APP) and are linked with its cleavage to generate β‐amyloid peptide, a key player in the pathology of Alzheimer's disease. How APP switches between adaptors at different stages of the secretory pathway is poorly understood. Here, we show that tyrosine phosphorylation of Mint1 regulates the destination of APP. A canonical SH2‐binding motif (202YEEI) was identified in the N‐terminus of Mint1 that is phosphorylated on tyrosine by C‐Src and recruits the active kinase for sequential phosphorylation of further tyrosines (Y191 and Y187). A single Y202F mutation in the Mint1 N‐terminus inhibits C‐Src binding and tyrosine phosphorylation. Previous studies observed that co‐expression of wild‐type Mint1 and APP causes accumulation of APP in the trans‐Golgi. Unphosphorylatable Mint1 (Y202F) or pharmacological inhibition of Src reduced the accumulation of APP in the trans‐Golgi of heterologous cells. A similar result was observed in cultured rat hippocampal neurons where Mint1(Y202F) permitted the trafficking of APP to more distal neurites than the wild‐type protein. These data underline the importance of the tyrosine phosphorylation of Mint1 as a critical switch for determining the destination of APP.Mint/X11 is one of the four neuronal trafficking adaptors that interact with amyloid precursor protein (The regulation of amyloid precursor protein (APP) trafficking is poorly understood. We have discovered that the APP adapter, Mint1, is phosphorylated by C‐Src kinase. Mint1 causes APP accumulation in the trans‐Golgi network, whereas inhibition of Src or mutation of Mint1‐Y202 permits APP recycling. The phosphorylation status of Mint1 could impact on the pathological trafficking of APP in Alzheimer's disease. ADAlzheimer's diseaseAPLPAPP‐like proteinAPPamyloid precursor proteinPTBphosphotyrosine bindinget al. et al. et al. Alzheimer's disease (AD) is caused by the toxicity of soluble β‐amyloid oligomers that arise from the cleavage of amyloid precursor protein (APP) by secretase enzymes  domains and link APP to a host of factors, including sorting, signalling and motor proteins (Kawasumi 202EEI) that is phosphorylated by C‐Src and mediates recruitment of the kinase and the sequential phosphorylation of two adjacent Mint1 tyrosine residues. Stabilisation of APP metabolism by Mint1 is abolished by mutation of the C‐Src phosphorylation site or inhibition of Src, thus revealing a regulatory role for the Mint1 N‐terminus in interactions with APP. Furthermore, trafficking of APP to distal synapses in neurons is also dependent on Mint‐Y202. We propose that the phosphorylation status of Mint1‐Y202 acts as a switch to regulate the destination of APP.In a search for a function of the N‐terminus of Mint1 we identified a canonical SH2 ligand  was purchased from BD Bioscience . Rabbit monoclonal anti‐myc and rabbit polyclonal anti‐FLAG and anti‐Src pY416 were from Cell Signaling Technologies . Rat monoclonal anti‐HA (clone 3F10) was from Roche . Rabbit anti‐TGN46 was a gift from Dr Daniel Ungar (University of York). Alexa‐fluor‐conjugated secondary antibodies were from Invitrogen . The Src family kinase inhibitor, PP2, was obtained from Tocris‐Cookson . Rabbit polyclonal anti‐GFP (green fluorescent protein) was from Insight Biotechnology . Affinity‐purified anti‐Mint‐PY202 rabbit polyclonal antibody, raised to the peptide CGLQEHVpYEEIGDA, was custom made by Genscript . Unless stated otherwise, all other reagents were from Sigma .Escherichia coli, pGEX4T‐1‐PTP1B‐3C‐His‐Src(Δ80), and expressing C‐Src‐FLAG in mammalian cells have been described elsewhere  obtained from GeneService . Mammalian epitope‐tagged Mint1 expression plasmids comprised pMH‐Mint(1‐314)‐HA and pMH‐myc‐Mint1(1‐431), pMH‐myc‐Mint1(1‐842) and pmCerulean‐N1‐Mint(1‐842). A plasmid encoding human APP(1‐751)‐FLAG in a pEGFP‐N1 backbone was prepared using IMAGE clone 6152423. Bacterial expression plasmids included pGEX‐6P1‐Mint1(1‐314), pGEX‐6P1‐Mint1(1‐431), pGEX‐6P1‐Mint3(1‐180) and pQE30‐myc‐Mint(1‐842). The Mint1‐Y202F mutation was introduced into the relevant Mint1 plasmids using the Quikchange system  according to the manufacturer's instructions. pGEX2T‐Mint2(1‐341) and pQE30‐Munc18 were generous gifts from the laboratory of Prof Bob Burgoyne, University of Liverpool, UK. Plasmids for expressing recombinant active His‐C‐Src in et al. l‐glutamine, 35 mM glucose, 100 units/mL penicillin, and 0.1 mg/mL streptomycin (Invitrogen). Cytosine arabinoside (2.4 μM) was added to the cultures 2–4 days after plating to inhibit the proliferation of non‐neuronal cells. Hippocampal neurons were transfected with Lipofectamine 2000 according to the manufacturer's instructions (Invitrogen).Flp‐in T‐REx HeLa cells stably expressing Tet‐inducible APP‐FLAG and COS‐7 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% foetal calf serum and penicillin/streptomycin (Invitrogen). Cells were transfected 24 h after plating with EcoTransfect transfection reagent according to the manufacturer's instructions . Hippocampal and cortical neurons were prepared from newborn Wistar rat pups of either sex (Harlan) as described previously . Reactions were initiated by the addition of pre‐warmed ATP  to 10 μg protein substrate and 100 nM C‐Src, incubated at 30°C for 3 h and terminated by the addition of 2× SDS sample buffer (Sigma).Phosphorylation reactions were prepared in kinase reaction buffer  was separated from the other assay components of a similar mass [Src kinase and bovine serum albumin (BSA)] by 2D gel electrophoresis using a first dimension of pH 3–10. The Coomassie‐stained Mint protein spot was sampled in four locations, each with increasing mass and decreasing pI. Following sequential tryptic and chymotryptic digestion, peptides were subjected to reversed phase LC‐MS/MS using a Dionex Ultimate HPLC with a polystyrene–divinylbenzene monolithic column coupled to either an Applied Biosystems QSTAR Pulsar I or Bruker HCT ultra‐mass spectrometer. Collision‐induced dissociation mass spectra were searched against a small custom database containing the Mint1 sequence. Phosphorylation sites were assigned using Mascot scores. ‘Delta score’ differences  were lysed in ice‐cold radioimmunoprecipitation assay buffer and incubated overnight at 4°C with 25 μL Protein G resin (Genscript) and 3 μg of the appropriate antibody. Beads were washed three times in radioimmunoprecipitation assay buffer and proteins eluted in SDS sample buffer and processed for western blotting.Transfected COS‐7 cells grown in 10 cm plates (10d‐lysine for neurons) prior to transfection. Culture media containing 1 μg/mL doxycycline was applied to Flp‐in T‐REx HeLa cells to induce expression of APP‐FLAG for the indicated times. Cells were fixed in 4% paraformaldehyde with 4% sucrose and then permeabilised for 30 min in phosphate‐buffered saline (PBS) with 1% BSA and 0.1% Triton X‐100. Primary antibody diluted in PBS with 1% BSA was applied for 1 h at 25°C. After three washes in PBS, Alexa‐fluor‐conjugated secondary antibodies (1 : 500) were incubated for 30 min. Following three further washes in PBS, coverslips were mounted in Mowial mountant. Images were captured using a Zeiss LSM 710 Meta confocal microscope in the Technology Facility, Department of Biology, University of York or a Nikon TE200 epifluorescence microscope equipped with a Rolera‐XR CCD camera .COS‐7, HeLa cells or hippocampal neurons were cultured on No. 1 glass coverslips . Furthermore, the N‐terminus of Mint1 is tyrosine rich, suggesting the potential for multiple phosphorylation events by tyrosine kinases. In vitro kinase assays using recombinant C‐Src demonstrated tyrosine phosphorylation of the Mint1 N‐terminus (1‐314), comparatively weak phosphorylation of the equivalent sequence in Mint2, and no phosphorylation of Mint3  was resolved by 2D gel electrophoresis and the protein spot corresponding to tyrosine‐phosphorylated protein  by C‐Src  wild‐type or a Y202F mutant. We found that in resting cells we could not detect phosphorylation of immunoprecipitated myc‐Mint1(1‐431) by immunoblotting with a phosphotyrosine antibody ‐HA and C‐Src‐FLAG were incubated in the presence or absence of pervanadate and then subjected to immunoprecipitation with anti‐HA antibodies and processed for western blotting. In agreement with the lack of resting phosphorylation of Mint1 . Twenty‐four hours after APP induction, cells expressing Mint1‐Y202F and APP alone had a similar APP distribution to 3 h, with 90% and 78% of APP, respectively, being cytoplasmic. In contrast, 26% of cells expressing wild‐type Mint1 had large perinuclear puncta  and the total number of APP puncta concomitantly decreased . The antibody detected phosphorylation of recombinant Mint1 N‐terminus expressed in tyrosine kinase expressing TKX bacteria Fig. a and in 02F Fig. a. As obs02F Fig. b. The ph02F Fig. b.To discover whether Mint1‐Y202 regulates APP trafficking in neurons, the co‐expression of APP and Mint1 plasmids was replicated in cultured hippocampal neurons. Here the effect was more striking as co‐expression of wild‐type Mint1 prevented APP from entering neurites and caused accumulation in the cell soma Fig. a. Again,We have discovered a novel sequential tyrosine phosphorylation of the Mint1 N‐terminus by C‐Src kinase. The specific downstream consequences of these phosphorylation events are the recruitment of active C‐Src and the regulation of APP sorting at the TGN. Our data identify Y202 as a determinant for Mint1‐dependent APP accumulation in the TGN and could have implications for the pathological trafficking of APP in AD.in vitro was only detected on Y202, Y191 and Y187 by mass spectrometry. Other tyrosine residues in Mint1 have been shown to be phosphorylated in brain samples in phosphoproteomic screens ‐Y202F in vanadate treated COS‐7 cells (Fig. et al. Phosphorylation of the Mint1 N‐terminus by C‐Src et al. et al. et al. et al. et al. The principal effects of preventing tyrosine phosphorylation of Mint1 were a decrease in the accumulation of APP in the TGN and enhanced transport of APP along neurites in neurons. Using inducible expression of APP, we demonstrated that the accumulation of APP likely arises after its endocytosis rather than prior to its initial secretion. Following internalisation, APP is sorted from early endosomes either to the TGN by the retromer complex or to the late endo‐lysosomal system (Vieira in vitro kinase assay using recombinant C‐Src. The lack of Mint3 tyrosine phosphorylation suggests Src phosphorylation of Mints 1 and 2 is a neuronal adaptation of its function and hence consistent with its role in neuronal APP trafficking. A previous study focussing on Mint2 tyrosine phosphorylation, also found the extent of Mint1 phosphorylation by C‐Src in vitro and in cells was many fold higher than that of Mint2 (Chaufty et al. et al. et al. et al. We observed very weak phosphorylation of the Mint2 N‐terminus and no phosphorylation of the Mint3 N‐terminus in our et al. et al. (et al. cas (Cary et al. How might multisite tyrosine phosphorylation of Mint1 by C‐Src regulate APP at the molecular level? First, Mint1 phosphorylation could allosterically regulate the interaction between its PTB domain and the APP C‐terminus. There are conflicting data regarding this mode of regulation, for example, deletion of the Mint1 N‐terminus inhibits the effect of the full length protein on APP processing (Mueller . et al.  showed t. et al. . It is a. et al. . In addiet al. et al. et al. et al. et al. et al. While the signalling adaptors Shc and Grb2 are recruited to the tyrosine‐phosphorylated tail of APP (Tarr


Here, we show that depletion of [Ca2+]i augments PMA- and EGF-induced phosphorylation of β4-T1736 and that this is caused by inhibition of the calcium-sensitive protein phosphatase calcineurin and augmentation of ERK1/2 activation. We also show that in keratinocytes the PMA-stimulated phosphorylation of β4-T1736 primarily is mediated by PKD2 activation downstream of PKCδ. On the other hand, both the EGF-stimulated phosphorylation of T1736 and the EGF-induced dissolution of HDs are dependent on a functional MAPK signaling pathway, and treatment with the RSK inhibitor BI-D1870 prevented EGF-stimulated phosphorylation of β4-T1736. Moreover, phosphorylation of β4-T1736 is enhanced by overexpression of wild-type RSK1, while it is reduced by the expression of kinase-inactive RSK1 or by siRNA-mediated depletion of RSK1. In summary, our data indicate that different stimuli can lead to the phosphorylation of β4-T1736 by either PKD2 or RSK1.The integrin α6β4, a major component of hemidesmosomes (HDs), stabilizes keratinocyte cell adhesion to the epidermal basement membrane through binding to the cytoskeletal linker protein plectin and association with keratin filaments. Disruption of the α6β4-plectin interaction through phosphorylation of the β4 subunit results in a reduction in adhesive strength of keratinocytes to laminin-332 and the dissolution of HDs. Previously, we have demonstrated that phosphorylation of T1736 in the C-terminal end of the β4 cytoplasmic domain disrupts the interaction of β4 with the plakin domain of plectin. Furthermore, we showed that β4-T1736 can be phosphorylated by PKD1  Hemidesmosomes (HDs) are junctional protein complexes that are crucial for maintaining firm adhesion of keratinocytes to the underlying basement membrane and thus for epithelial tissue integrity ,2. The iStudies with cultured keratinocytes revealed that the formation of HDs is critically dependent on the binding of plectin to the cytoplasmic domain of β4 –10. Mutain vitro and that this kinase is activated in keratinocytes upon PMA stimulation i chelator BAPTA-AM and the calcium ionophore A23187 on the phosphorylation of T1736 and S1364. Treatment of PA-JEB/β4 keratinocytes, grown in complete KGM, with BAPTA-AM resulted in a 2–3 fold increase in the phosphorylation of β4 at T1736. BAPTA-AM did not increase the already high basal level of S1364 phosphorylation, probably because this phosphorylation was already maximal  to one with a high calcium concentration (DMEM) 24h before analysis . Here we maximal . In contThe changes in phosphorylation of T1736 in PA-JEB/β4 keratinocytes treated with BAPTA-AM or A23187 paralleled to some extent those by ERK1/2, which may point to a possible role of the MAPK signaling pathway in the phosphorylation of this residue. However, compared to the phosphorylation of S1364, which is known to be controlled by the MAPK signaling pathway i with BAPTA-AM enhances and prolongs EGF-induced ERK1/2 activation in mouse embryo fibroblasts [2+]i on the activation of ERK1/2 is caused by the induction of Ca2+-dependent expression of MAP kinase phosphatases [Calcineurin is a serine/threonine phosphatase that is activated by the calcium/calmodulin complex . Recent roblasts . Howeverroblasts . We confphatases , the appphatases ,36.Our finding, that RSK regulates the interaction between β4 and plectin via phosphorylation of T1736, combined with previous observations that ERK1/2 and RSK regulate the interaction between β4 and the ABD of plectin, implicate the ERK/MAPK signaling pathway as a crucial signaling cascade in keratinocyte migration through controlling HD stability. Indeed, inhibition of the MAPK pathway by UO126 prevents EGF-induced HD dissolution. Recent evidence suggests that HD stability may also be controlled by the ERK-MNK2 pathway through the phosphorylation of plectin at S4642 . This phIn conclusion, we provide evidence, that depending on the stimulus, PKD2 or RSK1 mediates phosphorylation of β4-T1736, a site that regulates the interaction of β4 with the plakin domain of plectin.


Surprisingly, the equivalent site on AMPK-α2, Ser491, is not an Akt target and is modified instead by autophosphorylation. Stimulation of HEK (human embryonic kidney)-293 cells with IGF-1 caused reduced subsequent Thr172 phosphorylation and activation of AMPK-α1 in response to the activator A769662 and the Ca2+ ionophore A23187, effects we show to be dependent on Akt activation and Ser487 phosphorylation. Consistent with this, in three PTEN (phosphatase and tensin homologue deleted on chromosome 10)-null tumour cell lines , AMPK was resistant to activation by A769662. However, full AMPK activation could be restored by pharmacological inhibition of Akt, or by re-expression of active PTEN. We also show that inhibition of Thr172 phosphorylation is due to interaction of the phosphorylated ST loop with basic side chains within the αC-helix of the kinase domain. Our findings reveal that a previously unrecognized effect of hyperactivation of Akt in tumour cells is to restrain activation of the LKB1 (liver kinase B1)–AMPK pathway, which would otherwise inhibit cell growth and proliferation.The insulin/IGF-1 (insulin-like growth factor 1)-activated protein kinase Akt  phosphorylates Ser 487, leading to reduced Thr172 phosphorylation and activation by LKB1. We establish a molecular mechanism for this effect and show that it causes down-regulation of AMPK in several PTEN-null tumour cell lines.Akt phosphorylates the α1 subunit of AMPK at Ser Incubations were stopped and analysed by autoradiography of membranes after electrophoretic transfer to detect 32P incorporation, followed by probing with the indicated antibodies.GSK3β , AMPK , were incubated with the indicated amounts of Akt in a final volume of 20 μl for 10 min at 30°C in the presence of 5 mM MgCl487) was incubated with the amount of LKB1 indicated in the Figure legends in a final volume of 20 μl for 10 min at 30°C in the presence of 5 mM MgCl2 and 200 μM ATP. AMPK activity was subsequently determined by transferring 5 μl from this reaction mixture to an AMPK assay, as described below. The remaining 15 μl was retained for analysis by Western blotting.AMPK . Stable cell lines were generated and cultured as described previously .HEK-293 cells, stably expressing AMPK  were grown to ≈80% confluence and then serum-starved for 16 h. Cells were then treated as described in the Figure legends. Pre-treatments with MK2206 (3 μM) were for 30 min. Pre-treatments with rapamycin (100 nM) were for 45 min. Incubations in the presence of 30 ng/ml IGF-1 were for 20 min. Treatments with A769662 (300 μM) were for 40 min (unless otherwise indicated) and those with berberine (300 μM) for 60 min.6-tag followed by a KpnI site, allowing insertion into pGEXKG . Cultures were grown at 37°C until the D600 reached ∼0.6, when cultures were induced with 1 mM IPTG and kept at 20°C overnight. The bacteria were pelleted by centrifugation , lysed under liquid N2 using a pestle and mortar, and resuspended in 50 mM Tris/HCl, pH 8.1, 500 mM NaCl, 20 mM imidazole and the EDTA-free protease inhibitor cocktail (Roche). The protein was purified using a HisTrap FF column . Fractions containing protein were pooled and incubated for 30 min at 30°C with 5 mM MgCl2 and 200 μM ATP-γ-phosphorothioate in the presence or absence of His6-tagged Akt. The mixture was then applied to a 1 ml GST FF column . After washing, the column was loaded with thrombin protease (Sigma) in 50 mM sodium/Hepes, pH 8, and 200 mM NaCl, capped, and left overnight at 4°C. Flow-through fractions containing cleaved phosphorylated or non-phosphorylated peptide was collected.DNA encoding residues 466–525 from human AMPK-α1 were amplified by PCR to include an N-terminal XhoI site and a C-terminal HisFor analysis of ACC (acetyl-CoA carboxylase) phosphorylation, SDS/PAGE was performed using precast Novex NuPAGE Tris-Acetate 3–8% gradient polyacrylamide gels in the Tris-Acetate SDS buffer system. For analysis of all other proteins, SDS/PAGE was performed using precast Novex NuPAGE Bis-Tris 4–12% gradient polyacrylamide gels in the Mops buffer system (Invitrogen). Proteins were transferred to nitrocellulose membranes (Bio-Rad Laboratories) using the Xcell blot module. Membranes were blocked in LI-COR Odyssey blocking buffer for 1 h, and detection performed using the appropriate secondary antibody coupled with IR680 or IR800 dye. Membranes were scanned using the LI-COR Odyssey IR imager.Unless stated otherwise, statistical significance was tested using GraphPad Prism 5 by one-way ANOVA, with Bonferroni's multiple comparison tests of the selected datasets as shown in the Figures.487 in human α1, and the equivalent site on α2 (Ser491). Both of the latter have a serine residue at P-2, with Akt having a preference for the serine or threonine residue at this position triphosphate) and Akt (thiophosphate being resistant to protein phosphatases), or left unphosphorylated. Consistent with our hypothesis, the phosphopeptide caused a concentration-dependent inhibition of activation and Thr172 phosphorylation of the bacterially expressed α1β2γ1 complex by LKB1, although we were unable to generate the peptide at high enough concentrations to fully saturate inhibition. By contrast, the unphosphorylated peptide had no effect , in the context of the recombinant human α1β2γ1 complex used in 7 by Akt D.485 on rat AMPK-α1 by Akt, within a rat α1β1γ1 complex, caused a 40% reduction in the rate of subsequent phosphorylation of Thr172 and activation by LKB1 in cell-free assays, an effect that was abolished by a non-phosphorylatable S485A substitution [487) on the human α1β2γ1 complex  is an extremely poor substrate for Akt, and that the very low level of α2 phosphorylation obtained using Akt is not affected by an S491A mutation (491. That Ser491 is modified instead by autophosphorylation is shown by the following findings: (i) substantial phosphorylation of Ser491 occurred with the recombinant human α2β2γ1 complex in the absence of exogenous kinases (491 phosphorylation did not occur with an inactive (D157A mutant) complex  n rat α1 D; and  does not enhance Ser487 autophosphorylation. Our results show that Ser487 in human α1, unlike Ser491 in α2, is a good substrate for Akt. Although only phosphorylated in cell-free assays at approximately 10% of the rate of Ser9 on GSK3-β , in the intact cells the net phosphorylation status would also be affected by the activity of protein phosphatases acting on Ser487. The results using MK2206 in 487 is phosphorylated in four distinct cell lines in an Akt-dependent manner.Although Sere assays A, increaerberine C, sugges487 on the human α1β2γ1 complex caused a 40% reduction in subsequent phosphorylation of Thr172 both in active .Surprisingly, we found that phosphorylation of Akt at the mTORC2 site, Ser172 phosphorylation induced by the activator A769662. However, this effect was reversed by the addition of MK2206, a selective inhibitor of Akt activation that also reduced or abolished the phosphorylation of AMPK-α1 on Ser487, of GSK3β on Ser9 and of Akt itself on Thr308 and Ser473. These effects could also be reversed in DBTRG-05MG cells by re-expressing WT PTEN, but not a phosphatase-inactive (C124S) mutant. Our results suggest that a previously unrecognized effect of PTEN loss is to reduce the potential restraint on cell growth and proliferation provided by activation of AMPK. This mechanism would also be expected to operate in tumour cells in which Akt is hyperactivated due to activating mutations in subunits of PI3K, or mutation or overexpression of upstream receptors [To place our studies in the context of tumour cells, we also addressed whether AMPK activation was down-regulated in three PTEN-null tumour cell lines derived from human cancers. Interestingly, in two glioblastoma cell lines and a breast cancer cell line in which Akt was hyperactivated due to loss of PTEN, AMPK was rather resistant to activation and Threceptors . Previoueceptors . In that487 inhibits subsequent phosphorylation of Thr172, and hence activation, by upstream kinases. In a partial α1β2γ1 complex containing rat α1, which was expressed in bacteria and where Ser485 was therefore most likely unphosphorylated, the ST loop from Glu469 to Val524 was not resolved, indicating that it was mobile within the crystal [487 is phosphorylated, hindering the ability of upstream kinases to gain access to Thr172. This hypothesis is supported by the results in 466 to Asp525 on human α1 inhibits activation and Thr172 phosphorylation of an α1β2γ1 complex by LKB1, but only when phosphorylated on the residue corresponding to Ser487. This is an extremely specific effect, because the phosphopeptide had no effect on the activation or phosphorylation by LKB1 of BRSK2, which (with BRSK1) has the kinase domain most closely related to AMPK-α1 and AMPK-α2 within the human kinome.Finally, our results suggest a molecular mechanism by which prior phosphorylation at Ser crystal . Our hyp487) and five threonine residues, most of which are conserved in α1 subunits from other vertebrates and in C. elegans . It has been shown recently that GSK3β phosphorylates the ST loop at multiple sites, with site-directed mutagenesis suggesting that the initial phosphorylation was at Thr481, followed by Ser477 and perhaps Thr473 . Thr481 phosphorylation was proposed to inhibit net Thr172 phosphorylation by enhancing its sensitivity to dephosphorylation [487 on rat AMPK-α1 might promote phosphorylation of Thr481, although not by conventional priming because the residue spacing is not appropriate, and because phosphorylation was not affected by a GSK3β mutation that reduces phosphorylation of primed substrates [487 may lead to additional phosphorylation events within the ST loop. This might explain why we observed a larger effect on AMPK activation and Thr172 phosphorylation by modulation of Akt in intact cells than in cell-free assays  interact with multiple phosphate groups attached to the ST loop, thus anchoring the ST loop to the kinase domain and blocking access of Thr172 to upstream kinases. Interestingly, although at least one of these (Arg64 or Lys71) is conserved in all 12 AMPK-related kinases, none are conserved in the archetypal serine/threonine kinase domain of PKA. Consistent with our hypothesis, a human α1β2γ1 complex containing an ‘AAA’ mutation (R64A/K71A/K74A) was completely resistant to the ability of previous Akt phosphorylation to reduce the rate of Thr172 phosphorylation by LKB1 (As an extension of this hypothesis, we propose that the side chains of three basic residues located in the αC helix of the small lobe of the kinase domain (Arg by LKB1 D. Also c by LKB1 C. Final 487 phosphorylation on replication of the hepatitis C virus [Since AMPK activators such as AICAR or metformin can overcome the inhibitory effects of Ser C virus , our pre C virus ,48, alth


GPS collared polar bears (Ursus maritimus) that move seasonally between sea ice and terrestrial habitats. We found the concentration of sea ice that bears experience at a local level, along with the duration of exposure to these conditions, was most associated with individual migration timing. Our results corroborate studies that assume thresholds of >50% sea ice concentration are necessary for suitable polar bear habitat; however, continued periods  of exposure to suboptimal ice concentrations during seasonal melting were required before the proportion of bears migrating to land increased substantially. Time‐to‐event models are advantageous for examining individual movement patterns because they account for the idea that animals make decisions based on an accumulation of knowledge from the landscapes they move through and not simply the environment they are exposed to at the time of a decision. Understanding the migration behavior of polar bears moving between terrestrial and marine habitat, at multiple spatiotemporal scales, will be a major aspect of quantifying observed and potential demographic responses to climate‐induced environmental changes.Migration phenology is largely determined by how animals respond to seasonal changes in environmental conditions. Our perception of the relationship between migratory behavior and environmental cues can vary depending on the spatial scale at which these interactions are measured. Understanding the behavioral mechanisms behind population‐scale movements requires knowledge of how individuals respond to local cues. We show how time‐to‐event models can be used to predict what factors are associated with the timing of an individual's migratory behavior using data from  Variation in animal distribution is largely a consequence of how species respond to temporal distributions of key resources. Measurements of population distributions relative to static or dynamic habitat characteristics can be achieved at numerous scales. Quantifying how and when animal populations respond to seasonal environmental shifts or long‐term habitat change often involves measurements of interacting factors over large geographic areas or landscapes  typically do not display gregarious behavior and therefore may have higher degrees of individual variability in the timing of migration events.In its simplest form, migration is a round‐trip movement between isolated areas at different times of the year  were deployed on adult females with either cubs‐of‐the‐year or 1‐year‐old cubs. Collars were programmed to obtain 1 GPS location every 4 h.Polar bear captures occurred on land in autumn 2004–2008 between Churchill and the Nelson River  and onset of risk was defined as May 1 of each year because this is immediately before sea ice generally begins to melt in Hudson Bay . Using a single GPS location per day ensured the temporal and spatial resolution of the collar data matched the SSM/I sea ice data. Mean springtime movement rate for adult female polar bears in western Hudson Bay is 31.2 km/day . Our time‐to‐event model used a daily time scale, and we subsampled the first available GPS location per bear each day. Local sea ice concentration at each bear location was approximated from daily Special Sensor Microwave/Imager (SSM/I) passive microwave data from the National Snow and Ice Data Center , it was necessary to consider the potential shape of our underlying baseline hazard function . We used Akaike's information criterion analysis, corrected for small sample size (AICc), to evaluate relative support for exponential models examining effects of environmental covariates. All combinations of main effects involving environmental covariates were tested. We also examined 2‐way interactions for environmental covariates; however, interactions were only added to models including both corresponding main effects. Once the best‐fitting model using environmental covariates was identified, we used this model in a second stage of AICc analysis that examined combinations of proposed individual characteristics to determine whether model fit was improved.r| >0.65. We also visually inspected plots of Martingale residuals for each covariate to assess covariate functional forms and ensure our data met proportional hazard assumptions . Based on our projection of reproductive status, 13 bears had 1‐year‐old cubs and 8 bears were alone during migration events. The mean date bears began to head for shore was July 13 . There was an average of 5.8 (SE = 0.9) days between the date polar bears began a directional migration toward shore and their first recorded location on land. When polar bears began a directional migration toward shore, they were a mean distance of 80.4 km  from the coastline.P < 0.001, 95% CI = 0.90–0.96).We found similar support ∆AICc <2) for the time‐to‐event model examining environmental covariates that used local sea ice concentration by itself and models that combined local sea ice concentration with either distance to shore or sea ice rate of change Table . However for the time = 1.04, SE = 0.03, P = 0.09, 95% CI = 0.99–1.10) or the global model assessing effects of individual characteristics , which supports our assumption of an underlying exponential baseline hazard. In addition, the coefficients from global exponential models had overlapping 95% confidence intervals with those of equivalent semi‐parametric Cox proportional hazards models, indicating that we attained well‐fitting parametric models.Examination of the Martingale residuals indicated the functional forms of the covariates did not require transformation to meet proportional hazard assumptions for our exponential model  movements at different spatial scales and suggest that understanding which scales animals respond to provides insight into mechanistic reasons for movement between habitat types. For polar bears, fine‐scale and daily measurements of sea ice rate of change may vary rapidly and temporarily based on localized wind events and habitat heterogeneity, making the rate at which ice changes at the local level less likely to influence the timing of individual migration. In contrast, the rate of sea ice change at the landscape scale is likely a good indicator of how soon the population's marine habitat will be completely ice free.In contrast to research examining polar bear migration at the landscape scale (i.e., Cherry et al. Our measurements of daily distance to shore also did not influence the predictability of a polar bear's migration toward land. Sea ice in Hudson Bay mostly occurs along the western and southwestern coasts during later stages of breakup (Wang et al. Neither age nor reproductive status affected variation in timing of directional movement toward land. Our findings are similar to other studies that suggest female polar bears with older offspring do not have significantly decreased mobility, even during the breakup period (Ferguson et al. In our study, we demonstrate the importance of examining how individuals respond to localized daily environmental conditions and thus provide a more mechanistic understanding of spatial dynamics at the population level. For western Hudson Bay polar bears, continued monitoring of the relationship between exposure length to various environmental conditions and individual migration timing may be a key aspect of quantifying behavioral changes as a result of future climate change. Additional monitoring of polar bear movement behavior with improved GPS collar technology will also allow for an increased sample size and provide an opportunity to incorporate a higher number of potential environmental factors into time‐to‐event models. The longer‐term projections for sea ice conditions in Hudson Bay suggest the duration of the on‐land period will increase with negative demographic consequences (Molnár et al.


The engraved trails of rocks on the nearly flat, dry mud surface of Racetrack Playa, Death Valley National Park, have excited speculation about the movement mechanism since the 1940s. Rock movement has been variously attributed to high winds, liquid water, ice, or ice flotation, but has not been previously observed in action. We recorded the first direct scientific observation of rock movements using GPS-instrumented rocks and photography, in conjunction with a weather station and time-lapse cameras. The largest observed rock movement involved >60 rocks on December 20, 2013 and some instrumented rocks moved up to 224 m between December 2013 and January 2014 in multiple move events. In contrast with previous hypotheses of powerful winds or thick ice floating rocks off the playa surface, the process of rock movement that we have observed occurs when the thin, 3 to 6 mm, “windowpane” ice sheet covering the playa pool begins to melt in late morning sun and breaks up under light winds of ∼4–5 m/s. Floating ice panels 10 s of meters in size push multiple rocks at low speeds of 2–5 m/min. along trajectories determined by the direction and velocity of the wind as well as that of the water flowing under the ice. Racetrack Playa in Death Valley National Park, is well known for the phenomenon of tracks left by hundreds of rocks plowing across the nearly flat playa surface . Rock moAll authors agree that rocks are most likely to move when the playa surface is wet, creating a slick surface, and that wind must be involved. The first scientific study of the Racetrack suggested that rock motion was driven by dust devils Other authors, led first by Stanley In a test of the ice sheet hypothesis, R. Sharp and Carey To describe the meteorological conditions on the playa and the velocities and timing of rock motion we installed a weather station adjacent to Racetrack Playa, several time lapse camera systems overlooking the southeast corner of the playa The weather station was obtained from the Sweeney Granite Mountains Desert Research Center  and continuously recorded wind speed at 1 second intervals, along with temperature, insolation, rainfall, and GPS location. We anchored the weather station to the alluvial fan surface with sand bolts at N36.6823, W117.5515, northeast of the largest concentration of rocks on Racetrack Playa . This loCustom-built GPS loggers engineered by Interwoof were placed in limestone blocks of varying sizes and located on Racetrack Playa to the northeast of the largest concentration of natural stones . Instrumwww.erh.noaa.gov/box/tables/snowfall-meltwater.html), the total precipitation was ∼5.64 cm. The resulting pond repeatedly froze as nighttime temperatures dipped below freezing for most days to the end of our observations on January 9, 2014 when the pond still covered ∼1/4 of the playa surface. The pond eventually evaporated completely by the second week in February, 2014.We recorded rock movement associated with a shallow pond (∼10 cm maximum depth) that existed on Racetrack Playa between late November 2013 to early February 2014 . Our weaObserved rock movement occurred on sunny, clear days, following nights of sub-freezing temperatures. Steady light winds and morning sun caused floating ice to break-up near mid day, accompanied by widespread popping sounds from fragmenting ice panels. Ice initially broke into floating panels tens of meters in size that became increasingly fragmented and separated by open rippled water as melting continued. Floating ice sheets driven by wind stress and flowing water, pushed rocks resting on the playa surface, in some cases moving >60 rocks in a single event .Rocks move slowly and somewhat episodically during move events. For instance, on January 9, 2014, a rock was observed to move at ∼1–2 m/minute for about 18 seconds at 12:50 pm . This roOur instrumented rocks recorded movement events on December 4 and December 20, 2013 . Two rocFloating ice often fragments upon encountering rocks, producing wakes of open water filled with ice chips downstream of both stationary and moving rocks . Ice panIce movement produces rock trails that can be startlingly similar as seen in the December 20 event . Rock trA surprise is the thinness of ice involved in rock movement. Ice sheets 3–6 mm thick are insufficient to float rocks off the playa surface, as proposed in some models Rock movement is correlated with wind-driven transfer of water from the southern zone of the playa to the northern margin under sustained light winds of 3.0–4.5 m/s . These wTime-lapse images acquired since 2007, typically from late November to early March, show that the conditions we have observed are quite rare. Snowfall on January 2–3, 2011 and February 27, 2011 blanketed the playa for a couple of days but did not lead to flooding. Hills near the playa received a couple of dustings of snow in early 2012, but the south end of the playa did not experience more than a brief sheen of moisture. Winter 2012–2013 was likewise essentially dry. Although a ∼30 day period of flooding occurred in the late winter of 2010 A persistent pond can support multiple movement events; our GPS instrumented rocks have recorded at least two separate move events with total trail lengths for a single stone of up to 224 m . Our weaA surprising finding is the power of even thin sheets of ice to move large stones, without buoyant uplift A necessary condition for the rock motion we observed is the existence of a playa pool deep enough to submerge the southern section of the playa, yet shallow enough to leave many rocks partly exposed at the pond surface. Other repeating features of rock movement events that we observed include the presence of floating ice, temperatures and sunlight sufficient to create melt pools in the ice, and light breezes that are steady enough to drive floating ice. Although the ice breaks up around rocks, even thin moving ice sheets can generate sufficient force to drive rocks across the pool. All observed rock movement events occurred near mid-day when sufficient ice melting had occurred to allow ice break-up. Creation of rock trails is difficult to observe because trails form below the ice-covered pool surface where they are often not evident until the ice has melted, and liquid water has been removed. In addition, rock movement is slow and relatively brief—our GPS instrumented stones traveled at speeds of 2–5 m/minute for up to 16 minutes—so casual observation is likely to miss rocks in motion. Weather station data show that the freezing temperatures necessary for ice formation, and winds in excess of 3–5 m/s are common phenomena at Racetrack Playa during the coldest few weeks of winter. Therefore, the extremely episodic occurrence of rock motion (years to decades) is likely due to the infrequency of rain or snow events sufficient to form winter ponds.Table S1Weather data collected from Racetrack Playa, Death Valley National Park. Data period: Nov-20-2013 to Jan-9-2014. Records of hour-total rainfall (column 2), as well as hourly average insolation (column 3), air temperature (column 4), and wind velocity (column 5) with the time stamp given in column 1. The record of maximum wind gust strength (in column 7) is calculated to the nearest minute with a time stamp given in column 6. Station located at N36.6823, W117.5515. Instrument package specifications reported in the text and table header.(CSV)Click here for additional data file.Table S2Movement data for GPS-instrumented rocks on Racetrack Playa, Death Valley National Park. Data obtained for three rocks  that recorded position and velocity. For each rock, movement data are date (column 1), time stamp , latitude , longitude , and velocity . Rocks A3 and A6 had total trail lengths longer than recorded by their GPS instrument packages Click here for additional data file.


Based upon a thorough review of published clinical observations regarding the inhibitory system, I hypothesize that this system may play a key role in the pathogenesis of a variety of neuromuscular and neurological diseases. Specifically, excitatory overstimulation, which is commonly reported in neuromuscular and neurological diseases, may be a homeostatic response to inhibitory overstimulation. Involvement of the inhibitory system in disease pathogenesis is highly relevant, given that most approaches currently being developed for treating neuromuscular and neurological diseases focus on reducing excitatory activity rather than reducing inhibitory activity. In contrast, neurological diseases manifest primarily as a progressive decline in cognitive function. Interestingly, the clinical manifestations of neuromuscular and neurological diseases also overlap; this overlap is summarized in29. The clinical features shared between the neuromuscular disease ALS and the neurological disease FTD exemplify this overlap, as late-stage ALS can lead to the manifestation of FTD; conversely, FTD can progress to ALS, leading to the manifestation of FTD-ALS8.The pathogenesis of most neuromuscular and neurological diseases is poorly understood, despite their devastating impact on quality of life and the fact that they were first described more than a century ago. Clinically, neuromuscular diseases manifest as progressive muscle weakness together with a general set of motor symptoms, including speech-related difficulties, impaired mobility, and reduced fine motor skills29. Glutamatergic  overstimulation induces excitotoxicity in cultured neurons and is believed to be an important factor in the pathogenesis of both neuromuscular and neurological diseases29. Glutamate-induced excitotoxicity can result in the decay of neuronal pathways that innervate muscles and other physiological systems29. This decay gives rise to the loss of physiological function and is considered to lead to the clinical manifestations that present with both neuromuscular disease and neurological disease29. I hypothesize that these increased glutamate levels are actually a homeostatic response to an overstimulated inhibitory nervous system. This novel hypothesis is based upon the observation that the clinical findings in neuromuscular and neurological diseases can be explained by inhibitory activity, as discussed below.A key observation gleaned from analyzingSince they were first diagnosed more than a century ago, the clinical manifestations of neuromuscular and neurologic diseases have been well described. Strikingly, however, the consequences of one key clinical feature of these diseases—the absence of an elevated risk of seizure activity—have been largely overlooked.9. Moreover, a thorough search of PubMed for articles published from 1966 through 2016 using the key words “seizure” or “epilepsy” in combination with “amyotrophic lateral sclerosis” or “ALS” confirms the striking absence of epilepsy and/or seizures in ALS patients. This finding is consistent with the absence of seizures and/or epilepsy in review articles describing the clinical manifestation of ALS6.This is exemplified for ALS in which a broad, detailed retrospective study of the medical records of 657 ALS patients revealed that none of the patients presented with epilepsy as a co-morbid condition23. Importantly, increased glutamate levels are generally associated with epileptic seizures31. Thus, given the increased glutamate levels typically measured in the CSF of ALS patients, one would logically expect that the prevalence of epilepsy in ALS patients should be elevated relative to the general population. However, despite this expectation, epileptic seizures are simply not reported among ALS patients.A key observation that makes the absence of seizure activity in ALS even more remarkable is increased glutamate levels in the cerebrospinal fluid (CSF) of patients with ALS; on average, glutamate levels in the CSF of ALS patients are increased by 100%, and some ALS patients can have an increase of up to 800%15, even though all eight diseases present with elevated glutamate levels in the CSF29. With respect to Alzheimer’s disease, patients in the early stages of the disease occasionally develop seizures32; however, this seizure activity decreases as the disease progresses from the early stages to more advanced stages33.Strikingly, in addition to ALS, none of the other seven diseases listed in23 and complete muscle inhibition in the end stages. Although fasciculation and/or cramps can be observed in ALS patients4, these features occur in debilitated muscles as they progress from a fully functional state toward a fully inhibited state.A second key observation is that neuromuscular and neurological diseases have an inhibitory effect on muscle function, rather than being excitatory. The diseases listed ini) functions to oppose the glutamatergic excitatory neurotransmitter system,ii) inhibits muscle activity by reducing the firing rate of motor neurons, andiii) exerts strong anticonvulsive activity31.One possible explanation for these seemingly contradictory findings is a second system that exerts strong anticonvulsive activity in both neuromuscular and neurological diseases. Importantly, such a system should be as widespread throughout the nervous system as the glutamatergic system, and the inhibitory system fulfills these requirements. Specifically, the inhibitory (GABA) system35. In addition, fatal respiratory depression can be induced by administering an overdose of the GABAergic benzodiazepine midazolam36. Chronically stimulating the inhibitory system can cause chronic muscle disuse that can lead to muscle atrophy37. Moreover, ingestion of alcohol (another GABAergic inhibitory compound38) impedes coordination and causes slurred speech (dysarthria), which are features of neuromuscular and neurological diseases. In cats, dysphagia  can be either induced or reversed using GABA agonists or GABA antagonists, respectively39. Dysphagia has also been reported in humans following the administration of either benzodiazepines42 or alcohol43. Administration of benzodiazepines reduces voluntary saccadic eye movement function44 and increases EEG beta-wave activity44, clinical manifestations that also occur in neuromuscular and neurological diseases46. Increased GABAergic inhibitory activity can also cause bladder48 and gastrointestinal dysfunction50, both of which can manifest in neuromuscular and neurological diseases21. Interestingly, Bravoet al. reported that chronically feeding mice the lactic acid bacteriumL. rhamnosus increases expression of GABA receptors, suggesting a link between GABAergic activity and CNS disorders51. Strikingly, GABAergic activity can also explain the overlapping clinical manifestations between Alzheimer’s disease and alcohol-related dementia52, and it can explain the increase in dementia-like symptoms observed after the administration of the benzodiazepine diazepam53. Inhibitory activity can also explain neuromuscular and neurological disease predisposition in the elderly, as the sensitivity to GABA inhibitory activity is known to increase with age54. Finally, GABAergic activity has been implicated in cognitive dysfunction57, which is a hallmark feature of neurological diseases and is often observed in late-stage neuromuscular disease7. Taken together, these findings support the notion that the clinical features associated with neuromuscular and neurological diseases can be induced by activating the inhibitory system.A third key observation is that the clinical manifestations of neuromuscular and neurological diseases can be induced using interventions that increase GABAergic  activity . For exa58. A similar effect was reported in patients with ALS who received the GABAergic compound topiramate59. GABAergic action can also explain the more rapid disease progression of ALS in clinical trials in which patients received the antibiotic minocycline60, which has GABAergic activity61. Finally, GABAergic involvement can explain the observed efficacy of the taurine conjugate form of ursodeoxycholic acid (UDCA) in ALS patients62, as UDCA inhibits GABAergic action63.In addition to mimicking the majority of clinical manifestations observed in neuromuscular and neurological diseases, GABAergic activity can also explain the more rapid disease progression of ALS reported in clinical trials in which patients received GABAergic compounds. For example, in two trials gabapentin increased the rate of disease progression in patients with ALSA fourth key observation is that the clinical manifestations associated with neuromuscular and neurological diseases can be attributed to the activity of either simple inhibition (SI) or recurrent inhibition (RI) pathways. Specifically, I postulate that differences between muscles under the control of SI and/or RI underlie the important—yet poorly understood—manifestations of neuromuscular and neurological diseases.64. The RI system controls physiological functions that play a role in counteracting gravitational forces and other external forces acting on the body. During locomotion and/or to counteract the effects of gravity, RI uses a negative inhibitory feedback loop , bulbar symptoms (bulbar-onset ALS), or respiratory symptoms (respiratory-onset ALS), respectivelylvement . Patientlvement . Thus, S4. As discussed above, increased activity of both the SI and RI pathways leads to fatal respiratory depression, the principal cause of early death in patients with ALS.The differential involvement of the SI or RI system can also explain the observed differences in life expectancy between patients who present with limb-onset, bulbar-onset, or respiratory-onset ALS. Specifically, patients with respiratory-onset ALS generally have the shortest life expectancy following diagnosis4, which lies in the middle of the spectrum.Increased activity of either the SI or RI pathway—but not both—can also lead to respiratory depression, albeit not to fatal levels. Under these conditions, respiratory function, though impaired, can be maintained by either SI or RI pathway activity. However, due to impaired respiratory function resulting from either SI or RI overstimulation see, these p7. Alternatively, if SI overstimulation precedes RI involvement, FTD is the initial diagnosis, and the disease can progress to FTD-ALS when the RI pathway becomes involved. Moreover, the division between SI and RI can also explain the overlap between subcategories of ALS and FTD with respect to impaired cognition and altered behavior that involve SI, and movement dysfunction that involves RI.Differences between the effects of SI versus RI involvement can explain the fact that although ALS and FTD generally involve two distinct systems, these two diseases have a certain degree of overlap with respect to their clinical manifestations see. Thus, iAlthough it is generally considered one disease, ALS can present with a wide spectrum of clinical manifestations, and this spectrum can be explained by the involvement of SI and/or RI pathways. For example, SI overstimulation can lead to bulbar, cognitive, and frontotemporal dementia-related manifestations without causing severe muscle wasting or respiratory malfunction . On the other hand, RI overstimulation can lead to locked-in syndrome, a state in which the patient retains cognitive and emotional function but becomes “locked” in their body, with all of the muscles that counteract gravity and other external forces rendered essentially dysfunctional. Interestingly, the only muscles that are spared in locked-in syndrome—and the only way in which end-stage patients can communicate with the outside world—are the muscles that control eye movement. This is an important observation, given that the muscles that control eye movement are not controlled by RI pathways see. The dis66. With split-hand syndrome, the abductor pollicis brevis (APB) and first dorsal interosseous (FDI) muscles are affected, whereas the abductor digiti minimi (ADM) muscle is relatively spared. This syndrome is particularly puzzling, as these muscles are innervated identically, yet are affected differently66. I propose that split-hand syndrome can be attributed to differences in the extent to which RI pathways innervate the hand muscles that are involved in precision gripping, versus muscles that also play a role in power gripping. With precision gripping , the fingers and thumb press against each other; this type of grip does not involve lifting a relatively heavy object67. In contrast, power gripping  uses the fingers, palm, and thumb to clamp down on a heavy object in order to lift and control the object67. Napier67 used this distinction to distinguish muscle activities that are involved in body locomotion and/or posture from muscle activities that do not involve locomotion or posture. Thus, Napier’s separation also categorizes muscle activities into those that are controlled by RI and those that are controlled by SI. Because the primary function of the ADM muscle is to move the little finger  away from the hand, the ADM muscle is only involved in precision gripping and would therefore not be affected by RI overstimulation. This is consistent with the reported absence of RI in motor neurons that innervate the ADM68. On the other hand, the APB and FDI muscles are involved in the opposition and extension of the thumb and are therefore involved in power gripping64; thus, these two muscles are affected by RI overstimulation.Split-hand syndrome is common among patients with ALS69. However, because these tremors occur at rest (and not during active motion or while countering the effects of gravity), the muscles involved are likely innervated by SI pathways, making rest tremors an SI-specific phenomenon. This is further illustrated by the finding that rest tremors resolve when the affected body part becomes involved in locomotion, stance, or maintaining inertia69, actions that involve muscles that are controlled by RI64. Interestingly, the hand tremor that is most specific to patients with Parkinson’s disease—the so-called “pill-rolling tremor”—also results from muscles that are innervated solely by SI pathways. Specifically, the pill-rolling tremor involves muscles that play a role in precision gripping but not in power gripping, a distinction that is highly reminiscent of split-hand syndrome in ALS (see above). Furthermore, involvement of the inhibitory system in Parkinson’s disease rest tremors is supported by the observation that the rest tremors observed in restless legs syndrome resolve after the administration of quinine , a compound that reduces inhibitory activity70.In Parkinson’s disease, rest tremors arise from involuntary rhythmic oscillatory movements of a body part at rest; these tremors stop when the patient actively moves the affected body part. The pathways that underlie rest tremors have not been identified, and the fact that rest tremors resolve during voluntary movement is one of the most puzzling observations associated with Parkinson’s diseaseThe differentiation between SI and RI summarized inDifferential SI and RI involvement can also account for the wide variety of clinical manifestations in neuromuscular and neurological diseases during disease progression. As a group, neuromuscular and neurological diseases present with a wide spectrum of clinical manifestations see, and stiFinally, the differentiation between SI and RI can help explain the differences in life expectancy among patients with various neuromuscular and neurological diseases. As discussed above, increased activity of both the SI and RI pathways leads to fatal respiratory depression see, the pri31. Neurons that receive excessive excitatory stimulation can subsequently become overstimulated by inhibitory transmission, and vice versa. This raises the intriguing question of which system in neuromuscular and neurological diseases is overstimulated first, and which system becomes overstimulated as a homeostatic response. This question has been addressed with respect to epileptic seizures30. With respect to neuromuscular and neurological diseases, it is important to note that the administration of glutamatergic excitatory compounds does not lead to the clinical manifestations summarized incan give rise to the clinical manifestations in30.Taken together, the wealth of observations discussed above suggest that the opposing excitatory and inhibitory systems may play a role in the pathogenesis of the same disease. This phenomenon has precedent, as inhibitory/excitatory homeostasis processes are also involved in seizure activity72. Thus, inhibitory activity can occur without inducing pronounced sedation. This is supported by reports that benzodiazepine-induced dysphagia can occur even in non-sedated patients42. Second, the absence of seizures despite high glutamate levels could be due to a slow, but non-epileptogenic increase in glutamate levels during the progression of neuromuscular and neurological diseases. However, even small increases in glutamate levels can increase glutamatergic synchronization of a small subset of critical neurons, thereby leading to epileptic activity31. Moreover, epileptic seizures are simply not reported among ALS patients, an observation that cannot be explained by a slow increase in glutamate levels, as glutamate does not cause inhibitory activity29. Furthermore, slow increasing levels of glutamate in the absence of seizure activity may reflect the involvement of an inhibitory homeostatic process31. Finally, the differentiation between SI and RI depicted inOther possible interpretations of the findings summarized inbefore neurons have undergone cell death. The implication of this possibility is that decreasing inhibitory activity may be beneficial in terms of slowing—or even preventing—the progression of neuromuscular and neurological diseases. Compounds that can reduce inhibitory activity are currently available; unfortunately, however, these compounds can induce seizure activity and are therefore not used therapeutically. Nevertheless, their potential for preventing the pathogenesis of neuromuscular and neurological diseases suggests that compounds that target the inhibitory system could be developed for clinical applications. For example, the average life expectancy of a patient with ALS is 3–5 years after onset, and most neuromuscular and neurological diseases are severe and ultimately fatal. With respect to Alzheimer’s disease and Parkinson’s disease, dysphagia and respiratory depression–related aspiration pneumonia are the most common causes of death17. Neuromuscular diseases also present with the severe and potential fatal clinical manifestations listed in21 thus, the ability to prevent these symptoms could significantly prolong the life expectancy of these patients. From a treatment perspective, it is interesting to note that the selective GABA antagonist SGS-742 has been shown to be both clinically feasible and safe55.From a clinical perspective, an important consequence that emerges from the inhibitory overstimulation hypothesis is that the clinical manifestations summarized inBased upon the plethora of observations regarding the inhibitory system, I hypothesize that this system plays an important role in the pathogenesis of both neuromuscular and neurological diseases. Importantly, overstimulation of the inhibitory system can explain both the absence of epileptic seizures despite the elevated glutamate levels and the pharmacological induction of symptoms present in patients with neuromuscular and neurological diseases. Moreover, the separation between SI and RI can account for the various categories of clinical manifestations observed in these patients. Specifically, I hypothesize that increased glutamate levels in neuromuscular and neurological diseases are actually a homeostatic response to an overstimulated inhibitory system. Implicating the inhibitory system in the pathogenesis of neuromuscular and neurological diseases is highly relevant, given that the majority of approaches being developed for treating these diseases focus on reducing glutamatergic activity, rather than reducing inhibitory activity. Moreover, this putative connection between the inhibitory system and neuromuscular/neurological diseases may have long-reaching implications, including the need to develop therapies designed to reduce inhibitory overstimulation in neuromuscular and neurological patients. I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. The opinion paper by Tuk provides an overview of the inhibitory system involved in neural signal transduction. Based on an extensive review of the literature the author provides a number of challenging but well-reasoned hypotheses about the impact of this system on both neuromuscular and neurological diseases. This potential connection may have important implications for the understanding and future treatment of these so far  untreatable diseases. Only few minor points should be addressed.  The use of the term ‘inhibitory system’ is somewhat confusing – the context is often not specified (inhibitory system of what…) although the author means neural signal transduction – however,  this should be mentioned where relevant and certainly in the title.et al. (2011).The link between GABA production by intestinal bacteria and gut GABA receptor stimulation could be further addressed as it provides a way how other factors such as food or the gut microbiome may modulate GABA response . Based on the enormous increase of glutamate levels in ALS patients and the absence of elevated risk for epileptic activity in that population, there is still good support for postulating the theory of the inhibitory system as a plausible trigger for these diseases, but I propose that that section of the article and Table 1 be revised to reflect that nuance. I recommend that the article be indexed, with the minor revision outlined above.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.


CYP3A4 is recognized as the main enzyme involved in the metabolism of drugs and xenobiotics in the human body and its inhibition may lead to undesirable consequences. Stilbenes, including resveratrol, belong to a group of dietary health-promoting compounds that also act as inhibitors of CYP3A4. The aim of this study was to examine the use of computer modeling of enzyme-ligand interactions to analyze and predict the inhibition of structurally related compounds. To this end, an aldehyde group was attached to resveratrol and the interactions of CYP3A4 with resveratrol, its aldehyde analogue (RA) and a known synthetic inhibitor were studied and compared in two biological models. Specifically, the metabolism of testosterone was examined in a human intestine cell line (Caco-2/TC7) and in rat liver microsomes (RLM). The results demonstrated a weak inhibitory effect of RA on CYP3A4, as compared to resveratrol itself, in both biological models. Human CYP3A4 was more susceptible to inhibition than the commonly used model isozyme from rat. Modeling of the binding site of CYP3A4 revealed a combination of three types of interactions: hydrophobic interactions, electrostatic interactions and hydrogen bonds. A docking simulation revealed that the RA lacked an important binding feature, as compared to resveratrol, and that that difference may be responsible for its lower level of affinity for CYP3A4. Software analysis of binding affinity may serve as a predictive tool for designing new therapeutic compounds in terms of inhibition of CYP3A4 and help to reveal the biochemical nature of the interactions of dietary compounds, herbal compounds and drugs whose metabolism is mediated by this enzyme. The cytochrome P450 enzymes (P450s) are responsible for the metabolism of a wide range of endogenous compounds , as well as xenobiotics including drugs, environmental pollutants and dietary products –3. Of tht-resveratrol  was purchased from . All other chemicals and reagents were purchased from Sigma Aldrich, unless otherwise noted.Resveratrol , testosterone (99%) and ketoconazole (98%) standards were purchased from Sigma Aldrich, Israel. Testosterone  and 1.5 mM NADPH. Resveratrol, RA and ketoconazole were first dissolved in 30% methanol and sonicated, then stocks solutions were prepared in reaction buffer (maximum methanol concentration in each well was 0.95%). The different treatments were added to the wells to achieve final concentrations of 2, 5, 10, 25, 50, 75 and 100 μM for resveratrol and RA, and 2.5, 25, 50, 100 and 200 μM for ketoconazole. All treatments were assayed in triplicate with the following controls: a buffer control containing the reaction buffer instead of the inhibitor (no treatment), a methanol control containing 0.95% methanol instead of the inhibitor (to account for possible effects of this solvent) and an RLM control without any microsomes, to provide the background value. Reactions were then incubated for 4 h with continuous shaking at the optimal temperature of 37°C. The addition of 1 ml of ethyl ether followed by vortex mixing was used to stop the reactions. Ether and aqueous layers were allowed to separate for 10 min, and then transferred to -80°C for 15 minutes for a complete freeze of the aqueous phase. The aqueous phase was left for 1 h in the chemical hood for a complete evaporation of ether traces. The aqueous phase was shacked with 2 volumes (400 μl) of dextran coated (0.5%) charcoal slurry  for 1 min and centrifuged at 3000 g at 4°C for 5 min. The supernatant (300 μl) was collected into scintillation vials and mixed with 5 ml scintillation fluid. 3H was measured as disintegration per 5 min using a liquid scintillation β counter .The interaction of CYP3A4 with testosterone was examined using the tritiated water assay adapted from González and Piferrer . ReactioThe crystal structure of human CYP3A4 was determined with ketoconazole , availabTestosterone metabolism mediated by CYP3A4 was assessed based on the quantification of testosterone retained in the medium after incubation with Caco-2/TC7 cells. Testosterone metabolism was initially followed over a 24-h incubation period  and a 6-The effects of resveratrol and RA on the viability of Caco-2/TC7 cells were measured as described. Those measurements revealed that resveratrol did not affect the cells’ viability within the examined concentration range, but that amending the medium with 100 μM RA did reduce cell viability. None of the other examined concentrations of RA reduced cell viability . Thus, 5CYP3A4-mediated metabolism of testosterone in Caco-2/TC7 cell was recorded in 2-h intervals and during 6-h periods, based on the pattern shown above . Both re3H(N)] mediated by CYP3A in the RLM. The inhibition rate is expressed as a percentage relative to that of the control. The ketoconazole treatment represents the highest inhibition capacity  served as a model for that of resveratrol and RA. In order to validate the suitability of the selected docking model, we first docked ketoconazole and ensured that its bonding to the CYP3A4 binding site was restored to the initial state, as in the original 2V0M structure. According to the ketoconazole docking simulation, the binding site can be thought of in terms of three main features: a solid hydrophobic region, H-bond donors/acceptors and electrostatic interactions. The hydrophobic cluster includes the alkyl and alkyl-pi interactions between CYP3A4 residues Leu210, Phe241, Ile301, Ala305 and Leu482 and the chlorinated aromatic ring and imidazole ring of the ketoconazole molecule. The main H-bond donor residue is CYP3A4 Arg372, which interacts with the ketonic oxygen atom of ketoconazole; whereas the oxygen atoms of Ala370 serve as H-bond acceptors. The nitrogen atom of the heme group provides further anchoring to the molecule through its electrostatic interactions (anion-pi) with the imidazole ring of the ketoconazole Figs . A resvet-resveratrol on CYP3A4, both in vitro and in vivo, are well documented in the literature [50 values and greater affinity for CYP3A4, as compared to the parent resveratrol and its glucosides [Among the members of the P450 enzyme family, CYP3A4 is recognized as the main enzyme involved in the metabolism of drugs in the liver and, no less importantly, in the gut , 8, 52. terature –22 and cterature . A previucosides . QSAR anucosides , 35, 55.50 value of resveratrol (28 μM) was found to be lower than that of RA (51 μM) and ketoconazole exhibited the strongest inhibitory effect (IC50 = 14 μM). The IC50 value of resveratrol for CYP3A falls in the range found in the literature for RLM models [50 values of ketoconazole (3.7–13 μM) have been reported previously [50 values were 14 μM for ketoconazole and 28 μM for resveratrol. Piver et al. reported a similar conclusion when the resveratrol IC50 value for CYP3A-mediated testosterone 6β-hydroxylation in RLM was observed to be much higher than that in human liver microsomes (20 versus 4 μM) [Results of the examination of the inhibition of testosterone metabolism in the Caco-2/TC7 cells line reveal inhibitory effects of both resveratrol and RA on CYP3A4. The residence time of substances from ingested foods, drugs and others is estimated to be 1.5–2 h in the Jejunum and 5–7 h in the Ileum . The metM models , 57 and eviously , 58, 59.us 4 μM) . Furtherus 4 μM) –62.50 values for CYP3A4 and resveratrol and its analogs. In that work, methoxy-stilbenes, resveratrol and resveratroloside were found to have partition coefficients of 2.51, 1.87 and 0.89 and IC50 values of 0.47 μM, 1.4 μM and >40 μM, respectively [There are no remarkable differences between the logP values of ketoconazole, resveratrol and RA . Howeverectively , 63.50 values and CDOCKER energies, which were found to be in reverse order to their logP values. In line with the logP values, ketoconazole had the highest lipophilicity and the highest number of hydrophobic interactions in the binding site of CYP3A4  may prove to be quite useful for predicting CYP3A4 inhibition by candidate therapeutics, as well as interactions between consumed phytochemicals and prescribed drugs. Moreover, integration of results from various enzymatic models and computer modeling of docking promotes comprehensive understanding of these interactions.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file.


Knowledge of glomerular filtration rate (GFR) is required to detect and follow impairment of renal function, to allow correct dosage of drugs cleared by the kidneys, and for the use of nephrotoxic contrast media. Correct determination of GFR requires invasive techniques, which are expensive, slow and not risk-free. Therefore, GFR-prediction equations based solely upon cystatin C or creatinine and anthropometric data or upon cystatin C, creatinine and anthropometric data have been developed. The combined prediction equations display the best diagnostic performance, but in several easily identifiable clinical situations  prediction equations based upon either cystatin C or creatinine are better than the combined equations. In Lund, where cystatin C has been used as a GFR-marker in the clinical routine since 1994, a strategy based upon this knowledge has therefore been developed. This comprises simultaneous use of a cystatin C-based and a creatinine-based GFR-prediction equation. If the GFRs predicted agree, the mean value is used as a reliable GFR-estimate. If the GFRs predicted do not agree, clinical data is evaluated to identify reasons for not using one of the two prediction equations and the GFR predicted by the other one is used. If no reasons for the difference in predicted GFRs are found, an invasive gold standard determination of GFR is performed. If the GFRs predicted agree for a patient, the creatinine value is reliably connected to a specific GFR and can be used to follow changes in GFR of that patient. However, these so called gold standard techniques cannot be generally applied because they are labour-intensive, expensive and not entirely free of risk for the patient. The plasma or serum concentrations of endogenous substances, particularly creatinine, have therefore been used as markers for GFR for almost a century. But it has become evident that the creatinine level alone is far from ideal as a GFR marker because it is significantly influenced not only by GFR but also by muscle mass, diet, gender, age, drugs and tubular secretion [Knowledge of glomerular filtration rate (GFR) is required to detect and follow impairment of renal function, to allow correct dosage of drugs cleared by the kidneys and for the use of potentially nephrotoxic radiographic contrast media. GFR in humans cannot be measured directly and invasive techniques based on measuring the plasma or renal clearance rate of injected substances , were somewhat higher than that between creatinine and GFR [www.ncbi.nlm.nih.gov/pubmed in February 2010 generated 1031 hits.Cystatin C was first suggested as a new marker for GFR in 1979, when it was observed that the plasma level of cystatin C was up to 13 times higher in patients on haemodialysis than in healthy persons . One of  and GFR . However and GFR  and the The main advantage of cystatin C compared to creatinine as a GFR-marker is that it is less dependent upon the body composition of a patient than creatinine. For example, while muscle mass strongly influences creatinine, it does not, or only marginally, affects cystatin C . Muscle Since creatinine alone has clear drawbacks as a GFR-marker, it is widely considered that it should be replaced by GFR-prediction equations based not only upon creatinine, but also upon anthropometric and/or demographic data such as sex, age and ethnicity, to compensate for the influence of muscle mass on the creatinine level . Most of–B can be used both for adults and children [Since the level of cystatin C is less dependent upon anthropometric data than that of creatinine, simpler cystatin C-based GFR-prediction equations of the type GFR = A × cystatin C children ,33,46–48children , this muchildren .A considerable number of creatinine- or cystatin C-based GFR-prediction equations have been described . The reaAlthough some creatinine- or cystatin C-based GFR-prediction equations produce estimated GFR-values 80–85% of which are between ± 30% of GFR measured by invasive gold standard methods in some studies, the highest percentages of estimated GFR-values between ± 30% of measured GFR-values are obtained using GFR-prediction equations based upon both cystatin C and creatinine ,49,56–60Although GFR-prediction equations based upon both cystatin C and creatinine clearly seem to have better diagnostic performance than prediction equations based upon only one of these GFR-markers, such combined equations are not optimal in all clinical situations. To give some examples: in a patient suffering from paralysis with very low muscle mass, the combined prediction equation will be less reliable than a prediction equation based on cystatin C alone; in a patient treated with high doses of glucocorticoids, the combined prediction equation will be less reliable than a prediction equation using creatinine alone and anthropometric data. Thus, a strategy for GFR-estimation based upon automatic use of a combined prediction equation using both creatinine and cystatin C would consequentially have worse diagnostic performance than a strategy in which both a cystatin C-based and a creatinine-based prediction equation are used, concomitantly taking clinical data into account.2) is estimated both by a prediction equation based upon cystatin C alone as well as by a prediction equation based upon creatinine alone and anthropometric data. The two estimates are compared and, if they are in agreement within specified limits, the arithmetic mean of the two estimates is used. Diagnostically the arithmetic mean of the two estimates has been shown to perform at least as well as more complex ways of combining the two estimates [In Lund, where cystatin C has been available for clinical use since 1994 , the folstimates . The speTherefore in practice, we consider concordant cystatin C and creatinine-based estimates of GFR to be at least as reliable as GFR measured by invasive gold standard procedures. It has previously been shown that the practical execution of gold standard invasive determinations of GFR is not always reliable .If the GFR-estimate based upon cystatin C only does not agree with that based upon creatinine, the clinical situation is considered, e.g. concerning the presence of an abnormal muscle mass or use of high doses of glucocorticoids. When obvious reasons for not using either the cystatin C- or the creatinine-based estimate are found, only the estimate based upon the other, appropriate, prediction equation is used.If no obvious reasons for a discrepancy between the GFR-estimate based upon cystatin C alone and upon creatinine alone, respectively, can be found, GFR is measured by an invasive gold standard procedure.When the GFR of a patient has been estimated according to this strategy, changes in GFR can safely be monitored by determination of creatinine, since this strategy connects a reliable GFR-value to the creatinine level of that particular patient. But if the muscle mass of the patient significantly changes, the strategy involving two GFR-estimates has to be repeated.www.egfr.se and this site can also be used to implement it and to calculate absolute GFR from relative GFR, which might be required, e.g. for dosing of medicines cleared by the kidneys.The strategy outlined above does not require any particular cystatin C-based or creatinine-based prediction equation to be used. Characterization of the population served by a hospital may be required to select the best prediction equations for that hospital. In Lund we have chosen a cystatin C-based equation working for both children and adults  and a cr


Bacillus subtilis to phosphorylate each other in vitro and interact with each other in vivo. The interactomics data suggested a high degree of connectivity among all types of kinases, while phosphorylation assays revealed equally wide-spread cross-phosphorylation events. Our findings suggest that the Hanks-type kinases PrkC, PrkD, and YabT exhibit the highest capacity to phosphorylate other B. subtilis kinases, while the BY-kinase PtkA and the two-component-like kinases RsbW and SpoIIAB show the highest propensity to be phosphorylated by other kinases. Analysis of phosphorylated residues on several selected recipient kinases suggests that most cross-phosphorylation events concern key regulatory residues. Therefore, cross-phosphorylation events are very likely to influence the capacity of recipient kinases to phosphorylate substrates downstream in the signal transduction cascade. We therefore conclude that bacterial serine/threonine and tyrosine kinases probably engage in a network-type behavior previously described only in eukaryal cells.Bacteria possess protein serine/threonine and tyrosine kinases which resemble eukaryal kinases in their capacity to phosphorylate multiple substrates. We hypothesized that the analogy might extend further, and bacterial kinases may also undergo mutual phosphorylation and activation, which is currently considered as a hallmark of eukaryal kinase networks. In order to test this hypothesis, we explored the capacity of all members of four different classes of serine/threonine and tyrosine kinases present in the firmicute model organism  Mycobacterium tuberculosis  to produce the 6xHis-tagged fusions of proteins. Strep-tagged versions of proteins were obtained using a pQE-30 vector with His6-tag replaced by a strep-tag  were used for vector construction and protein synthesis, respectively. Cells were routinely grown in LB medium supplemented with appropriate antibiotics when necessary (ampicillin 100 μg/ml and kanamycin 25 μg/ml). Protein synthesis and purification were carried out as described previously , or Strep Tactin affinity chromatography (Novagen), respectively. For the insoluble proteins, PrkD K54D and YabT K55D, the inclusion bodies were dissolved in the buffer containing 6 M guanidine hydrochloride, 50 mM Tris pH 7.5, 100 mM NaCl, 5 mM MgCl2, and 5% glycerol. The purification was performed as mentioned before but in the buffer with additional 6 M guanidine hydrochloride. To refold the proteins, the concentration of guanidine hydrocloride was lowered to 0.2 M. Purified proteins were desalted on PD-10 columns , and stored at −80°C in 10% glycerol.The following kinase and substrate genes were PCR-amplified using the 2, 5% glycerol, 50 μM ATP, and 20 μCi/mmol [γ-32P]-ATP. Each phosphorylation reaction contained one wild type active kinase and one catalytically deficient kinase. The assay was assembled using the following final protein concentrations: 3 μM RsbT WT, 3 μM HprK WT, 5 μM RsbW WT, 5 μM PtkA WT with 5 μM TkmA, 2 μM PrkD WT, 1 μM YabT WT, 1 μM PrkC WT, 5 μM SpoIIAB WT, 9 μM RsbT N49A, 4 μM HprK K159M, 9 μM RsbW N53A, 3 μM PtkA K59D, 3 μM PrkD K54D, 2 μM YabT K55D, 3 μM PrkC K40D, 9 μM SpoIIAB N50A. Phosphorylation reactions were incubated for 1 h at 37°C, stopped by boiling at 100°C, and samples were separated on an 8–12% SDS-polyacrylamide gel. Autoradiography signals were revealed using the FUJI phosphoimager.Phosphorylation reactions were incubated in a buffer containing: 50 mM Tris pH 7.5, 100 mM NaCl, 5 mM MgClProteins  were incubated in a buffer containing 50 mM Tris pH 7.5, 100 mM NaCl, 5 mM MgCl2, 5% glycerol, and 1 mM ATP to perform the phosphorylation reactions. The reactions were incubated for 1 h at 37°C, stopped by boiling at 100°C, and samples were separated on an 8–12% SDS-polyacrylamide gel. Signals from phosphorylated protein have been revealed by Pro-Q® Diamond phosphoprotein stain (Life Technologies). After two fixation steps in a solution containing 50% methanol and 10% acetic acid (30 min each), the gel was stained by the Pro-Q® Diamond phosphoprotein stain for 90 min. The gel was de-stained by three 30-min washes in a solution containing 20% acetonitrile and 50 mM sodium acetate, pH 4.0. The gel was washed twice with ultrapure water for 5 min, before scanning.In vitro phosphorylation reactions of PrkD K54D, YabT K55D, PtkA K59D, SpoIIAB N50A, and HprK K159M phosphorylated by PrkC, PtkA, PrkC, PrkD, and PrkD, respectively, were performed as described above, with the only difference of using only non-radioactive ATP. Denaturation of the samples was performed by buffer exchange to 6 M urea and 2 M thiourea in 10 mM Tris-HCl pH 8.0. Mass spectrometry analysis of phosphorylation sites was performed essentially as described previously     on their respective substrates: Hpr, RsbS, RsbV, and SpoIIAA Figure . All mutB. subtilis cell . Therefore, such complementary approaches should be combined whenever possible.To explore the potential consequences of cross-phosphorylation on the recipient kinase function, we focused on several cases representing different types of cross-talk and identified the phosphorylated residues on recipient kinases by mass spectrometry. The examined “recipient” kinases: PtkA, PrkD, YabT, SpoIIAB, and HprK, comprised representatives of all four kinase types. After identifying the phosphorylation sites, we mapped them onto the resolved kinase structures or homology-based structural models, in order to evaluate the possible impact of cross-phosphorylation on the recipient kinase function.First we examined the phosphorylation of the Hanks-type kinase YabT by the BY-kinase PtkA. We detected three phosphorylated tyrosine residues on YabT Figure . Two of PrkD is a cytosolic Hanks-type serine/threonine kinase with no transmembrane helix. For its transmembrane paralogs, PrkC and YabT, the activation mechanism by respective binding of muropeptides  revealed a high degree of connectivity among different classes of kinases, kinase activators, substrates, and phosphatases. Here we have explored whether the connectivity at the protein-protein interaction level may point to a functional interaction, i.e., cross-phosphorylation among the four classes of B. subtilis serine/threonine and tyrosine protein kinases. Our data suggest an even higher degree of connectivity than the interactomics studies, with all tested kinases engaging in cross phosphorylation, either as donors or recipients. Moreover, the identity of residues phosphorylated on recipient kinases in most cases supports the notion that phosphorylation of one kinase by another has a functional/regulatory consequence. The phosphorylated residues are situated in, or immediately adjacent to, protein-protein or protein-DNA interaction surfaces, activating loops or the kinase active sites. It is therefore likely that bacteria also possess kinase cascades similar to those described in eukarya (Marshall, B. subtilis (Macek et al., in vivo studies.Our global interactomics screen of the regulatory network based on serine/threonine/tyrosine phosphorylation in The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Individuals with birch pollinosis may show allergic reactions after consumption of soybean-containing food. This is caused by cross-reaction of IgE directed against the major birch pollen allergen Bet v 1 with its homologue Gly m 4 from soybean. Birch pollen allergic subjects without soybean allergy often present IgE binding to Gly m 4.To identify IgE epitopes of Gly m 4 in birch pollen-allergic subjects with and without allergy to soybean.E. coli. Secondary and tertiary structures were assessed by CD and NMR spectroscopy. IgE binding of variants was analyzed by competitive immunoblotting and inhibition ELISA.Sera from 33 birch pollen-allergic patients with and without clinical reactivity to soy as determined by oral food challenge or convincing history of soy allergy were included in the study. Specific IgE against Bet v 1 and Gly m 4 was determined by ImmunoCAP. IgE binding of the sera to rGly m 4 and soy extract was tested in western blot analysis. To predict putative IgE epitopes, 20 IgE-binding phage-displayed peptide mimics and heptamers representing the total Gly m 4 amino acid sequence were bioinformatically mapped onto the molecular surface of Gly m 4. To verify predicted epitopes, Gly m 4 variants with substituted epitope residues were expressed in and purified from A/L in sera of both patient groups. CAP values were above 3.5 kUA/L in 18 sera (86%) of patients with clinical soy allergy and in 7 sera (58%) of patients sensitized, but without clinical reactivity to soy, respectively. Intensity of IgE binding in western blot corresponded to ImmunoCAP analysis. Bioinformatic mapping resulted in four distinct epitopes on Gly m 4 surface. Several amino acid residues were chosen for generation of substitutional variants. Purified rGly m 4 variants showed a typical Bet v 1-like structure according to CD spectra and 1D-1H-NMR spectroscopy. IgE binding to several substitutional variants was reduced, indicating a multiple number of epitopes on Gly m 4 surface.Gly m 4- and Bet v 1-specific IgE levels ranged from 0 to >100 kUNo significant differences in Gly m 4-specific IgE binding could be observed between birch pollen-allergic patients with and without clinical soy allergy in ImmunoCAP and immunoblot analysis. However residual IgE reactivity persists in substitutional rGly m 4 variants, indicating the presence of further IgE epitopes not yet identified.