leads CO2 by 1900 years, and the correlation between CO2 and δD is weaker (r2 = 0.57), as determined from the EDC core8.Precise knowledge of the relationship between changes in temperature, atmospheric CO2 variations10. A box model, for example, estimated a ca. 60% increase in CO2 during TI that is attributable to direct and indirect temperature effects, such as changes in sea ice cover and vertical mixing in the Southern Ocean9. On millennial time scales, a multi-proxy study suggests that an antiphased hemispheric temperature response to ocean circulation changes resulted in Antarctic temperatures leading global temperatures and CO2 during TI11. On orbital time scales, the latitudinal temperature differences between Antarctica and the surrounding ocean may arise from annual mean insolation (AMI) variations because the amplitude of AMI variations is largest in the polar regions and smallest in the mid-latitude regions16. The AMI is paced only by the 41-kyr periodicity of the Earth’s obliquity17. Indeed, a strong 41-kyr cycle that is likely related to the local AMI variations was recognized in the Antarctic temperature record obtained from the Vostok ice core in the 1980s15. However, uncertainties in the age models of the ice cores and ocean sediments limit our understanding of the potentially different time lags between Antarctic and Southern Ocean temperatures relative to AMI changes. Therefore, well-dated surface temperature records with high temporal resolution from the Southern Ocean provide crucial information.Although the mechanisms underlying the coupling and the phase lags remain unclear, the Southern Ocean region, rather than Antarctica, is thought to play the central role in regulating COTsite), it is also affected by the extent of fractionation-associated rainout from the water vapour as it is transported from its moisture source in the ocean to the site where the precipitation occurs. After correcting the δD variations for this moisture source effect, the ΔTsite record shows a stronger correlation with CO2 over the past 350 kyr18, suggesting a potential bias in the use of δD as a temperature proxy. However, the effects of moisture source corrections are unknown prior to 420 kyr BP19. The lack of long records prevents elucidation of the potential influence of the 400-kyr eccentricity cycle on the relationship among AMI, CO2 and Antarctic and ocean temperatures.In addition to these climatic mechanisms, the validity of δD as a temperature proxy should be considered. While this proxy depends on Antarctic site temperatures  using new δD and deuterium excess records from the Antarctic Dome Fuji (DF) ice core, which spans the past 720 kyr. The new ΔTsite and ΔTsource records enable us to compare the pattern and timing of temperature variations between the Antarctic Plateau and its oceanic moisture source using the same age scale from that of the DF ice core. Our data extend the longest (420-kyr) record of ΔTsite19 by 300 kyr, and they reveal the impact of moisture source corrections during the lukewarm interglacials. The correlation between ΔTsource and CO2 is stronger than that between δD and CO2. Over the entire periods covered by the extended records, changes in local solar insolation, paced by obliquity, strongly affects ΔTsite. At the obliquity periodicity, the changes in ΔTsite and ΔTsource are delayed with respect to AMI. Further, the magnitude of the phase lag is minimal during the period of low orbital eccentricity, suggesting that the phase modulation is related to secular changes in the global carbon cycle and ocean circulations.To investigate the relationship between temperatures and CO20 to obtain a 720-kyr deuterium excess (d = δD–8δ18O) record. The δD and δ18O values of the ice between the depths of 2399.5 and 3035.1 m (297–715 kyr BP) were measured at 10-cm intervals. The new DF2 data were combined with earlier data from the first Dome Fuji core (DF122) and were plotted on the AICC2012 time scale23. The relationships among δD, d, ΔTsite and ΔTsource were quantified using a mixed cloud isotopic model (Methods). In this study, temperatures are expressed as deviations, Δ, from the modern value (the average of the past 2 kyr). ΔTsource is a proxy for temperature in moisture source regions22, and the major moisture source for modern precipitation at DF is estimated to lie in the South Atlantic and Indian Oceans between 38 and 65˚ S, based on backward-trajectory calculations26 2  over the past 720 kyr. With their higher sampling resolution (10 cm vs. 55 cm), the DF data depict the paleoclimate signals in greater detail than the EDC core. However, the deepest part appears to have been smoothed by ice diffusion, a process also observed in the 11-cm samples from the EDC core that correspond to Marine Isotope Stage (MIS) 11  to 4.0 °C (MIS 9.3) when 2-kyr mean values are used , as suggested by the EDC δD variations2.The DF Δord Fig. . Millenn °C MIS 1.2 to 4.0d record is shown in terms of the logarithmic definition of d . The use of these values produces glacial–interglacial variations in ΔTsource that are larger than those presented by previous studies29, resulting in good agreement between the DF ΔTsource record and a stacked SST record30 over the past 150 kyr22. Here, to evaluate the reliability of our ΔTsource estimates over multiple glacial cycles, we constructed a stacked sea surface temperature (SST) record based on 17 individual deep-sea sediment cores obtained in the source region , and these correlations are significantly stronger than that between δD and CO2 . The only exception for the strong coupling between ΔTsource and CO2 is observed during MIS 12.2, during which the ΔTsource shows exceptional cooling  is stronger than that between δD and CO2 . Rather, the correlation between CO2 and ΔTsource suggests a close link between surface temperature changes in the Southern Ocean and the global carbon cycle.The similarities and differences between temperatures and CO δD Fig. . Indeed,CO2 Fig. . Within  δD Fig. . These rTsource and CO2, ΔTsource lags CO2 slightly in most frequency bands   and the moisture source region (38–66° S). The ΔTgrad record is negatively correlated with the obliquity  Fig. . A decreity Fig. . This reTsite and ΔTsource was evaluated by filtering the records in the obliquity band . Thus, the difference is significantly reduced, and roughly consistent with the ΔTsite/ΔTsource ratio. Remaining difference would be balanced by other compensation mechanisms, such as the transport of heat from the low to the high latitudes36, and latitudinal contrasts in the cloud feedback, which is negative near 60° S and positive at low latitudes37.The impact of the differences in AMI on the amplitudes of Δand Fig. . The ran °C Fig. , which inge Fig. . Althougion Fig. . AccountTsite is delayed with respect to AMI (or obliquity) by 4.1 ± 2.7 kyr at the obliquity period  over the past 100–720 kyr23. Large uncertainties were expected during MIS 11-12 because of the lack of age markers23. However, an abrupt decrease in δ18O values seen in Chinese stalagmites dated independently using the U/Th technique38 coincides with an abrupt increase in EDC CH439 at Termination V, which supports the accuracy of the AICC2012 age scale.As seen in the Δcle Fig. . On aver18O record also shows a similar varying phase with respect to obliquity40 11. Thus, the timing at which CO2 begins to rise during a termination would be determined by when the Northern Hemisphere ice sheet begins to melt. When eccentricity is small, the summer insolation maxima are small. Thus, if obliquity rises beyond the threshold of melting, a moderate climate forcing could cause warming enough that the southern margin of the North American ice sheet begin to retreat41. Therefore, from 350 to 450 kyr BP, the obliquity component of CO2 began to rise rapidly without a phase difference relative to obliquity.The phase modulation of COity Fig. , which r2 to increase, occurs at the time of low eccentricity45 , which is a proxy for deep-water ventilation in the South Atlantic Ocean, shows weaker deep-water ventilation during glacial times with a clear 100-kyr glacial cycle;49 this gradient also displays a secular 400–500-kyr-long trend in the glacial minima from MIS 6 to MIS 14  during the next 10 kyr would cause temperatures on the East Antarctic Plateau to decrease by 1–2 °C in the obliquity frequency band. The obliquity-paced solar radiation is also important as a driver of changes in glacier mass balance in Antarctica52. Thus, it is crucial to understand the climate mechanisms that underlie the apparent 400-kyr phase modulation of AMI relative to temperatures, CO2 and ice volume.Our temperature data reveal that the asynchrony between Antarctic temperature and CO53 at the DF station in East Antarctica. The drilling site is located at a summit of the East Antarctic ice sheet and has a current annual mean air temperature of −55 °C. Extensive analyses of the deepest part of the ice (3000.0–3035.0 m) indicate that the bottom ice originated from meteoric water with altered chemical compositions54.The DF2 ice core was drilled to near bedrock at a depth of 3035.2 m18O values were performed for 6349 samples, which were obtained at depths ranging from 2399.5 to 3035.0 m. Each sample was 10 cm in length, which corresponds to a mean temporal resolution of ~70 yr. To remove surface contamination, 13 wt% of the surface was removed from each 24-g ice sample with a ceramic knife. The ice samples were melted at room temperature in sealed polyethylene bags. The water was then transferred to glass bottles and preserved in a frozen state. Both δD and δ18O values were measured using an automated equilibration method55 at the National Institute of Polar Research, Japan. The δ18O values of the ice samples (δ18Oice) were used in this study, whereas the δ18O values of ‘sawdust’ samples (δ18Osaw) were used in the previous DF article20. The sawdust was collected during ice cutting at the DF site. The average difference (=δ18Osaw–δ18Oice) and its variations (±1σ) were 0.31 ± 0.18‰ (n = 15), respectively. The small enrichment in δ18Osaw was caused by sublimation during sampling and/or transport of the samples from Antarctica to Japan. In addition, the resolution of this study is five times higher than that of the 50-cm sampling used in the previous DF2 δ18Osaw data20.Measurements of δD and δδ notation: δ = Rsample/RVSMOW−1, where Rsample and RVSMOW are the isotopic ratios (D/H and 18O/16O) of the sample and Vienna Standard Mean Ocean Water (VSMOW), respectively. To improve the precision of the measurements, 45% of the samples were measured two to four times. The analytical precisions (1σ), which are based on the differences between the duplicate analyses of individual samples, were 0.44‰ (δD), 0.05‰ (δ18O) and 0.52‰ (d).The isotope data are given using conventional 58, the resolution was ca. 400 years, and some parts were discontinuous. The overlapping portion (2400–2500 m) of the DF1 and DF2 records exhibited remarkable similarity59 because the horizontal distance between the two sites was only 43 m. The combined record was obtained by replacing the DF1 data58 with the DF2 data within the depth interval where the two cores overlap22 and adding the DF2 record. In this study, the DF records were plotted on the AICC2012 age scale60. To transfer the DF data to the AICC2012 age scale23, volcanic tie points between DF and EDC were used for 0–216 kyr BP61. For 216–720 kyr BP, the isotopic profiles were synchronized using the Match software package62. The glacial inceptions were excluded from the predetermined tie points20 because of potential differences in the patterns of the two isotopic profiles63. The largest mismatch between DF2 and EDC was found during 600–610 kyr BP. Isotope matching was impossible for this part of the record because of substantial differences in the observed trends.To obtain a complete isotopic record over 720 kyr, we used published data sets from the first Dome Fuji (DF1) core for shallow depths ranging from 0.0 to 2399.5 m. For the portion of the record derived from the DF1 core64. However, the δD and δ18O variations in Antarctic ice cores2 depend on the extent of fractionation-associated rainout from atmospheric water vapour along the pathway from the oceanic moisture source to the precipitation site, as well as the site temperature (ΔTsite). To estimate the site air temperature (ΔTsite), including a correction for the temperature of the moisture source regions (ΔTsource), a linear inversion method was used. This method has previously been applied to the Vostok core29, the EDC core65 and the DF core22. The sensitivities of the δD and d values to DF site air temperatures (ΔTsite) and the temperatures within the moisture source region (ΔTsource) were estimated using a mixed cloud isotopic model66. The multiple linear regression is expressed using the following equations:Traditionally, δD is used as a proxy for surface air temperatures, based on the spatial linear correlation between δD measurements and Antarctic temperatures18Osw)67 using the Rayleigh equation68. For the temperature sensitivity parameters, γsite, γsource, βsite and βsource, we used the values given by Uemura et al.22. Based on Monte Carlo simulations performed using the Vostok core, the uncertainties in the glacial–interglacial magnitudes of ΔTsite and ΔTsource are 1.1 °C and 0.8 °C, respectively29. The uncertainties for the DF site should be similar to those at Vostok because both are located in the inland portion of East Antarctica.where Δ represents the deviation from the modern , and the subscript ‘corr’ indicates the ice core record corrected for the isotopic composition of ocean water  by the t-value (97.5% confidence level). The stacked SST profile and uncertainty intervals are shown only for the period during which multiple data records (4 or more) were available . The albedos of the DF region and the moisture source region are 0.78 and 0.46, respectively.The albedo climatology was derived from data collected by the TIROS-N satellite. This data set was obtained from the climate data archive of the Joint Institute for the Study of the Atmosphere and Ocean  of CO2 with δD, ΔTsite and ΔTsource are 0.68–0.73, 0.76–0.80 and 0.75–0.79, respectively. For the lukewarm period, the ranges of the correaltion of CO2 with δD, ΔTsite and ΔTsource are 0.60–0.70, 0.72–0.79 and 0.77–0.83, respectively. Thus, the correlation between ΔTsite (and ΔTsource) and CO2 is significantly stronger than that between δD and CO2. The correlation between ΔTsource and CO2 is slightly higher than that between ΔTsite and CO2, but it is statistically insignificant.The uncertainty of the linear correlation coefficient was estimated using the Fisher 87  with a bandwidth of 0.00488).Spectral analyses were conducted with the Blackman–Tukey method 30% lag) using the Analyseries software package0% lag us87 Fig. . The ampσphs). The relative lags between the temperature proxies were as reliable as possible because they were estimated from the same ice sample. The phase analysis between ΔTsite and ΔTsource . Determining the exact phase lag between CO2 concentrations and temperatures was complex because the air was younger than the surrounding ice. A phase analysis between the temperature proxies and CO2  Ice-age versus orbital-age phase analysis error (σorb). To compare with the variation in obliquity, the absolute age uncertainty of AICC2012 for ice must be considered. The uncertainty of AICC2012 is ±2.6 kyr on average during the past 100–720 kyr23. The phase lag and confidence intervals (90%) of ΔTsite, ΔTsource and ΔTgrad with respect to obliquity were 4.1 ± 0.7, 7.5 ± 1.3 and 1.9 ± 0.3 kyr, respectively. For example, a combined error estimate for ΔTsite that is based on the root mean squared error is ±2.7 kyr. Therefore, the 4.1-kyr lag of ΔTsite against obliquity was significant during most of the studied period. The error in the gas-age versus orbital-age phase analysis is similar to σorb because the uncertainty of the AICC2012 age scale for gas is ±2.6 kyr, on average, during the past 100–720 kyr23.The uncertainties associated with the phase analysis were of three types: (i) ice-age versus ice-age phase analysis error . The data are also available from the authors.The raw data used in this study are available at the NOAA’s National Centers for Environmental Information (NCEI), which is formerly the National Climate Data Center (NCDC) data archive (Supplementary Information


Coinciding with global warming, Arctic sea ice has rapidly decreased during the last four decades and climate scenarios suggest that sea ice may completely disappear during summer within the next about 50–100 years. Here we produce Arctic sea ice biomarker proxy records for the penultimate glacial (Marine Isotope Stage 6) and the subsequent last interglacial (Marine Isotope Stage 5e). The latter is a time interval when the high latitudes were significantly warmer than today. We document that even under such warmer climate conditions, sea ice existed in the central Arctic Ocean during summer, whereas sea ice was significantly reduced along the Barents Sea continental margin influenced by Atlantic Water inflow. Our proxy reconstruction of the last interglacial sea ice cover is supported by climate simulations, although some proxy data/model inconsistencies still exist. During late Marine Isotope Stage 6, polynya-type conditions occurred off the major ice sheets along the northern Barents and East Siberian continental margins, contradicting a giant Marine Isotope Stage 6 ice shelf that covered the entire Arctic Ocean. Coinciding with global warming, Arctic sea ice has rapidly decreased during the last four decades. Here, using biomarker records, the authors show that permanent sea ice was still present in the central Arctic Ocean during the last interglacial, when high latitudes were warmer than present. Ice significantly reduces the heat flux between ocean and atmosphere; through its high albedo it has a strong influence on the radiation budget of the entire Arctic. Furthermore, the sea-ice cover strongly affects biological productivity, as a more closed sea-ice cover reduces primary production due to low light influx in the surface waters. As sea ice is sensitive to atmospheric and oceanic variability, and because it is involved in several key climate feedbacks , sea ice plays a substantial role in the global climate system variability, known as polar amplification3.Sea ice with its strong seasonal and interannual variability Fig.  is a ver2 increase, Arctic sea ice has significantly decreased in its extent , i.e., Marine Isotope Stage (MIS) 5e (Eemian), lasting from about 130 to 115 ka and often proposed as a possible analog for our near-future climatic conditions on Earth12. Based on proxy records from ice, terrestrial and marine archives, the LIG is characterized by an atmospheric CO2 concentration of about 290 ppm, i.e., similar to the pre-industrial (PI) value13, mean air temperatures in Northeast Siberia that were about 9 °C higher than today14, air temperatures above the Greenland NEEM ice core site of about 8 ± 4 °C above the mean of the past millennium15, North Atlantic sea-surface temperatures of about 2 °C higher than the modern (PI) temperatures16, and a global sea level 5–9 m above the present sea level17. In the Nordic Seas, on the other hand, the Eemian might have been cooler than the Holocene due to a reduction in the northward flow of Atlantic surface water towards Fram Strait and the Arctic Ocean, indicating the complexity of the interglacial climate system and its evolution in the northern high latitudes19.The last time that Arctic temperatures were significantly higher than today was the Early Holocene Thermal Maximum23. In order to test and approve climate models for simulation and prediction of Arctic climate and sea ice cover28, however, precise (semi-quantitative) proxy records about past sea ice concentrations are needed. Such records may be obtained using a quite recently developed biomarker approach that is based on the determination of a highly branched isoprenoid (HBI) with 25 carbons (C25 HBI monoene)29. This biomarker is only biosynthesized by specific diatoms living in the Arctic sea ice and thus named ‘IP25’ (= ice proxy with 25 carbons)30. That means, the presence of IP25 in the sediments is a direct proof for the presence of past Arctic sea ice. Meanwhile, this biomarker approach has been used successfully in many studies dealing with the reconstruction of Arctic sea ice history during the last glacial to Holocene time interval, i.e., the last about 30 ka37. Furthermore, this biomarker seems to be quite stable as it was found in sediments as old as the late Miocene8. By combination of this sea ice proxy IP25 with (biomarker) proxies for open-water phytoplankton productivity such as brassicasterol, dinosterol or a specific tri-unsaturated HBI (HBI-III)41, a more precise (semi-quantitative) reconstruction of present and past Arctic Ocean sea ice conditions from marine sediments are now available , including the extent of Arctic sea ice cover, we will have additional confidence in their representation of Arctic processes and their projections for the future49. Our proxy records are compared with climate model simulations using a coupled atmosphere-ocean general circulation model.Here we produce these biomarker proxy records of sea ice distribution in the central Arctic Ocean for the time interval of late MIS 6–MIS 5. Including open-water phytoplankton biomarkers as well as micropaleontological data, we demonstrate (1) that a permanent sea ice cover existed during MIS 6 and (2) that during the LIG sea ice was still present in the central Arctic Ocean during the spring/summer season even under  boundary conditions significantly warmer than the present. Seasonal open-water conditions, on the other hand, occurred along the Barents Sea continental margin during the interstadials of MIS 5  but also during the preceding glacial MIS 6. The latter finding—although still based on a low-resolution record—appears to contradict the hypothesis of a thick ice shelf covering the entire Arctic Ocean during MIS 6 as proposed by Jakobsson et al.25, open-water phytoplankton biomarkers, and terrestrial biomarkers from four selected sediment cores  to LIG MIS 5) conditions, we determined the sea ice biomarker proxy IP conditio25 as well as brassicasterol concentrations are zero or close to zero throughout the time interval from MIS 6 to MIS 5 in the two high-Arctic cores PS2200-5 and PS51/038-3 . Based on these data and looking at the position of the data points within the IP25 vs. brassicasterol diagram . Based on these data, an extended but variable sea ice cover with closed sea ice to ice-edge conditions occurred during late MIS 6 . The model experiments were driven by orbitally-induced insolation and greenhouse gas concentrations in the atmosphere  with phytoplankton and sea ice algae production, subglacial meltwater outflow and the deposition of suspended material on the slope at site PS2138-2. Furthermore, the seasonal open-water conditions along the Barents Sea continental margin might have been fostered by the inflow of Atlantic Water , the MIS 6 situation might have been similar to that of the LGMter Fig.  that was1-1 Fig. 58.53 has existed at this time  as proposed by Niessen et al.ime Fig. . Evidenceet Fig. , resultieet Fig. 57, 58. Sean Fig. 49. On th60 as well as the Arctic , however, are present during the LIG at both sites PS2200-5 and PS51/038-3  in the Eemian sediments of Core PS2200-5  or almost not (Core PS2200-5) survive zooplankton grazing, transport through the water column and degradation at the seafloor and in the sediment.As described above for the biomarker proxies of Core PS2200-5 and Core PS51/038-3 .Peak abundances of the small subpolar planktic foraminifer species 58 and (at least in the early-mid MIS 5e) driven by solar insolation  reach very similar minimum numbers of about 0.2 and less . We relate this sea ice minimum to the strong inflow of warm Atlantic Water as indicated by the contemporaneous prominent maxima in open-water phytoplankton biomarker concentrations , total numbers of dinoflagellate cysts and—especially—the dinoflagellate species O. centrocarpum  as clearly reflected in the semi-quantitative PIPess Fig. , i.e., npum Fig.  as well mum Fig. , but is 20.Towards the end of the LIG, i.e., during the early stage of the last glacial inception after 115 ka and coinciding with a minimum of the northern summer insolation, sea ice along the Barents Sea continental margin started to extend by a factor of 2–3 in comparison to the mid-LIG/Eemian minimum Fig. . Contemp64—may have resulted from a reduced total oceanic heat flux towards the north, as suggested from (compared to the PI control runs) reduced AMOC patterns during LIG-130 and LIG-125  near 120 ka  Figs.  and 8 an ka Fig. . During 28 indicate in model experiments that the height of the Greenland Ice Sheet also affects the sea ice cover in the Arctic Ocean. That means, a reduced Greenland Ice Sheet would result in a significantly reduced sea ice cover as shown in our LIG-130 simulation  and the RCP6 (808 ppm CO2eq) . Nevertheless, our findings already provide important groundtruthing data of sea ice conditions during the penultimate glacial and the LIG/Eemian of the central Arctic Ocean and the Siberian-Barents Sea continental margin, that may help to further test and improve models for simulation and prediction of future climate change. In follow-up research, such a study should be extended to a high-resolution approach to be carried out on thicker, well-dated MIS 6/MIS 5 sequences recovered from key areas of the Arctic Ocean and adjacent marginal seas. Such sedimentary sequences with the essential spatial and temporal resolution, however, are not available yet and have to be cored in the future. These new sea ice proxy records are needed (1) to fully prove the scenarios of a succession from an extended ice shelf to polynya/open-water conditions  have mainly been identified by oxygen and carbon isotope stratigraphy, paleomagnetostratigraphy, and abundances of planktic foraminifers and coarse fraction  have mainly been identified by lithostratigraphy, paleomagnetostratigraphy, and correlation with other dated sediment cores   have mainly been identified by 69). For quantification internal standards, 7-hexylnonadecane , squalane (2.4 µg per sample), and cholesterol-d6 , were added prior to analytical treatment. Separation of the hydrocarbon and sterol fractions was carried out via open column chromatography ). The latter fraction was silylated with 200 µl bis-trimethylsilyl-trifluoroacet-amide (BSTFA) . IP25, tri-unsaturated HBI , and sterols were analyzed by gas chromatography (GC) using an Agilent Technologies 7890B  coupled to an Agilent Technologies 5977A Extractor mass selective detector . GC analyses were performed with the following temperature program for the hydrocarbons: 60 °C (3 min), 150 °C (rate: 15 °C/min), 320 °C (rate: 10 °C/min), 320 °C  and for the sterols: 60 °C (2 min), 150 °C (rate: 15 °C/min), 320 °C (rate: 3 °C/min), 320 °C . The injection volume was 1 µl splitless. Helium was used as carrier gas (1 ml/min constant flow). Component assignment was based on comparison of GC retention times with those of reference compounds and published mass spectra 3) and β-sitosterol (as 24-ethylcholest-5-en-3β-O-Si(CH3)3) were calculated to be 1.018 and 1.077 , respectively.Extraction of 5–10 g of freeze-dried sediments was carried out using an accelerated solvent extractor  with dichloromethane:methanol  as a solvent. For comparison, a set of samples was also extracted by ultra sonic techniques giving similar results  in relation to the abundant fragment ion m/z 266 of the internal standard (7-HND) were used . The different responses of these ions were balanced by an external calibration33. The Kovats Index calculated for IP25 is 2086. The detection limit for quantification of IP25 using the GC–MS system described is 5 ng mL−1. Brassicasterol and β-sitosterol were quantified as trimethylsilyl ethers using the molecular ions m/z 470 and m/z 486, respectively, in relation to the molecular ion m/z 464 of cholesterol-D6.For the quantification of IP25 is absent under a permanent sea ice cover limiting light penetration and, as a consequence, sea ice algal growth . As IP25 is only produced within the sea ice matrix, ice-free conditions also result in zero IP25 concentrations. Thus, the two extremes, ice-free vs. thick closed sea ice cover, cannot be distinguished solely by using the IP25 biomarker. By considering also a phytoplankton biomarker indicative for open-water primary production, these extremes can be easily separated as under a permanent sea ice cover the phytoplankton biomarker is absent but reaches maximum concentrations under open-water conditions . The balance factor c is needed due to the significant concentration difference between IP25 and brassicasterol (or dinosterol). For the calculation of the mean concentration values of a specific core  zero concentrations should be excluded. The coupling of IP25 with phytoplankton biomarkers such as brassicasterol or dinosterol proves to be a viable approach to determine (spring/summer) sea ice conditions as is demonstrated by the good alignment of the PIP25-based estimate of the recent sea ice coverage with satellite observations38. More recently, Smik et al.41 introduced the HBI-III alkene as a phytoplankton biomarker replacing the sterols in the PIP25 calculation. This modified PIP25 approach is far less dependent on the balance factor c and based on biomarkers from the same group of compounds  with more similar diagenetic sensitivity. In a study of sediment cores from the western Barents Sea and the northern Norwegian Sea, Belt et al.37 have calculated PIP25 values using brassicasterol (ʽPBIP25’) and HBI-III (ʽPIIIIP25’) as a phytoplankton biomarker, respectively. Importantly, these authors could demonstrate that both approaches yielded similar outcomes if the core-specific balance factors were used  scenarios RCP4.5 (583 ppm CO2eq) and RCP6 (808 ppm CO2eq)27.We simulate four distinct periods, three from the LIG corresponding to LIG-130 (130 ka), LIG-125 (125 ka), and LIG-120 (120 ka) with greenhouse gas and orbital values derived from ice core recordshttps://doi.org/10.1594/PANGAEA.874357.All data generated or analyzed within this study are included in this published article (and its supplementary information files) and available at Supplementary InformationPeer Review File.