Smooth muscle strips from gastric fundus were isometrically contracted with ET-1 which induced an increase in monophosphorylation from 9 ± 1 % under resting conditions (PSS) to 36 ± 1 % determined with 2D-PAGE. Electric field stimulation induced a rapid, largely NO-mediated relaxation with a half time of 8 s, which was associated with an initial decline in RLC phosphorylation to 18 % within 2 s and a rebound to 34 % after 30 s whereas relaxation was sustained. In contrast, phosphorylation of RLC at Ser19 probed with phosphospecific antibodies declined in parallel with force. LC/MS and western blot analysis with phosphospecific antibodies against monophosphorylated Thr18 indicate that Thr18 is significantly monophosphorylated during sustained relaxation. We therefore suggest that (i) monophosphorylation of Thr18 rather than Ser19 is responsible for the phosphorylation rebound during sustained EFS-induced relaxation of mouse gastric fundus, and (ii) that relaxation can be ascribed to dephosphorylation of Ser19, the site considered to be responsible for regulation of smooth muscle tone.Nitrovasodilators and agonists, via an increase in intracellular cyclic nucleotide levels, can induce smooth muscle relaxation without a concomitant decrease in phosphorylation of the regulatory light chains (RLC) of myosin. However, since cyclic nucleotide-induced relaxation is associated with a decrease in intracellular [Ca Structural studies suggest that the actin-activated MgATPase activity of unphosphorylated myosin is low because of an asymmetric interaction between the two myosin heads  of myosin at Ser19 is a prerequisite for the actin activation of MgATPase activity of smooth muscle myosin  the rate of relaxation can be accelerated even at basal or near basal levels of RLC phosphorylation  within seconds. It is well established that EFS elicits relaxation by activating non-adrenergic non-cholinergic (NANC) neurons of the plexus myentericus which in turn release NO and vasointestinal peptide (VIP) leading to an increase in cGMP and cAMP levels in the fundus smooth muscle cells  from Perbio Science/Thermo Fisher Scientific, Germany; and the Phos-tag kit from Wako Chemicals GmbH, Germany. All other chemicals were from Sigma or AppliChem, Germany.The used primary antibodies were rabbit polyclonal anti-phospho-myosin light chain Ser-19 (pSer19), Rockland, PA, USA; rabbit polyclonal anti-phospho-MLC20 Thr-18 (pThr18), Santa Cruz, CA, USA; anti-phospho-myosin light chain 2 pThr18/pSer19, Pierce Biotechnology, Rockford, IL, USA; and mouse monoclonal anti-MLC20 antibody for total MLC20 (RLC), Sigma, Germany. The secondary antibodies were IRDye conjugated goat anti-mouse (IRDye680) and goat anti-rabbit (IRDye800), LI-COR, NE, USA and horse radish peroxidase-conjugated donkey anti-mouse and donkey anti-rabbit both from Dianova, Germany. Thermolysin came from R&D Systems, Minneapolis, USA. The Sypro2HPO4, 1.2 MgCl2, 0.16 CaCl2, 24 Hepes, 10 glucose, pH 7.4 at 37 °C and bubbled with 100 % O2. Murine gastric fundi were freed of mucosa, cut in the direction of the circular muscle layer into strips (2 mm wide) and mounted vertically in Ca2+-free PSS in an organ bath between two platinum electrodes. Slack length was determined in Ca2+–free PSS, the fundus strips were then stretched to 167 % of slack length in low calcium PSS. Calcium was slowly increased up to 1.6 mM and a control contraction was induced by depolarization with 45 mM K+, replacing Na+ in the PSS solution. Force was recorded with a type Q11 transducer . Adrenergic/cholinergic transmission as well as prostaglandins were inhibited by addition of atropine (10 μM), propranolol (1 μM), phentolamine (1 μM) and indomethacin (1 μM) to PSS for 20 min. This inhibitor cocktail was present throughout the experiment. Contractions were elicited with 5 nM endothelin-1 (ET-1). Electrical field stimulation (EFS) was performed with custom-made stimulators with trains of pulses at 10 Hz  for 30 s. The strips were snap frozen within 100 ms with liquid nitrogen precooled tongs at distinct time points of EFS  and fixed in dry-ice precooled 15 %TCA/acetone.Male mice, 12 weeks old, were killed by cervical dislocation following procedures approved by the Institutional Animal Care and Use Committee of the University of Cologne. The stomach was quickly removed and transferred to PSS with low calcium (in mM): 118 NaCl, 5 KCl, 1.2 Na2+], the muscle strips were mounted horizontally in a myograph on the stage of an inverted Zeiss microscope  using the cuvette of the confocal wire myograph system 120CW . The strips were equilibrated as above. After the K+-induced test contraction, the strips were loaded with fura-2 for 4 h with 1 μM acetoxy methylester fura-2 (fura-2 AM) dissolved in DMSO premixed with pluronic FF127  140 KCl, 2 EGTA, 24 HEPES, 1.2 MgCl2, 1.2. Na2HPO4, pH 6.8). Then 10 mM Ca2+ was added to obtain the maximal ratio, Rmax, followed by addition of 20 mM MnCl2 to record background values. From these values, [Ca2+] was calculated using the formula and an apparent dissociation constant of the Ca2+–fura 2 complex of 224 nM given by Grynkiewicz et al.  . The lysates were subjected to 2D-PAGE and the silver or Sypro Ruby®, and the desmin band was evaluated densitometrically using the Odyssey Infrared Imaging System (LI-COR).TCA was removed as above. For separation by SDS-PAGE, the strips were prepared and subjected to SDS-PAGE as described by Lubomirov et al. . The sepFor separation by Phos-tag gel electrophoresis, the fundus strips were agitated for 2 h in 120 μl sample buffer B , boiled and centrifuged as described above. The protein concentration was determined in the supernatant according to Bradford  using BSNitrocellulose membranes were first probed with antibodies against pSer19, pThr18, or RLC total followed by incubation with the appropriate secondary antibody and visualization of the immunoreactive signal with enhanced chemiluminescence ECL. Blocking of non-specific binding sites and densitometric evaluation of immunoreactive bands were performed as in Lubomirov et al. . When us4HCO3 and 5 mM NH4HCO3/50 % acetonitrile (ACN) (v/v) for 10 min and dried in vacuo. In-gel digestion of proteins was performed for 2 h at 60 °C using 2 ng thermolysin dissolved in 5 μl of 50 mM NH4HCO3. Proteolytic peptides were extracted by incubating the gel spots twice with 10 μl of 95 % ACN (v/v)/0.1 % trifluoroacetic acid (TFA) (v/v) for 15 min. Extracts were combined and dried in vacuo. For mass spectrometric analysis, peptides were re-dissolved in 15 μl 0.1 % TFA (v/v).Following staining with colloidal Coomassie Brilliant Blue G-250, protein spots of interest were cut from the gel, destained by alternated incubation with 20 μl of 10 mM NH® PepMap μ-Precolumn Cartridge; 0.3 mm × 5 mm, particle size 5 μm) and an Acclaim® PepMap RSLC analytical column . Peptides were preconcentrated on the precolumn and washed for 5 min using 0.1 % (v/v) TFA at a flow rate of 30 μl/min. Subsequently, peptides were separated at a flow rate of 300 nl/min using a binary solvent system consisting of solvent A [0.1 % formic acid (v/v)] and solvent B . The following gradient was used: 5–40 % solvent B in 30 min and 40–95 % solvent B in 5 min. The column was then washed for 5 min with 95 % solvent B and equilibrated with 5 % solvent B for 15 min.Online reversed-phase nano-HPLC separations were performed using the UltiMate 3000 RSLC System  equipped with two precolumns (Acclaimm/z 370 to 1700 and acquired in the Orbitrap with the Automatic Gain Control (AGC) set to 5 × 105 ions and a maximum fill time of 500 ms. The five most intense multiply charged ions were selected for fragmentation by multistage activation (MSA). MSA scans were performed in the linear ion trap with an AGC set to 10,000 ions and a maximum fill time of 400 ms. Fragmentation was carried out using the following parameters: normalized collision energy, 35 %; activation q, 0.25; activation time, 30 ms; ion selection threshold, 2500; dynamic exclusion, 45s; multistage activation enabled; and listed neutral losses m/z 98, 49, 32.6.The LTQ Orbitrap XL instrument was equipped with a nanoelectrospray ion source (Thermo Fisher Scientific) and distal coated SilicaTips . The instrument was externally calibrated using standard components. The general mass spectrometric parameters were as follows: spray voltage, 1.5 kV; capillary voltage, 45 V; capillary temperature, 200 °C; and tube lens voltage, 120 V. For data-dependent MS/MS analyses, the software XCalibur 2.0.7 (Thermo Fisher Scientific) was used. Full scan MS spectra were recorded from Mus musculus, 143,284 sequences) using the MASCOT algorithm v. 2.3.02 . The level of significance was set at p < 0.05.All data are mean ± SEM; n = 41), and to EFS with a TTX sensitive relaxation. The relaxation amplitude increased with increasing frequencies and was maximal at 10 Hz, which relaxed the ET-1 precontracted strips by 80 ± 2 % (n = 16). This frequency was used throughout. The time course of relaxation was biphasic starting with a lag period of ~1.2 s followed by an exponential decay  was 8.0 ± 0.13 s, and relaxation was stably maintained for at least 30 s. ET-1-induced contraction was associated with an increase in intracellular [Ca2+] from ~142 nM under basal conditions (PSS) to ~310 nM. Upon EFS, intracellular [Ca2+] decreased in parallel with relaxation and remained low during maintained relaxation  phosphorylation  under resting conditions (PSS) to 36 ± 1 % of RLCtotal . The time point chosen was such that it represented the phosphorylation status just preceding EFS-induced relaxation, i.e. during the tonic phase of the contraction. EFS-induced relaxation was preceded by a decline in m-phosphorylation to 18 ± 1 % of RLCtotal (p = 0.004) within 2 s after starting EFS. Thereafter, RLC were rephosphorylated  and 12 ± 1 in 30 s EFS-treated tissues (n = 4) of RLCtotal) did not differ significantly between the different treatments suggesting that only a small amount of RLC was di-phosphorylated. This conclusion was confirmed by western blots probed with phosphospecific antibodies directed against di-phosphorylated RLC (Thr18/Ser19), in which no immunoreactive signal was detected  with a half time of ~30 s. RLC m-phosphorylation during sustained relaxation induced by DEA–NO and VIP was not significantly different from the value of ET-1 contracted fundus . Thus, although relaxation induced by exogenously added neurotransmitters was much slower, it was also uncoupled from RLC m-phosphorylation in a similar manner as with EFS-induced relaxation irrespective of whether the neurotransmitters acted through cGMP or cAMP. NO–cGMP signalling may predominate under our conditions since l-NAME inhibited relaxation by ~75 % (data not shown), which is in line with an earlier report showing that EFS-induced relaxation in PKG knock-out mice is significantly blunted  of the value before starting EFS in ET-1 contracted strips which was taken as 100 %. For comparison, under resting condition (PSS before addition of ET-1) it was 11 ± 4 % of ET-1 (n = 3). Using Phos-tag gels which allow to separate m- from di-phosphorylated phospho-species of RLC , i.e. RLC, was unambiguously identified in all spots with a sequence coverage of 89–97 %. Furthermore, using multistage activation as an effective method for fragmentation of phosphopeptides, no phosphorylation was detected in the protein spot assigned non-phosphorylated, whereas phosphorylated peptide species were reliably detected in the spots of phosphorylated RLC from both ET-1 and 30 s EFS-treated samples. In the ET-1 treated sample, Ser19 was determined to be the major phosphorylated residue in RLC by MS/MS  we do not know whether phosphorylation of Ser1 and 2 contribute to m-phosphorylation, (ii) the semiquantitative nature of western blots in particular with the pThr18 phosphospecific antibody, and (iii) that the phosphospecific antibodies only allow to determine the relative intensity changes between different treatments for a given antibody but not to reliably calculate the ratio between m-pThr18 and m-pSer19 during ET-1 and EFS. For instance, if one takes into account the relative changes of the immunoreactive signals during EFS, then the maximally ~1.7 fold increase in Thr18 would call for a ratio of pSer to pThr ~1:1 in ET-1 treated preparations which is clearly at variance with our MS/MS data and with previous investigations which showed that Thr18 was the minor phosphorylated site and reported a pSer19/pThr18 ratio of 6:1 in 2+] and whether EFS activates such a kinase. Of the Ca2+-independent RLC kinases currently in question, ILK rather than ZIP kinase appears to be an attractive candidate. This is because in Ca2+-sensitized ileal smooth muscle ILK was proposed to be downstream of PKC  or phosphatase(s) are responsible for m-phosphorylation of Thr18 at low intracellular [Ca2+-independent kinases and was mainly considered to be present in diphosphorylated RLC. Whereas diphosphorylated myosin enhanced actin-activated MgATPase activity in solution (Ikebe and Hartshorne, In recent years, pThr18 phosphorylation has gained attention as an index of the action of CaAssuming that pSer19 regulates attachment of crossbridges and that the remaining phosphorylation of Ser19 is below the threshold for activation of contraction, dephosphorylation of Ser19 is sufficient to induced relaxation and no additional regulatory mechanisms are required for switching off the contractile machinery. Of note, RLC phosphorylation was high before starting relaxation. However, relaxation by cAMP/cGMP has also been induced at low levels of RLC phosphorylation, i.e. during the latch state (Miller et al. In conclusion, the initial phase of cGMP/cAMP-mediated relaxation of gastric fundus smooth muscle induced by the release of inhibitory neurotransmitters from intrinsic neurons was associated with a decline in m-phosphorylation of RLC. However, during the sustained phase of relaxation phosphorylation rebound to the values before starting relaxation. This suggested that stress is uncoupled from RLC dephosphorylation as has been observed in vascular smooth muscle relaxed by NO (Rembold et al.


Changes in snow and ice conditions are some of the most distinctive impacts of global warming in cold temperate and Arctic regions, altering the environment during a critical period for survival for most animals. Laboratories studies have suggested that reduced ice cover may reduce the survival of stream dwelling fishes in Northern environments. This, however, has not been empirically investigated in natural populations in large rivers. Here, we examine how the winter survival of juvenile Atlantic salmon in a large natural river, the River Alta , is affected by the presence or absence of surface ice. Apparent survival rates for size classes corresponding to parr and presmolts were estimated using capture-mark-recapture and Cormack-Jolly-Seber models for an ice-covered and an ice-free site. Apparent survival (Φ) in the ice-covered site was greater than in the ice-free site, but did not depend on size class (0.64 for both parr and presmolt). In contrast, apparent survival in the ice-free site was lower for larger individuals (0.33) than smaller individuals (0.45). The over-winter decline in storage energy was greater for the ice-free site than the ice-covered site, suggesting that environmental conditions in the ice-free site caused a strong depletion in energy reserves likely affecting survival. Our findings highlight the importance of surface ice for the winter survival of juvenile fish, thus, underpinning that climate change, by reducing ice cover, may have a negative effect on the survival of fish adapted to ice-covered habitats during winter. In seasonal temperate environments, changes in snow and ice conditions are one of the most distinct impacts of global warming  is an Atlantic salmon river in Northern Norway (Jensen 3s−1 (1972–1986) to 29 m3s−1 (1988–2003)  is also commonly caught. In addition, Arctic char  is most common in the lower part, whereas whitefish (Coregonus lavaretus), European grayling  and minnow (Phoxinus phoxinus) are most common in the upper part. Flounder (Platichthys flesus), three-spined stickleback (Gasterosterus aculeatus), pike (Esox lucius), burbot (Lota lota), yellow perch (Perca fluviatilis) and eel (Anguilla anguilla) are occasionally found.The River Alta ..oN, 23oEJuvenile Atlantic salmon density in the upstream part of the reach accessed by Atlantic salmon decreased after the river was developed for hydropower production, but in recent years, there has been a partial recovery .Two study sites were selected: (1) a site that was ice-covered during winter (Gargia), ∼20 km distant from the hydropower reservoir; and (2) a site that was relatively ice-free during winter (Sautso), just ∼2 km downstream of the hydropower plant outlet . At Saut3s−1; ice-free, mean 29 m3s−1).Water temperatures and discharges were measured at both study sites . Water t3s−1) and wetted width approached the channel width of ∼60 m. Individuals were captured in 21 stations in the downstream ice-covered site and 16 stations in the upstream ice-free site. Each captured individual above 60 mm total length was classified as a parr or a presmolt according to size , based on the minimum observed length of smolting fish caught in a smolt trap (situated 11 km upstream from the river mouth) from another study  study was conducted to provide data for fitting Cormack-Jolly-Seber (CJS) models for estimating the survival over the winter of 2004–2005. Each site, of approximately 700 m in river length, was compartmentalized longitudinally into stations of 25 m in length, and individuals were then captured within each section by electrofishing along the river banks during mid-October 2004. Electrofishing was conducted by wading along one of the banks using backpack mounted Geomega FA 4 apparatus (Terik Technology). The bank that was selected was chosen as that which had most optimal conditions for electrofishing and the electrofishing switched banks when poor conditions (such as deep areas) were encountered. Approximately, 20–25% of the channel was electrofished. Discharge was rather high during the capture  to the nearest mm and weighed to the nearest 0.1 g. A scalpel was used to make a small incision in the fish abdomen, and a Passive Integrated Transponder  was gently pushed into the abdominal cavity through the incision .3s−1 and the mean water temperature was 0.9°C. The first recapture event in the ice-covered site took place a month later (1–7 May) at a somewhat higher discharge (mean 39 m3s−1), but the mean water temperature was 0.9°C, which was similar to the temperature at the ice-free site at first recapture.At the first recapture event in the ice-free site (1–6 April), the mean water discharge was 29 mP), which is the probability of recapture of a surviving individual  models were used to estimate (1) the apparent survival probability (Φ), which is the probability that an individual will both survive and remain within the study area, and (2) the apparent encounter probability :where Φγi is the probability of an individual being unavailable for capture at time i. Individuals can become unavailable for capture if they temporarily move out of the study area. In this study, apparent survival and apparent encounter probabilities between tagging in October and the first recapture event in spring were estimated. The second recapture event in spring allowed separate estimates for apparent survival and apparent encounter probabilities between October and the first recapture event to be obtained. As is typical for capture-mark-recapture methods, survival and encounter is confounded for the last sampling interval (between the first and second recapture event in spring) and separate estimates cannot be obtained for this last interval.where Cormack-Jolly-Seber (CJS) models were fitted using the package MARK (White and Burnham 5/body length[mm]3) was chosen as a metric of the relative well-being of the fish because it was possible to obtain without sacrificing the individual, thus enabling direct comparison of condition factor of an individual at release and recapture. To evaluate the validity of this, the relationship between condition factor and specific energy (J g−1) was determined using juveniles sampled pre and post winter at both sites, and during the winter at the upstream site  and specific energy , and%DB is the percentage dry body mass of the individual. To determine water content of the fish, the carcass, including intestines with fat deposits, but without stomach contents, was dried in a drying cabinet (70°C) until the weight stabilized. The relationship between individual condition factor and specific energy for each site and date was determined using Pearson's correlation.where U-tests.Distances moved between release and first recapture were calculated from GPS measurements at release and recapture. Measurements of distance moved were missed on some individuals due to error. Absolute distance moved for tagged and recaptured individuals according to site and size class was compared using Mann–Whitney P = 0.47) than in the ice-free site (P = 0.35). The model with second lowest AIC  ; medians 6.0% and 11.7 for presmolts in the ice-covered and ice-free site, respectively . Condition factor and specific energy in samples collected post winter were less than those collected prewinter in the ice-covered site. At the ice-free site (which could be sampled throughout winter due to lack of ice cover), condition factor and specific energy showed a fairly consistent decline throughout winter  and presmolts . Given this, the condition factors of groups at mark and first recapture can be considered to be a reasonable indication of their relative energy content.Condition factor declined between mark and recapture for both size classes at both sites (Mann–Whitney < 0.001) . The dect winter . ConditiU-test, P = 0.77)  between release and first recapture. Thirteen percent of first presmolt recaptures in the ice-free site were at a distance of greater than 200 m from release, whereas the corresponding figure for first parr recaptures was only 2.7% , but presmolts moved significantly further in the ice-free site than the ice-covered site (P < 0.001). There was a tendency to upstream movement at both sites  . In the nly 2.7% . No signth sites . Howeverth sites .Apparent survival probability was lower in the ice-free than the ice-covered site, according to our hypothesis. However, whereas both size-classes had equivalent apparent survival in the ice-covered site, that of presmolts was lower than that of parr in the ice-free site. Apparent survival depends on both mortality and emigration; hence, our results suggest a real survival minimum of 66% , 45%  and 33% . The higher mortality in the ice-free site is supported by previous observations of abundances at the two sites. Both sites are fully recruited and have similar 1 + juvenile abundances, but in the ice-free site juvenile abundances decline faster with increasing age from 1 + to 4 + age groups and presmolt density is approximately half that of the ice-covered site  due to operational reasons. Therefore, sampled individuals in the ice-free site were not experiencing mortality during April, a period that previous studies have shown to be critical for survival  and/or site-specific characteristics biasing the estimate of survival . Stranding mortality due to sudden drops in water discharge downstream of the power station has been suggested as a reason for the reduced juvenile densities in this area after regulation in 1987, but improved routines for operation of the hydropower station have reduced this problem considerably , red-breasted merganser (M. serrator) and seagulls (Larus spp.) found in this area during summer are usually absent during winter , but the mammalian predators American mink (Neovison vison) and otter (Lutra lutra) may occur. The most likely fish predator occurring on these stretches is large-grown brown trout . However, the general predation level on Atlantic salmon juveniles in this area during winter remains unknown.Additionally, reduced ice cover may increase the predation risk by birds and mammals. Bird predators such as goosander (−1 (Finstad et al. Parr did not have a lower apparent survival than presmolts, contrary to our hypothesis. The effect of size on apparent survival differed with ice cover condition, with equal apparent survival of both size classes in the ice-covered site, and presmolts actually having lower apparent survival than parr in the ice-free site. An energetic based difference in mortality cannot be ruled out as presmolts had a larger decline in condition factor in the ice-free site compared to the ice-covered site. The critical body energy level for winter survival of juvenile salmon in the River Alta is approximately 4400–4800 J gThe difference between the parr and presmolt groups is not only a size difference, but is also probably physiological. The presmolt group has likely started the smolting process in April and May, although they do not enter the sea before the end of June and beginning of July in this river (Davidsen et al. In the ice-free site, recaptured presmolts moved further than recaptured parr, but the greater distances moved were not only in a downstream direction, suggesting that these movements were not associated with an early downstream migration connected to the smolt run. It is possible that the greater movements of presmolts resulted from them seeking more appropriate habitat. During winter, juvenile salmon become nocturnal and seek refuge during the day to avoid diurnal predators (Valdimarsson and Metcalfe The Arctic Climate Impact Assessment ACIA  has conc


The insulin receptor substrate-1 (IRS1) is phosphorylated on serine 307  in response to insulin as part of a feedback loop that controls IRS1 phosphorylation on tyrosine residues by the insulin receptor. This in turn directly affects downstream signaling and is in human adipocytes implicated in the pathogenesis of insulin resistance and type 2 diabetes. The phosphorylation is inhibited by rapamycin, a specific inhibitor of mammalian target of rapamycin (mTOR) in complex with raptor (mTORC1). The mTORC1-downstream p70 ribosomal protein S6 kinase (S6K1), which is activated by insulin, can phosphorylate IRS1 at serine 307 in vitro and is considered the physiological protein kinase. Because the IRS1 serine 307-kinase catalyzes a critical step in the control of insulin signaling and constitutes a potential target for treatment of insulin resistance, it is important to know whether S6K1 is the physiological serine 307-kinase or not. We report that, by several criteria, S6K1 does not phosphorylate IRS1 at serine 307 in response to insulin in intact human primary adipocytes: (i) The time-courses for phosphorylation of S6K1 and its phosphorylation of S6 are not compatible with the phosphorylation of IRS1 at serine 307; (ii) A dominant-negative construct of S6K1 inhibits the phosphorylation of S6, without effect on the phosphorylation of IRS1 at serine 307; (iii) The specific inhibitor of S6K1 PF-4708671 inhibits the phosphorylation of S6, without effect on phosphorylation of IRS1 at serine 307. mTOR-immunoprecipitates from insulin-stimulated adipocytes contains an unidentified protein kinase specific for phosphorylation of IRS1 at serine 307, but it is not mTOR or S6K1. As part of insulin signaling, the insulin receptor substrate-1 (IRS1) is phosphorylated directly by the insulin receptor on specific tyrosine residues. IRS1 serves as a hub for transmitting the insulin signal, for example, to metabolic control of the cell via protein kinase B (PKB) and mitogenic control via MAP-kinases ERK1/2. IRS1 is also important as a target for modification of the insulin signal through crosstalk with other signaling pathways and through downstream feedback signals in response to insulin. Many of these modifying signals impinge on IRS1 through phosphorylation of serine and threonine residues, reviewed in in vivoMuch of the phosphorylation of IRS1 at serine/threonine residues has been associated with inhibition of insulin signaling, either through interference with insulin receptor phosphorylation of IRS1 at tyrosine, binding of downstream signaling intermediaries and/or through increased IRS1 degradation −/− mouse embryo fibroblasts Ser307 is found in a consensus sequence for phosphorylation by PKB or the p70 ribosomal S6 kinase (S6K1). Insulin induced phosphorylation at Ser307 is acutely inhibited by rapamycin The study was approved by the Regional Ethics Board at Linköping University; all patients obtained written information and gave their informed verbal approval before the surgery. Verbal approval was considered sufficient by the ethics board considering that a small piece of adipose tissue was obtained during elective surgery for other reasons, and that the procedure posed no discomfort or threat to the health of the patients. The procedure was documented as part of the surgical protocol and adipose tissues samples were anonymized.Antibodies against IRS1 phospho-Ser307 (#2384S), S6 ribosomal protein phospho-Ser235/236 (#2211S), S6 ribosomal protein , p70S6K (#9202), and mTOR (#2972) were from Cell Signaling Technology . Anti-beta-Tubulin (#T4026) was obtained from Sigma Aldrich , anti-IRS1 (06–248) from Millipore  and anti-Raptor (A300–506A) from Bethyl Laboratories, Inc. . PCR primers and restriction enzymes were obtained from Invitrogen Life Science . QuikChange Site-Directed Mutagenesis Kit was from Stratagene. PF-4708671 and the rest of the chemicals were from Sigma Aldrich unless otherwise indicated in the text.2.Subcutaneous adipose tissue was obtained during elective abdominal surgery from patients under general anesthesia. Patients were not diagnosed with diabetes and were within the body mass index range of 18–28 Kg/m·mL−1 adenosine deaminase for 20 min, before incubation with additions as indicated.Adipocytes were isolated by collagenase digestion  as described in Adenovirus expression plasmid pAD/CMV/V5-DEST carrying dominant negative S6K1 or S6K1-DN (T389A) was a generous gift from Jianping Ye, Pennington Biomedical Research Center, Louisiana. pAd/CMV/V5-DEST EGFP was from Invitrogen . Adenoviruses were amplified, harvested and titrated according to ViraPower Adenoviral Expression System protocol, Invitrogen.For transfection, the isolated adipocytes were incubated in DMEM supplemented with 1% BSA, 1% Penicillin-Streptomycin, 200 nM adenosine, 7.5 mM glucose and 10% fetal calf serum (Invitrogen) for 1 h at 37°C. Adipocytes were then transduced with S6K1-DN adenovirus (MOI 30) for 48 h with serum starvation for 18 h prior to incubation with10 nM insulin for indicated time periods. Untransfected and EGFP adenovirus infected cells were used as controls.Adipocytes were lysed in 2 vol lysis buffer  by passing three times through a 27 gauge needle. The lysate was immediately put on ice, and cell debris and fat was removed after centrifugation for 10 min at 500×g at 4°C. The infranatant was additionally centrifuged for 30 min at 20000×g, 4°C to remove the last traces of fat and then stored at −80°C. Lysate with 0.2 mg protein was pre-cleared on 25 µL Protein G PLUS-Agarose  for 30 min of rotation end-over-end at 4°C and then incubated overnight with 4 µL anti-mTOR antibody rotating end-over-end at 4°C. 25 µL Protein G PLUS-Agarose was then added to the anti-mTOR-incubated lysates and incubated for 4 h, rotating end-over-end at 4°C. Precipitates were washed three times with lysis buffer and then prepared for SDS-PAGE or for S307-kinase assay.Cell incubations were terminated by separating cells from medium by centrifugation through diisononylphtalate. To minimize post-incubation modifications of proteins, the cells were immediately dissolved in SDS and β-mercaptoethanol with protease and protein phosphatase inhibitors, and rapidly frozen. Cell samples were thawed by boiling, and immunoprecipitates boiled, in SDS-PAGE sample buffer for 3 min and resolved by SDS-PAGE. Proteins were transferred to PVDF membranes  and immunoblotted with indicated antibodies. The signal was detected by chemiluminiscence  imaging  and analyzed using imaging software MultiGauge V3.0 . In this study we examine acute effects of insulin on protein phosphorylation, and since the total amount of IRS1, S6K or S6 protein doesńt change  we choseAGCCATCACCGCCACCTCC5′-TCACGCACTGA and AGACATCACCGCCACCTCC5′-CGATCACGCACTGA, respectively . These primers also created an Eco 571 restriction site for identification. Positive clones were verified by sequencing. In order to create plasmids encoding GST-fusion proteins, cDNA with these mutations or the wild-type sequence, encoding aa 288–314 was amplified by PCR using the following primers: 5′-TGCGCCCGGGCCATCTCAACAATC and 5′-CCCAGAATTCTGGCCGGGGAGGT, cut with Xma I and Eco RI and subcloned into the pGEX-JD plasmid.Human IRS-1 S307A mutant was obtained by PCR and QuikChange Site-Directed Mutagenesis Kit from the plasmid pSIS2-hIRS-1-HA using the following primers: E. coli Y1090 and GST-fusion protein production was induced with 1 mM isopropyl β-thiogalactoside. Cells were lysed through sonication and lysate was cleared . Lysate was diluted in NET-N buffer  and GST-peptides were captured on GSH-sepharose beads for 30 min at 4°C. The beads were washed twice in NET-N buffer and once in 50 mM NH4HCO3 with 2 mM CaCl2. The peptides were cleaved off the GST-tag with 1 unit thrombin  in a total volume of 1 ml for 2 h at 22°C.The vectors were transformed into competent 2, 50 mM KCl and 25 mM Hepes at pH 7.4 for 2 h at 30°C, with or without 0.5 µM Torin , 20 µM PF-4708671 , or 3 µM Akti1/2 , as indicated. Incubations were terminated by isolating the peptides on a C18 ZipTip  followed by digestion with trypsin  for 2 h at 37°C. Trypsinated peptides were dried and resolved in 0.1% Formic Acid before being subjected to LC-MS/MS analysis.Immunoprecipitates of mTOR were incubated with the S307-peptide and 50 µM ATP in the presence of 10 mM MgClThe obtained peptide mixtures were analyzed by LC-MS/MS. Separation was done using nano-flow HPLC system  and data were acquired using on-line electrospray ionization ion trap“HCTultra PTM Discovery System” . Peptides were separated during 45 min by reverse phase chromatography on a 20 mm×100 µm C18 pre column followed by a 100 mm×75 µm C18 column  at a flow rate 300 nL/min. A gradient of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B) was distributed as follows: starting with 4% B; linear gradient 4%–20% B in 0–25 min; 20%–100% B in 25–35 min and 100% B in 35–45 min.Homo sapiens proteins in Swissprot database on a public Mascot server (www.matrixscience.com) with a second-pass Mascot error tolerant search to detect protein sequence variants with amino acid substitutions. The search parameters allowed mass errors up to 0.6 Da for MS data, and up to 0.6 Da for MS/MS data in case of CID of peptide ions and 1.5 Da in case of ETD of peptide ions. The charge states of the peptides were varied; and three missed cleavage sites were permitted. Serine and threonine phosphorylation were selected as variable modifications. The sequence of each peptide was verified by manual analysis of MS/MS spectra.Automated online tandem MS analyses were performed using alternating collision induced dissociation (CID) and electron transfer dissociation (ETD) of peptide ions. Peak lists were created from the raw data using DataAnalysis 3.4  and the resulting MGF files were used to search for For the comparative quantification of phosphorylation of the S307-peptide, peptide mixtures from the Ser307-kinase assay were analyzed directly by LC-MS/MS using 45 min LC-method. The extracted ion chromatograms acquired were computed for the selected peptide ions. Ion chromatograms of tryptic peptides originated from S307-peptide TESITATSPAR, with M/Z 567.3,  and corrThe ion chromatogram peak area represents the total ion abundance of the selected peptide. Analyses and determination of areas of each individual extracted ion chromatogram were performed using DataAnalysis 3.4 . The resulting data are expressed as a percentage relative control incubations with no additions (vehicle). Statistical difference between a treatment and the control group was analyzed using one-sample t-test and P<0.05 was considered to be statistically significant.We examined human primary mature adipocytes, which represent a primary cell type for insulin action and the development of insulin resistance in obesity. We first analyzed the time-course for phosphorylation of IRS1 at Ser307 in response to a maximal effective concentration of insulin, and compared it with that of the phosphorylation of the ribosomal protein S6 (S6) at serine 235/236, which is a bona fide physiological substrate of S6K1. The phosphorylation of IRS1 at Ser307 was rapid, reaching a quasi steady-state extent of phosphorylation after 5 min . In contCells were transfected with a dominant negative construct of S6K1 (S6K1-DN) with the mTORC1 phosphorylation site at threonine 389 substituted with alanine, which precludes activation of the S6K1-DN by insulin. Overexpression of S6K1-DN  inhibiteAs it is possible that the S6K1-DN may have different binding characteristics for different substrates, we next examined also the effect of the S6K1 specific inhibitor PF-4708671 The non-compatible time-courses, the selective effect of S6K1-DN, and the selective effect of the S6K1 inhibitor, each analyzes a very different aspect of signaling by S6K1 and each indicates non-compatibility with S6K1 phosphorylating IRS1 at Ser307. Taken together these findings therefore demonstrate that S6K1 is unlikely to be the physiological protein kinase for phosphorylation of IRS1 at Ser307 in response to insulin in human primary adipocytes.in vitro assay. We produced a peptide (S307-peptide) that contains amino acid residues 288–314 of human IRS1  and a low concentration of ATP (50 µM), phosphorylation of the S307-peptide was not consistently inhibited  at Ser307 in an in vitro-assay Figure S1Overexpression of adenovirus expressing EGFP does not affect S6 or IRS1 Ser307 phosphorylation. Isolated human primary adipocytes were infected with EGFP adenovirus. Untransfected cells were used as control. Cells were stimulated with 10 nM insulin for 40 min (A) or 10 min (B). Whole cell lysates were subjected to SDS-PAGE and immunoblotted against S6 phospho-ser235/236 (A) and IRS1 phospho-ser307 (B). Mean±SE (n = 3).(PDF)Click here for additional data file.


This decrease in Rh was a sum of the 42% decrease (4.1 mol C m−2 yr−1) in litter respiration and 3.2 mol C m−2 yr−1 more CO2 efflux from mineral soil in thinned sites compared with unthinned control. Increases in temperature, temperature sensitivity, alteration of litters, and microbial composition may be responsible for the contrary changes in Rh from mineral soil and litter respiration, respectively. These findings manifested that thinning management of larch plantations could enhance biomass accumulation and decrease respiratory efflux from soil, which resulted in the effectiveness improvement in sequestrating C in forest ecosystems.Thinning management is used to improve timber production, but only a few data are available on how it influences ecosystem C sink capacity. This study aims to clarify the effects of thinning on C sinks of larch plantations, the most widespread forests in Northeastern China. Both C influx from biomass production and C efflux from each soil respiration component and its temperature sensitivity were determined for scaling-up ecosystem C sink estimation: microbial composition is measured for clarifying mechanism for respiratory changes from thinning treatment. Thinning management induced 6.23 mol C m Carbon dynamics in forest plantations have become a hot topic for forest ecological researches due to their important role in global climate change , 2. In rChina is home to the world's largest plantation forests over 62 Mha), and selective thinning is one of the main management practices used in this region. Larch forests are widely distributed around the world at a latitude of 60 degrees north, and about 4.5 Mha of larch forests is distributed in Northeastern China , 10. Com2 Mha, anRh)) is often used to assess ecosystem carbon sink capacity as well as in international carbon trading [Rh needs to be distinguished from root autotrophic respiration, and few partitioned data is available for thinning management of forests. This makes it difficult to evaluate the effects of thinning on the ecosystem carbon sink. Given that a decrease of Rh after thinning practices, enhancement of forest carbon sink could be observed via decreasing CO2 efflux from soil. Or else, the biomass carbon gain from NPP may be offset by the increase in Rh. A long-term respiration separating Rh  and autotrophic respiration from roots may facilitate the quantification of Rh changes owing to thinning management [Owing to the large storage, a small change in soil carbon from soil heterotrophic respiration changes can reverse the direction of forest ecosystem balance . Instead trading , 13. To nagement , 14, 15.Forest management has been shown to have profound but inconsistent influences on soil respiration –18. ThisThe aims of this study were (1) to quantify the effects of long-term thinning on ecosystem carbon sink capacity via survey of biomass carbon and soil respiratory efflux and (2) to check the microbial and thermal changes for soil respiratory alteration from thinning treatment.L. gmelinii) being studied was afforested in 1969 at an initial planting density of 3300 plants·ha−1. Thinning was performed on 3 occasions following afforestation: at 11 years (1980), 20 years (1989), and 25 years (1994). The removal of the first and third thinnings was approximately 200 tree·ha−1, while the second thinning was approximately 300 tree·ha−1. During thinning, weak larches and other competing shrubs and saplings under the canopy were removed. The unthinned site is a long-term permanent plot located in the same larch plantation. This site was established in 1978 and has no artificial thinning except dead tree movement for timber utilization. Three replicating pairs of thinned and unthinned treatments (ca. 20 m ∗ 20 m) were selected in this paper. For each replicating pair, thinned plot and unthinned plot are neighbor for securing the data reliability. Area of the thinned site (2.5 ha) and unthinned site (0.5 ha) is approximately 3 ha.The study was conducted at Laoshan Experimental Station . The larch plantation (Rm) was measured, while outside the trenched box, soil respiration excluding recognizable litters (R−litter) and total soil respiration (Rt) were measured. Root respiration can be calculated as the difference between R−litter and Rm, while respiration from litter decomposition is the difference between Rt and R−litter. Duration of the measurement was in the growing season (end of April to early Oct) from April 2005 to September 2007. The measurements were carried out one time per month.Owing to its simple and cost effective and realistic respiration partitioning , 23, theAt both the thinned and unthinned sites, a thermometer probe (Li-6400 system) was used to measure soil temperature at a depth of 5 cm at the same time as soil respiration measurements were taken. Continuous soil temperature data were also recorded at 30 min intervals using a thermo Recorder mini Rt-21s  from 2005 to 2007. These data were used to compare thermal conditions at the thinned and unthinned sites and were scaled-up to estimate whole ecosystem respiration.L. gmelinii), birch (Betula platyphylla), and ash (Fraxinus mandshurica) was carried out at both the thinned and unthinned sites. Tree height (H) and diameter at breast height (DBH) were recorded, and tree density was calculated for each species. Similarly, measurements of the height, basal diameter, and tree density of 7 understory shrubs and saplings  were also carried out using 4 quadrats (5 m × 5 m) at both the thinned and unthinned sites. Following the arbor layer censuses, 10 to 18 different-sized individuals of each species were harvested to determine the biomass of each of plant parts ; no such distinction between different organs was made for the understory species. Oven-dried biomass (108°C) was used to determine the allometric relationship between biomass and DBH2H for each species . These relationships were then used to determine the difference in total biomass between the thinned and unthinned sites.An inventory of the arbor layer species larch (3) fumigation method, which was first proposed by Jenkinson and Powlson [Soil microbial carbon was measured using the chloroform , T is the measured soil temperature, and R0 and b are the best-fitting coefficients. R0 is theoretical soil respiration rate at 0°C. Q10 was then calculated using the expression exp(10∗b), to assess temperature sensitivity [The relationship between soil respiration and soil temperature was expressed by the exponential relationship: sitivity . The besThe differences of diameter, tree height, biomass of trees between different treatments, respiration between different treatments as well as among measured seasons were statistical analyzed by SPSS 17.0.P < 0.05)  than at the thinned site . The basal diameter and height of shrubs and saplings were 0.78 cm and 0.4 m smaller, respectively, at the thinned site than at the unthinned site  . DBH andned site .−1) was lower than that at the thinned site (231.1 Mg·ha−1) (P < 0.05), while the biomass of the shrub layer at the unthinned site (2.51 Mg·ha−1) was approximately 1.0 Mg·ha−1 higher than that at the thinned site (P > 0.05). By summing the biomass of the 2 layers, total biomass at the unthinned site was 38.9 Mg·ha−1 (approximately 20%) lower than that at the thinned site. In total, arbor biomass carbon at the thinned site was 1994 g C m−2 higher than that at the unthinned site (P < 0.05), while understory carbon at the thinned site was 50 g C m−2 lower than that at the unthinned site (P > 0.05)  and inventory data were used to calculate differences in biomass accumulation between the thinned and unthinned sites . The bio > 0.05) . By divis carbon .Microbial carbon at the thinned site was 8% lower than that at the unthinned site . SimilarThe effect of long-term thinning on soil temperature can be observed by comparing the continuous measurements and discrete measurements taken at the thinned and unthinned sites . On averRm was higher at the thinned site than at the unthinned site, while the other half showed the opposite pattern  showed that the unthinned site had higher respiration rates than the thinned site, while the remainder indicated that the unthinned site had lower or similar respiration rates compared to the thinned site  than at the unthinned site (58.8%), on average. In contrast, root respiration was generally higher at the unthinned site than that at the thinned site throughout the year, contributing by 15.2% of total respiration compared with 9.1% at the thinned site. The 3-year average litter respiration was the same at the unthinned site and the thinned site (0.69 μmol m−2 s−1). However, its contribution to total respiration was slightly higher at the unthinned site in spring and autumn but lower in summer. These resulted in a slightly higher overall average contribution at the unthinned site (26.2%) than at the thinned site (25.2%) (Table s2).By classifying the measurements by season, it was found that the respiration of soil microbes peaked in summer. In spring and autumn, soil microbes contributed more to the total soil respiration at the thinned site  than at the unthinned site (55.9% and 65.2%), while levels were similar during the summer (55.2% and 54.5%) (Table s2). This seasonal difference resulted in the Rm, R−litter, and Rt  is 10% lower than that at the thinned site (2.37). Higher Q10 values for R−litter and Rt at the thinned site were also observed; however, the percentage was less than 5%  in total heterotrophic respiration. Autotrophic respiration from roots in thinned site was 0.7–0.9 mol m−2 yr−1 higher than that at the unthinned site  could be captured annually by these managed forests compared with unthinned forests. During the same period (2005–2007), total industrial CO2 efflux was 670 Tg CO2 on average, and the annual increase was as high as 60 Tg CO2 in Northeastern China [2 efflux and 20% of its annual increase of this industrial efflux.Forest management practices such as tending and thinning can dramatically affect stand biomass and volume of harvested timber , 8, 27. thinning . Larch prn China . Thus, t2 efflux from soil was similar in the thinned site (45.4 mol m−2 yr−1) and the unthinned site (45.5 mol m−2 yr−1), the percentage of heterotrophic respiration and autotrophic respiration were altered  could be a practical silvicultural strategy to offset industrial CO2 emission fulfilling the requirements of the Kyoto Protocol [However, feasibility of this carbon increase used in tradeoff industrial emission depends on the ecosystem carbon sink, instead of biomass carbon alone , 13. Thu altered . On averg forest . Its effg forest , 16. In Pinus ponderosa plantation demonstrated a 13% reduction in soil respiration in the first year after thinning [Rm, R−litter, and Rt in any season using the instant measurements from 2005 to 2007 , and the soil microbial composition [Rm  with highly intensive agricultural exploitation . In shor2 efflux, although the short-term instant measurement may show diversified result. Scaled-up data manifested that biomass increase could trade off 21% of the annual increase of total emission of local industry, and inclusion of soil respiration can give another 14% increase in the sink size. These findings support the use of thinning practices in larch plantation management in Northeastern China for improving ecosystem carbon sink capacity.Our findings support the suggestion that long-term thinning of larch plantation in Northeastern China can improve forest carbon sink through both increase in biomass carbon and decrease in soil heterotrophic CO


Thus, neither chaperones nor interaction partners are required during folding of this protein to its catalytically active state.Auto-phosphorylating kinase activity of plant leucine-rich-repeat receptor-like kinases (LRR-RLK's) needs to be under tight negative control to avoid unscheduled activation. One way to achieve this would be to keep these kinase domains as intrinsically disordered protein (IDP) during synthesis and transport to its final location. Subsequent folding, which may depend on chaperone activity or presence of interaction partners, is then required for full activation of the kinase domain. Bacterially produced SERK1 kinase domain was previously shown to be an active Ser/Thr kinase. SERK1 is predicted to contain a disordered region in kinase domains X and XI. Here, we show that loss of structure of the SERK1 kinase domain during unfolding is intimately linked to loss of activity. Phosphorylation of the SERK1 kinase domain neither changes its structure nor its stability. Unfolded SERK1 kinase has no autophosphorylation activity and upon removal of denaturant about one half of the protein population spontaneously refolds to an active protein  Plant Receptor-like Kinases (RLK's) belong to a large gene family Arabidopsis thaliana, the Somatic Embryogenesis Receptor-like Kinase 1 (SERK1). SERK1 is part of a protein family consisting of five highly conserved leucine-rich-repeat receptor-like kinases that function in plant signalling pathways Here, we focus on an RLK from in vitroArabidopsis thalianaPrevious studies show that the kinase domain of SERK1 (SERK1-KD) is active SERK1 functions in an oligomeric complex with other RLK's such as BRI1, the main ligand binding receptor for BRs Arabidopsis thaliana proteome, it has been predicted that 29% of its proteins contain intrinsically disordered regions, and 8% of its proteome consists of completely disordered proteins Since RLK activation is often the first response to a signal, RLK's need to be kept in the non-responsive or “off”-state in absence of activating ligands. One way to achieve this would be to keep these RLK's disordered during synthesis and transport to their final location. Many proteins are intrinsically disordered and fold, in whole or in part, upon binding to their physiological targets An example of IDP's in plants is the GRAS-protein family of transcriptional regulators in vitro. These data show that SERK1-KD predominantly is a structured protein at ambient condition. Loss of this structure is intimately linked to loss of activity of SERK1-KD. In vitro, unfolded SERK1-KD spontaneously refolds to an active state in absence of transphosphorylating partners or chaperones.In this study we show that use of a disorder analysis computer program predicts that the kinase domain of SERK1 (SERK1-KD) possibly contains a disordered region. We report experimental data of folding, stability and folding-induced phosphorylation activity of SERK1-KD www.ebi.ac.uk/Tools/msa/clustalo/). Potential presence of disordered regions within these protein sequences was analysed using PONDR® VL-XT (www.pondr.com) www.dabi.temple.edu/disprot/) Kinase domains and C-terminal tails of SERK1 , SERK3/BAK1 , FLS2 , BRI1 , MPK3 , EFR (AT5G20480 residues 705–1031) and BSK1  were defined by sequence alignment using Clustal Omega (version 1.1.0) from the EBI web server (C CGG ACA GCT CAA GAG GTT TTC T and TTA CCT TGG ACC AGA TAA CTC AAC GGC. Escherichia coli BL21* (Invitrogen) cells containing plasmid were grown in LB at 37°C until OD600 = 0.8, after which the culture was cooled to 20°C and protein production was started by addition of IPTG. After 20 h cells were harvested. Cells were broken using a French Press. The protein was purified by running cell free extract over a HIS-pure cobalt column . Subsequently, protein was further purified through use of a SourceQ-15 column. Final purification was achieved by analytical gel filtration on a Superdex 75 10/300 GL .SERK1-KD (residues 285–625) was cloned into the pET151 bacterial expression vector and contains an N-terminal His-tag (Invitrogen). Primers used for cloning were CA0 GL see . Purific°C. ATP was subsequently removed using a P-10 desalting column (Amersham Biosciences). For determination of phosphorylation status, SERK1-KD after autophosphorylation was subjected to immunoblotting with anti-Phosphoserine, anti-Phosphothreonine and anti-Phosphotyrosine antibodies (BD biosciences).Phosphorylated SERK1-KD was obtained by incubating 24 µM SERK1-KD with 3 mM ATP for 45 min at 30Fluorescence was measured using a Varian Cary Eclipse Fluorescence Spectrophotometer.°C in 10 mm quartz cuvettes.For denaturant induced unfolding, 0.24 µM SERK1-KD was incubated for 6 h at room temperature at various concentrations of urea, after which fluorescence spectra were recorded. For refolding, 12 µM SERK1-KD was first unfolded in 4.5 M urea for a period of 6 h. Subsequently, the sample was diluted 50 fold with buffer containing no urea and refolding took place during a period of 30 min at room temperature. Fluorescence emission was acquired from 290 to 600 nm, using an excitation wavelength of 280 nm with emission and excitation slits set to 10 nm. For each measurement, 5 scans were averaged and every nm a data point was collected. All spectra were recorded at 20°C to 70°C at a rate of 1°C/min. Data points were collected every 0.5°C. Protein concentration of both phosphorylated and native SERK1-KD was approximately 0.24 µM. Excitation was at 280 nm, and fluorescence emission was recorded at 340 nm. Emission and excitation slits were set to 10 nm. The thermal midpoint of unfolding as reported by fluorescence spectroscopy was determined by using a two state model of unfolding Fluorescence emission of SERK1-KD was also recorded during its temperature induced unfolding, which was achieved by increasing temperature from 15°C using 1 mm quartz cuvettes. All spectra were corrected by subtracting spectra of corresponding blank solutions. Buffer is 10 mM NaPi, pH 7.4, unless stated otherwise.Far-UV CD data were acquired on a Jasco J-715 Spectropolarimeter. Spectra were recorded at 20For acquisition of far-UV CD spectra of native and phosphorylated SERK1-KD, samples had a protein concentration of 5 µM and 5.5 µM respectively. Spectra were obtained by averaging 20 wavelength scans acquired from 185 to 260 nm. A data point was collected every 0.2 nm.For acquisition of far-UV CD spectra of native SERK1-KD in 0.45 M urea, 1.5 µM protein was incubated with 0.45 M urea. For spectra of unfolded SERK1-KD, 2.4 µM protein was incubated with 4.5 M urea. Both samples were kept at room temperature for 6 h before measurements commenced. For spectra of refolded SERK1-KD, native SERK1-KD at a concentration of 17 µM was first unfolded by incubation with 4.5 M urea for 6 h at room temperature. Subsequently, the sample was diluted 10 fold with buffer containing no urea and refolding took place during a period of 30 min at room temperature. Far-UV CD spectra were obtained by averaging 20 wavelength scans acquired from 210 to 260 nm. Below 210 nm the presence of urea causes considerable scatter of the CD signal. A data point was collected every 0.2 nm.°C to 80°C at a rate of 1°C/min and a data point was collected every 0.5°C. The thermal midpoint of unfolding as reported by far-UV CD spectroscopy was determined by using a two state model of unfolding Thermal unfolding of 5 µM native and 5.5 µM phosphorylated SERK1-KD was followed at 210 nm. Temperature was increased from 15For each measurement of SERK1-KD autophosphorylation, 0.5 µM SERK1-KD was incubated at a particular concentration of urea (ranging from 0 M to 3 M urea). All samples were kept at room temperature for 6 h before the assay was started. For refolding, 8 µM SERK1-KD was first unfolded in 3 M urea for 6 h at room temperature. Subsequently this sample was diluted with buffer containing no urea to a final concentration of 0.18 M urea and 0.5 µM SERK1-KD. Refolding took place during a period of 30 min at room temperature.32P-γATP (Perkin Elmer) was added to the samples. Final concentration of ATP in each sample was 50 µM. Phosphorylation took place for 30 min at 30°C and was stopped by addition of SDS-loading buffer. Samples were subsequently boiled for 3 min at 100°C and separated by SDS-PAGE. Incorporated radioactive phosphate was determined using a PhosphorImager. Relative intensities of radioactive bands were analysed using ImageJ software (ImageJ 1.45 s). Background intensity was subtracted and intensity at 0 M urea was set to 100%.To measure kinase phosphorylation activity, cold ATP and 500 µCi To identify possible regions of disorder within SERK1-KD, we use disorder predictors PONDR® VL-XT, VSL2B and IUPred. SERK1-KD consists of the catalytic kinase domain (residues 295–581) and a C-terminal region  of 45 amino acids. As a comparison, disorder predicted for several other plant kinases is also analysed. Sequence alignment identified the catalytic kinase domain and the C-terminal tail of these proteins. BAK1 (SERK3) is a close paralog of SERK1. Its disorder profile  stronglyBRI1, FLS2 and EFR are the main ligand binding LRR-RLK's for brassinosteroid, flagellin and EF-Tu respectively BSK1 and MPK3 are cytoplasmic kinases functional in brassinosteroid and flagellin signalling, respectively Protein phosphorylation is thought to occur predominantly within disordered regions of a protein E. coli is referred to here as native SERK1-KD. To assess whether phosphorylation of the kinase domain of SERK1 alters protein structure, and to obtain insight into potential control mechanisms of kinase activity in planta, we acquired far-UV CD spectra of native SERK1-KD and of in vitro autophosphorylated SERK1-KD. Phosphorylation status of SERK1-KD is assessed with antiphospho Ser/Thr/Tyr-antibodies. Native SERK1-KD turns out to be only marginally phosphorylated compared to in vitro autophosphorylated proteins  is an important parameter determining refolding efficiency, since aggregation highly depends on protein concentration. This concentration is highest in the refolding sample used for activity determination, explaining the lower refolding efficiency observed  comparedArabidopsis thaliana is an inherent property of its kinase domain. Our results show that SERK1-KD regains its catalytic activity upon refolding in vitro, indicating that the protein has the intrinsic capacity to become active. Because in planta SERK1 RLK is a non-ligand binding co-receptor functioning in several signalling pathways, maintaining the inactive status of the kinase domain is necessary to prevent unscheduled signalling. Inactivation of the kinase domain could be achieved by either intra- or intermolecular inhibitory elements. One way by which such an inhibitory element could act is by maintaining parts of the protein disordered until activity is required.In this work we addressed whether the catalytic activity of LRR-RLK SERK1 from in vitro kinase phosphorylation activity and in planta function of SERK1 in plantaThe bioinformatics tool PONDR® VL-XT predicts that SERK1-KD is largely structured, with one extended region of disorder. This disordered region comprises part of its C-terminal tail and 41 amino acids in domains X and XI, regions previously identified as essential for both in planta are in predicted structured parts of the protein. Four of these sites are found in the activation loop of SERK1-KD , of which phosphorylation is known to be important for kinase activity Phosphorylation sites are often found in disordered regions of proteins, probably due to easy access of these sites in vitro. The observed coincidence of loss of activity and three-dimensional protein structure upon denaturant induced unfolding demonstrates that properly folded structure is essential for SERK1-KD to be active. Upon lowering denaturant concentration, unfolded SERK1-KD is able to refold to its native structure and regains catalytic activity. Thus, SERK1-KD folds to active kinase, while its cellular context comprising chaperones, membrane components and interaction partners, is absent. However, to counteract aggregation and make folding more efficient in the hugely crowded cellular context in vivo.Fluorescence, far-UV CD and activity data presented in this paper show that SERK1-KD is largely structured and active in planta SERK1 already contains an active kinase domain before it is transported to its proper location, the plasma membrane in planta to keep kinase activity of SERK1 in check. For other plant RLK's candidate inhibitory proteins have indeed been identified, such as BKI1 or protein phosphate 2A for BRI1 in planta, until its activity is required.For organisms it is important to keep RLK's inactive until their signalling is required to properly respond to signals. For plant RLK's this issue has not received much attention. We observe that SERK1-KD autonomously folds to its active state within 30 minutes. This observation suggests that Figure S1SDS-PAGE and Western blot of SERK1-KD. Left: SDS-PAGE of aliquots taken at different steps during SERK1-KD purification. Lane 1, cell extract from BL21* E. coli cells expressing 6xHIS-SERK1-KD; lane 2; eluate from HIS-pure cobalt column; lane 3, eluate from SourceQ-15; lane 4, eluate from Superdex 75 10/300 GL ; lane 5, Marker. Right: Western blot of final purified protein, using anti- His-tag antibodies. Lane 1, purified SERK1-KD; lane 2, Marker.(TIFF)Click here for additional data file.Figure S2SERK1-KD phosphorylation properties. Left: Autophosphorylation of SERK1-KD (1 µg); Right: Transphosphorylation of casein (1 µg) by SERK1-KD (0.2 µg). Aliquots are taken at the time points indicated (in minutes) and subsequently separated by SDS-PAGE. Incorporation of 32P-yATP is visualized using a PhosphoImager.(TIF)Click here for additional data file.Figure S3Phosphorylation status of native and autophosphorylated SERK1-KD. Anti- Phosphoserine, -threonine and -tyrosine antibodies are used to probe the phosphorylation status of SERK1-KD. Lane 1, 0.75 µg of SERK1-KD after 45 min of incubation with ATP; lane 2, 0.75 µg of SERK1-KD as purified from E. coli; M, marker.(TIF)Click here for additional data file.Figure S4Far-UV CD spectrum of refolded SERK1-KD. SERK1-KD was first heated to 85°C and subsequently cooled to 15°C. The far-UV CD spectrum of this protein differs from the corresponding spectrum of native SERK1-KD shown in (TIF)Click here for additional data file.


Pasteurella multocida toxin (PMT) is the main virulence factor to induce P. multocida-associated progressive atrophic rhinitis in various animals. PMT leads to a destruction of nasal turbinate bones implicating an effect of the toxin on osteoblasts and/or osteoclasts. The toxin induces constitutive activation of Gα proteins of the Gq/11-, G12/13- and Gi-family by deamidating an essential glutamine residue. To study the PMT effect on bone cells, we used primary osteoblasts derived from rat calvariae and stromal ST-2 cells as differentiation model. As marker of functional osteoblasts the expression and activity of alkaline phosphatase, formation of mineralization nodules or expression of specific transcription factors as osterix was determined. Here, we show that the toxin inhibits differentiation and/or function of osteoblasts by activation of Gαq/11. Subsequently, Gαq/11 activates RhoA via p63RhoGEF, which specifically interacts with Gαq/11 but not with other G proteins like Gα12/13 and Gαi. Activated RhoA transactivates the mitogen-activated protein (MAP) kinase cascade via Rho kinase, involving Ras, MEK and ERK, resulting in inhibition of osteoblast differentiation. PMT-induced inhibition of differentiation was selective for the osteoblast lineage as adipocyte-like differentiation of ST-2 cells was not hampered. The present work provides novel insights, how the bacterial toxin PMT can control osteoblastic development by activating heterotrimeric G proteins of the Gαq/11-family and is a molecular pathogenetic basis for understanding the role of the toxin in bone loss during progressive atrophic rhinitis induced by Pasteurella multocida.The 146-kDa  Pasteurella multocida causes as a facultative pathogen various diseases in men and animals. One induced syndrome is atrophic rhinitis, which is a form of osteopenia, mainly characterized by facial distortion due to degradation of nasal turbinate bones. Strains, which especially affect bone tissue, produce the protein toxin P. multocida toxin (PMT). Importantly, PMT alone is capable to induce all symptoms of atrophic rhinitis. To cause osteopenia PMT influences the development and/or activity of specialized bone cells like osteoblasts and osteoclasts. Recently, we could identify the molecular mechanism of PMT. The toxin constitutively activates certain heterotrimeric G proteins by deamidation. Here, we studied the effect of PMT on the differentiation of osteoblasts. We demonstrate the direct action of PMT on osteoblasts and osteoblast-like cells and as a consequence inhibition of osteoblastic differentiation. Moreover, we revealed the underlying signal transduction pathway to impair proper osteoblast development. We show that PMT activates small GTPases in a Gαq/11 dependent manner via a non-ubiquitously expressed RhoGEF. In turn the mitogen-activated protein kinase pathway is transactivated leading to inhibition of osteoblastogenesis. Our findings present a mechanism how PMT hijacks host cell signaling pathways to hinder osteoblast development, which contributes to the syndrome of atrophic rhinitis. Streptococcus mutans, Actinobacillus actinomycetemcomitans or Staphylococcus aureus inter alia. The mechanism of bacterial-induced bone damage may be caused by factors released from pathogens, which interact with bone matrix or affect bone cells, or by bacteria which directly invade bone cells to initiate pathological changes Pasteurella multocida, which causes various diseases in men and an`imals. As a commensal P. multocida is found mainly in the nasal/pharyngeal space of domesticated and wild animals and is frequently isolated from cat and dog bites P. multocida is directly or as a supportive factor connected to several diseases like haemorrhagic septicaemia in hoofed animals, avian cholera or snuffles in rabbits P. multocida has a central role Bone tissue is a common target for bacterial infections. Diseases like caries, periodontitis or osteomyelitis are due to infections by Pasteurella multocida toxin (PMT), which is produced by capsular type D and some type A strains in vitro models the toxin inhibits osteoblastic differentiation and stimulates the differentiation of osteoclasts The causative agent of atrophic rhinitis is q/11-, Gα12/13- and Gαi-family Up to date a detailed analysis of PMT-activated signaling pathways in osteoblasts was hampered by the fact that the intracellular substrate of the toxin was unknown. Recently, we identified the molecular mechanism of PMT. The toxin stimulates heterotrimeric G protein signaling. In the switch II region of the α-subunit of heterotrimeric G proteins, PMT deamidates a specific Gln residue, which is involved in GTP hydrolysis s and Gαi signaling appear to control differentiation of bone cells in an opposite manner s and Gαi on osteoblasts depend at least partly on the regulation of adenylyl cyclase q blocked differentiation of osteoblasts. Transgenic mice, expressing this mutant in osteoblast progenitors, developed osteopenia Differentiation and activity of osteoblasts and osteoclasts are tightly regulated. Osteoblast differentiation is stimulated by various factors like BMP, PTH or growth factors as IGF or TGF, acting on different types of receptors Also the mitogen-activated protein kinase (MAPK) pathway contributes to bone development. However, the data available are inconsistent, because studies provide evidence for positive as well as for negative effects on bone cell development upon MAPK activation q/11, subsequent stimulation of RhoA/Rock pathway via p63RhoGEF and transactivation of MAPK cascade.Elucidation of the molecular mechanism of PMT and recent progress in the understanding of bone cell development prompted us to analyze the effects of PMT on osteoblasts and osteoblast precursors in more detail. Here we present evidence that PMT controls the differentiation of osteoblasts by constitutive activation of GαC1165S inhibited the osteoblast formation. Moreover, as determined by the concentration dependent blockade of ALP activity, the toxin was found to be a strong inhibitor of osteoblastic differentiation. 10 pM PMT nearly abolished osteoblastic differentiation (enzyme labeled fluorescence (ELF) 97, which specifically stains ALP. Control cells showed strong staining with ELF97, indicating proper osteoblast differentiation, whereas no alkaline phosphatase staining was visible in PMT-treated cells  or CCAAT/enhancer-binding family of proteins (C/EBPα). Congruently, quantification of mRNA expression levels of those markers showed no inhibitory effects of PMT .C1165S led to deamidation under these conditions y from q/11- and Gα12/13-family of heterotrimeric G proteins. Via different Rho guanine nucleotide exchange factors (GEF) like p115RhoGEF or p63RhoGEF the heterotrimeric G proteins are linked to RhoA q/11 dependent portion of PMT-induced RhoA activity, we utilized a specific inhibitor of Gαq/11 signaling, YM-254890 q/11 in PMT-induced RhoA activity prompted us to study the effect of Gαq/11 inhibition on osteoblast differentiation. Blockade of PMT-induced Gαq/11 activation by YM-254890 abrogated the toxins effect on ALP activity and expression in ST-2 cells  pathway has been implicated in regulation of osteogenesis perfringens large), a toxin produced by Clostridium perfringens. TpeL mono-O-GlcNAcylates specifically Ras and inhibits thereby Ras signaling and Ras-Raf interaction Next, we asked whether PMT-induced MAPK activation is involved in the inhibition of osteoblast differentiation. The MEK-1 inhibitor PD98059 effectively abrogated the PMT effect on osteoblastogenesis in ST-2 cells and primary osteoblasts as measured by ALP activity. PD98059 also blocked PMT-induced ERK phosphorylation . The MAPq/11- induced MAPK activation was studied by using the inhibitor YM-254890 and the p63RhoGEF knockdown cells. YM-254890 abolished PMT-induced ERK phosphorylation. Most interestingly, also the knockdown of p63RhoGEF decreased PMT-induced MAPK signaling in stromal ST-2 cells  exhibited no inhibitory effect on osteoblastic differentiation of ST-2 cells. The high potency of PMT is reflected by the low concentrations sufficient for impairment of cell differentiation.Atrophic rhinitis is characterized by increased bone resorption by osteoclasts and a lack of bone regeneration by osteoblasts Because of the high potency and effectiveness of PMT to block osteogenic differentiation, we were prompted to test, whether the toxin inhibits any kind of differentiation in stromal ST-2 cells; e.g. osteogenic and/or adipogenic differentiation. Therefore, we cultured stromal ST-2 in a medium forcing adipogenesis. Interestingly, PMT was not able to reduce differentiation of the adipocyte lineage. In line with these findings, the toxin was not able to reduce the mRNA levels of PPARγ and C/EBPα, which typically increase during adipogenesis of ST-2 cells. However, PMT even increased Oil Red O staining. Whether the observed rise in cell number completely accounts for increased Oil Red O staining or the increased expression of transcription factors like C/EBPα is additionally involved, should be clarified in further studies. If the toxin even enhances adipocyte development should also be addressed in a subsequent work.The data indicate that PMT is not a general inhibitor of any type of differentiation but a specific inhibitor of osteoblastic but not adipogenic differentiation in stromal cells like ST-2. In another cell culture model PMT inhibited adipogenesis Primary osteoblasts from newborn rat calvariae were used to verify the results obtained with the cell culture model ST-2. Martineau-Doizè and colleagues previously demonstrated that PMT-challenged rats develop atrophic rhinitis like symptoms in vitro systems. Mulan and coworkers performed studies with co-cultures of osteoblasts and osteoclasts. They discussed the question whether both osteoblasts and osteoclasts are directly affected by PMT or whether the toxin targets one cell type to induce effects in the other cell type in a paracrine manner PMT reportedly affects primary osteoblasts and osteoclasts in various Although PMT did not impair adipocytic differentiation of ST-2 cells, we could also verify the toxin activity under adipocytic conditions. Therefore, we conclude that PMT specifically inhibits the osteoblastic differentiation of stromal ST-2 cells but not adipocytic differentiation, although G proteins are deamidated under both conditions.12/13 and Gαq/11 and PMT was shown to stimulate RhoA via both G protein families q/11, YM-254890 q/11 but not on Gα12/13.To analyze the signal transduction pathway of PMT-induced blockade of osteoblastogenesis, the role of Rho proteins was studied. RhoA is a common effector of Gαq/11 inhibition on PMT-dependent blockade of osteoblast differentiation. Gαq/11 inhibition by YM-254890 abolished the inhibitory effect of PMT on the expression of osteoblast markers  during osteoblast differentiation. These results confirmed the important role of Gαq/11 in PMT-induced effects. Moreover, our results are in line with the studies of Ogata et al. showing that ectopical expression of a constitutive active mutant of Gαq impairs differentiation and induces osteopenia In a next step we studied the effect of GαIn addition, inhibition of Rock abrogated the effects of PMT on osteoblast differentiation in stromal ST-2 cells and primary osteoblasts reconfirming the pivotal role of active RhoA/Rock as a negative regulator of osteoblastic differentiation q/11 play pivotal roles in PMT-induced osteoblast impairment, we were encouraged to elucidate the missing link between these signaling factors. Heterotrimeric G proteins activate RhoA via RhoGEF proteins, e.g. p115RhoGEF (Gα12/13) or LARG  q/11 but not Gα12/13, to RhoA activation q/11-p63RhoGEF axis. Therefore the Gαα12/13-induced RhoA activation via other RhoGEFs may represent only a minor part of entire RhoA activity. Finally, the inhibitory effect of PMT on osteoblastogenesis in primary osteoblasts was compared to p63RhoGEF knockdown cells. Primary osteoblasts depleted for p63RhoGEF were not affected by PMT, whereas differentiation of control cells was inhibited by PMT intoxication. These results strongly suggest that Gαq/11 activity is inhibitory to osteoblast differentiation. Furthermore, RhoA stimulation due to the Gαq/11-specific p63RhoGEF is sufficient for this effect.Because RhoA and Gαq/11, Rock and knockdown of p63RhoGEF blocked the toxin-induced ERK phosphorylation. This indicates, that PMT-activated Gαq/11 leads via p63RhoGEF, RhoA and Rock to a transactivation of the MAPK cascade. Inhibition of Ras by TpeL toxin, which inactivates Ras by GlcNAcetylation The contribution of MAPK signaling in osteoblast differentiation is under discussion q/11q effector, involved in MAPK transactivation via RhoA. p63RhoGEF exhibits a restricted tissue distribution q/11 dependent signaling. Thus, we suggest that PMT affects pre-/osteoblasts by activating the Gαq/11-p63RhoGEF-RhoA axis. This leads to transactivation of the MAPK pathway resulting in inhibition of the osteoblastogenesis.Previously, RhoA-induced MAPK signaling has been implicated in osteoblastogenesis q/11 PMT activates α-subunits of the Gα12/13 and Gαi family q/11 pathway apparently prevails.Besides GαP. multocida and/or Bordetella bronchiseptica give rise to atrophic rhinitis B. bronchseptica and P. multocida are dermonecrotic toxin (DNT) and PMT, respectively. DNT activates small GTPases of the Rho family like RhoA, Rac and Cdc42 by deamidation or polyamination and impairs osteoblastogenesis q/11-p63RhoGEF, might account for the exacerbation of the disease in the case of coinfection. A single infection with B. bronchiseptica causes only moderate bone loss whereas coinfection with P. multocida induces more drastic effects called progressive atrophic rhinitis P. multocida as the causative agent of progressive atrophic rhinitis without any coinfection necessary The pivotal clinical symptom of atrophic rhinitis is the atrophy of nasal turbinate bones. Over the last decades it was discovered that infections with P. multocida as a strong functional interaction of bone and the immune system takes place P. multocida reduces serum IgA and IgG response to ovalbumin q/11-p63RhoGEF axis is associated with these previously described immune modulatory effects of PMT should be analyzed in further studies. This would present an important function of PMT in addition to the manifest destruction of bone tissue.Besides this obvious impact on bone tissue, the inhibition of osteoblastogenesis by PMT might result in a further pathogenetic advantage for q/11 activation impairs osteoblastogenesis via a RhoA – MAPK pathway . All other reagents were of analytical grade and purchased from commercial sources.Murine stromal ST-2 cells were obtained from the Leibniz Institute DSMZ (German Collection of Microorganisms and Cell Cultures) and cultivated in RPMI 1640 supplemented with 10% FCS. ST-2 cells were incubated to differentiate into adipocytes or osteoblasts in the corresponding induction medium for 10 days. The adipocyte differentiation medium (adm) consisted of RPMI 1640 (Gibco) supplemented with 10 µg/mL insulin (Sigma) and 40 ng/mL dexamethasone (Sigma). The osteoblast differentiation medium (odm) consisted of RPMI 1640 (Gibco) supplemented with ascorbic acid (284 µM) and β-glycerophosphate (200 µM). Medium including supplements was changed every two days.Primary osteoblasts were isolated from 2–3 day old rats using collagenase digestion 2, 1 mM EDTA, 1% Nonidet P-40, 0.5% (w/v) deoxycholate and 0.1% (w/v) SDS), containing complete protease inhibitor (Roche) and phosphatase inhibitor cocktail 2 and 3 (Sigma). Protein concentrations of the lysates were determined by Bradford measurement. Lysates with equalized amounts of protein were used for immunoblot analysis.Cell lysates were prepared as follows: Cells were grown to confluency and serum starved overnight. After 30 min preincubation with the indicated inhibitors the cells were incubated with PMT . Thereafter cells were lysed in RIPA buffer , 12 mM MgCl2, 0.1 mM ZnCl2 and 100 mM glycine-NaOH, pH 10.5) for 10 min at 37°C. The reaction was stopped by the addition of 200 mM NaOH. The absorption was determined at 405 nm.To perform the alkaline phosphatase assay cells were cultured for 10 (ST-2) or 4 (primary osteoblasts) days in odm. To measure alkaline phosphatase activity the cells were washed with PBS and then incubated with ALP assay solution  ELF97 (Life Techn.) in the absence of light for 15 min at 25°C. Again cells were washed twice with PBS and incubated for 1 h with a 0.02% SYTO Green/PBS solution. Cells were mounted with Mowiol 4–88 (Carl-Roth).et. al 2008 van Kossa staining: Cells were stained as described by Mukherjee Oil Red O staining: To detect lipid droplets, cells were stained with Oil Red O. Therefore, the cells were fixed in PFA and incubated with a saturated Oil Red O solution. Unbound Oil Red O was washed out with 70% (v/v) EtOH. To quantify the amount of Oil Red O bound to lipid droplets, the dye was extracted with a 4% (v/v) Nonidet P-40/isopropanol solution. The absorption was measured at 520 nm.−ΔΔCt.Total RNA was extracted from either ST-2 cells or primary osteoblasts with the RNeasy Mini Kit (Qiagen). cDNA was prepared using the QuantiTect Reverse Transcription Kit (Qiagen). All Kits were used following the manufacturer's manual. Quantitative PCR was performed using GoTaq qPCR Master Mix (Promega). The expression levels of the ribosomal Protein S29 (mouse) or HPRT (rat) were used as an internal control and fold changes were calculated using the ΔΔCt method. Values are shown as 2ATGGGTCACCAGCAGCTCTA, reverse-AGCCTATGTCCTTCGCGTACT, mALP: forward-AATGAGGTCACATCCATCCTG, reverse-CACCCGAGTGGTAGTCACAA, mPPARγ : forward-AAGACAACGGACAAATCACCA, reverse-GGGGGTGATATGTTTGAACTTG, mC/EBPα: forward-AAACAACGCAACGTGGAGA, reverse-GCGGTCATTGTCACTGGTC, rHPRT: forward-GACCGGTTCTGTCATGTCG, reverse-ACCTGGTTCATCATCACTAATCAC, rALP: forward-GCACAACATCAAGGACATCG, reverse-TCAGTTCTGTTCTTGGGGTACAT, rRUNX2: forward-CCACAGAGCTATTAAAGTGACAGTG, reverse-AACAAACTAGGTTTAGAGTCATCAAGC, rSP7: forward-CGTCCTCTCTGCTTGAGGAA, reverse-TGGAGCCACCAAACTTGCThe following primer pairs were used for analysis: mS29: forward-2O2) was used to detect binding of the second horseradish peroxidase-coupled antibody with the imaging system LAS-3000 (Fujifilm). Quantifications of immunoblots were done using MultiGauge software.For immunoblot analysis proteins were subjected to SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride-membrane. Anti RhoA-antibody (sc-418 (26C4)) was purchased from Santa CruzBiotech , anti p63RhoGEF-antibody (51004) from Proteintech, anti pERK-antibody (4370S) from New England Biolabs, anti tubulin-antibody (T9026) from Sigma Aldrich and anti p84-antibody (ab487) from Abcam. Deamidation specific antibody anti-Gαq Q209E (3G3) was kindly provided by Dr. Y. Horiguchi  To detect the levels of activated RhoA a Rhotekin pulldown assay was performed as described previously For adenoviral infection cells were seeded and directly supplemented with sh-virus suspension and 8 µg/mL polybrene. A specific p63RhoGEF shRNA was used and as control a specific GFP shRNA Results are presented as means ± S.E. Significance was assessed by paired Student's t test. p values<0.05 were considered statistically significant . Multiple group comparisons were analyzed by ANOVA followed by Student's t test.www.felasa.eu/guidelines.php) and the national animal welfare body GV-SOLAS (www.gv-solas.de). The animal welfare committees of the universities of Freiburg as well as the local authorities  approved all animal experiments.All animal experiments were performed in compliance with the German animal protection law (TierSchG). The animals were housed and handled in accordance with good animal practice as defined by FELASA Click here for additional data file.Figure S1Inactivation of Rac1 by TpeL. ST-2 cells were treated with Ras inactivating Clostridium perfringens toxin TpeL at indicated concentrations overnight. Thereafter, cells were lysed and subjected to immunoblot analysis. Rac1 was detected with glucosylation-sensitive antibody (non-glucosylated Rac1) or with glucosylation-insensitive antibody . TpeL modifies Rac1 only at concentrations higher than 5 nM. The Rac1 modifying Clostridium difficile toxin A (TcdA) was used as control (1 nM). Shown is a representative immunoblot. Quantification was done by using MultiGauge and demonstrated as fold induction normalized to untreated cells. The indicated Results are given as mean ± S.E. (n as indicated).(PDF)Click here for additional data file.


Missense mutants in the late endosomal Rab7 GTPase cause the autosomal dominant peripheral neuropathy Charcot-Marie-Tooth disease type 2B (CMT2B). As yet, the pathological mechanisms connecting mutant Rab7 protein expression to altered neuronal function are undefined. Here, we analyze the effects Rab7 CMT2B mutants on nerve growth factor (NGF) dependent intracellular signaling in PC12 cells. The nerve growth factor receptor TrkA interacted similarly with Rab7 wild-type and CMT2B mutant proteins, but the mutant proteins significantly enhanced TrkA phosphorylation in response to brief NGF stimulation. Two downstream signaling pathways (Erk1/2 and Akt) that are directly activated in response to phospho-TrkA were differentially affected. Akt signaling, arising in response to activated TrkA at the plasma membrane was unaffected. However Erk1/2 phosphorylation, triggered on signaling endosomes, was increased. Cytoplasmic phospho-Erk1/2 persisted at elevated levels relative to control samples for up to 24 h following NGF stimulation. Nuclear shuttling of phospho Erk1/2, which is required to induce MAPK phosphatase expression and down regulate signaling, was greatly reduced by the Rab7 CMT2B mutants and explains the previously reported inhibition in PC12 neurite outgrowth. In conclusion, the data demonstrate a mechanistic link between Rab7 CMT2B mutants and altered TrkA and Erk1/2 signaling from endosomes. Charcot-Marie-Tooth (CMT) disease is a family of inherited disorders that usually results in the degeneration of peripheral sensory and motor neurons Among the genes associated with CMT disease are those that encode proteins involved in the regulation of endocytosis and growth factor signaling, one of which is the Rab7 GTPase. Rab7 regulates transport of growth factors and other cargo from early to late endosomes and on to lysosomes by controlling membrane budding, cytoskeletal transport and fusion between endocytic compartments. Following nerve growth factor (NGF) stimulation of neurons, Rab7 forms a transient complex with the internalized NGF receptor called TrkA and thereby impacts receptor signaling and neurite outgrowth There are four mutations in the Rab7 gene that have been identified as disease-causal in CMT2B patients In the present study, we demonstrate that the Rab7 CMT2B mutants increase endosomal TrkA signaling leading to enhanced Erk signaling. The activated Erk proteins predominate in the cytoplasm in lieu of their normal nuclear localization following NGF stimulation of TrkA. These observations provide new insight into the molecular basis for the alterations in neurite outgrowth and axonal viability associated with the CMT disease state.The interaction of Rab7 with the receptor tyrosine kinase (RTK), TrkA is NGF dependent and furthermore the association is time dependent following nerve growth factor (NGF) stimulation Rab7 is known to control endosomal trafficking of TrkA as well as receptor signaling. To assess the effect of the Rab7 CMT mutants on NGF signaling, PC12 cells expressing the mutant proteins were briefly stimulated with NGF followed by removal of residual surface bound NGF  in regulating the level of phosphorylated Erk1/2. MKP3 expression is triggered by the translocation of the activated Erk to the nucleus, is maximal around 3 h after NGF stimulation and the upregulation can last as long as five days in PC12 cells In conclusion, our study throws light on the pathogenic mechanism leading to Charcot Marie Tooth Type2B disease by defining the enhanced Rab7-dependent activation of TrkA and downstream Erk signaling pathways.PC12 and BHK-21 cells were from American Type Culture Collection . PC12 cells were grown in DMEM supplemented with 10% horse serum, 2.5% FBS, 2 mM glutamine, 50 U/ml penicillin and 50 µg/ml streptomycin and BHK-21 were grown in complete G-MEM . Cell culture reagents were purchased from Invitrogen/Gibco . Restriction and modification enzymes were from New England Biolabs  and all chemicals used were from Sigma-Aldrich . Recombinant NGF was from Invitrogen.Rab7a used in these studies was Canis lupus familiaris (NM_001003316) GFP-Rab7 in the pEGFP-C3 vector. The plasmids were used as templates for PCR-based mutagenesis. All amino acid substitutions were generated by a one-step reverse cyclic PCR method using the appropriate base changes in the synthetic oligonucleotides 5′-ATTGACTTCGAAAACAGACAAGTGGC-3′, Rev-5′-CTTGTTTCCCAACACAACGAAAGGGA-3′),Rab7L129F ,Rab7K157N . Mutations were confirmed by sequencing the entire Rab7 gene.Rab7V162M (Fwd -Cell lines were cultured as described above and passaged on consecutive days to maintain in logarithmic growth phase immediately prior to transfection. Transfections of PC12 cells were performed using Lipofectamine 2000 (Invitrogen) according to manufacturer's instructions. Rab7 expression was maximal 16–24 h post-transfection and experiments were conducted during this time frame. Transfection efficiency of ∼35% was consistently observed.NGF (200 ng/ml) stimulation was performed as described previously Rabbit polyclonal antibody directed against Rab7 was used for immunoblotting, and immunoprecipitation assays as described 3VO4, pH 7.5 supplemented with protease inhibitor mixture from Calbiochem/EMD Chemicals, Gibbstown, NJ). Lysates were precleared by centrifugation at 13,000 rpm in an Eppendorf microcentrifuge for 10 min and pretreatment with protein A Sepharose ; 500 µg cell lysate/40 µl protein A Sepharose at 4°C for 3 h with mild rotation. The supernatant (precleared lysate) was transferred in a clean microfuge tube and incubated with 3 µg rabbit pAb directed against GFP (Invitrogen) for 2 h at 4°C. Antibody complexes were recovered by incubation with protein A Sepharose (40 µl) at 4°C for 3 h. Immunoprecipitates were washed in 0.1% NP-40, 25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM Na3VO4 supplemented with protease inhibitor and a final wash of ice cold, 10 mM Tris-HCl, and pH 7.5. The beads were boiled in Laemmli buffer and the proteins resolved by SDS-PAGE (10% gel).Sixteen hours (h) post-transfection, PC12 cells were washed in PBS and transferred to DMEM/BSA, starved for 5 h and stimulated with NGF for 10 min, followed by surface stripping of ligand with ice cold stripping buffer (0.2 M acetic acid and 0.5 M NaCl) 2 0.3% (v/v) NP-40, 1 mM PMSF, 1 mM Na3VO4, 10 mM NaF, 30 mM sodium β-glycerophosphate, 10 µg/ml chymostatin, 10 µg/ml leupeptin, 10 µg/ml antipain and 10 µg/ml pepstatin). Cells were incubated on ice for 5 min followed by centrifugation at 500×g for 5 min to pellet the nuclei. The cytosolic fraction separated in the supernatant. Nuclei pellets were washed in the same lysis buffer without NP40. The cytosolic and nuclear fractions were boiled in 2× Laemmli sample buffer and resolved on a 10% SDS polyacrylamide gel and probed with a phospho Erk1/2 antibody. The purity of the nuclear fraction was checked with an antibody directed against nuclear lamin B.Nuclear and cytosolic fractions were prepared as previously described with minor modifications 2) for 2–3 min, to allow optimal visualization of membrane structures and minimize cytosolic protein staining, and then fixed with 3% (w/v) paraformaldehyde in PBS. The coverslips were washed four times with PBS/saponin and mounted on glass slides in Mowiol 4–88 BHK21 cells were grown and processed for immunofluorescence staining using standard procedures. Briefly, transfected cells overexpressing Rab7 GFP and the Rab7 CMT2B mutants GFP were pre-permeabilized with 0.05% (v/v) saponin in PIPES buffer  Transiently transfected PC12 cells expressing GFP, GFP‐Rab7wt, GFP‐Rab7L129F, GFP‐Rab7K157N, GFP‐Rab7N161T and GFP‐Rab7 V162M were lysed 16 h post‐transfection and immunoblotted for Rab7 to probe for GFP tagged proteins as well as the endogenous Rab7 levels. (B) Films from three independent experiments were quantified using Image J analysis. Values of overexpressed (OE) GFP‐Rab7wt and GFP‐Rab7 CMT2B mutant protein expression were compared to endogenous (Endo.) Rab7levels. Error bars indicating mean ±S.E.M. n=3, *p<0.05; **p<0.01.(TIF)Click here for additional data file.