Hunter-gatherer societies have distinct social perceptions and practices which are expressed in unique use of space and material deposition patterns. However, the identification of archaeological evidence associated with hunter-gatherer activity is often challenging, especially in tropical environments such as rainforests. We present an integrated study combining ethnoarchaeology and geoarchaeology in order to study archaeological site formation processes related to hunter-gatherers’ ways of living in tropical forests. Ethnographic data was collected from an habitation site of contemporary hunter-gatherers in the forests of South India, aimed at studying how everyday activities and way of living dictate patterns of material deposition. Ethnoarchaeological excavations of abandoned open-air sites and a rock-shelter of the same group located deep in the forests, involved field observations and sampling of sediments from the abandoned sites and the contemporary site. Laboratory analyses included geochemical analysis , phytolith concentration analysis and soil micromorphology. The results present a dynamic spatial deposition pattern of macroscopic, microscopic and chemical materials, which stem from the distinctive ways of living and use of space by hunter-gatherers. This study shows that post-depositional processes in tropical forests result in poor preservation of archaeological materials due to acidic conditions and intensive biological activity within the sediments. Yet, the multiple laboratory-based analyses were able to trace evidence for activity surfaces and their maintenance practices as well as localized concentrations of activity remains such as the use of plants, metals, hearths and construction materials. Compared to agriculturalists, hunter-gatherer societies have more ephemeral site structures also due to the use of less durable materials in their activities e.g., –4). From. From4])Studying archaeological site formation processes involves the investigation of the anthropogenic factors—the processes driven by human activity forming and shaping the archaeological material record, and the natural and environmental factors—the geochemical processes altering or preserving the materials after their deposition . FollowiAlthough hunter-gatherer use of space has previously been studied through ethnoarchaeology at the macro-level  the study of material deposition patterns at microscopic scale that result from hunter-gatherer activity and 2) the investigation of natural post-depositional processes in tropical forests. The current sites are used as a pilot study to understand the use of space within hunter-gatherers habitation site as manifested by a set of different proxies . There is no suggestion that diverse contemporary hunter-gatherer groups are culturally homogenous or that these groups represent a relic of prehistoric hunter-gatherers. We embrace here an approach which examines how specific social practices, mainly mobility and immediacy, are expressed in the spatial distribution of microscopic and elemental signatures, and how they preserve after abandonment in a tropical environment.The study area is located in the forested hills of the Western Ghats mountain ridge in South India . This foFieldwork was conducted among a Nayaka hunter-gatherer group. The Nayaka were classified as immediate-return forest dweller hunter-gatherers by Bird-David (see selected publications –22). Birad hoc relationships between the people at the site.More recent work by Lavi e.g., , 25) exp exp25]) Based on these observations, the current geo-ethnoarchaeological study was carried out in order to test the preservation of anthropogenic activity markers related to hunter-gatherers way of living and to evaluate the post-depositional effect and processes on these markers in a tropical environment.Ethnographic study was carried out by Lavi and Friesem in a contemporary settlement located at the fringe of the forest. Permits for fieldwork were obtain through affiliation with the MS University of Baroda and the Spain-India collaborative NoGAP project. Observations and interviews were made through numerous visits to the same Nayaka site in 2010, 2012 and 2014, each time for a period of two, four and six months respectively. The informants have given their oral consent for the work and all the ethnographic data is reported anonymously. The ethnographic data supplied valuable information regarding peoples’ use of space and the material deposition patterns related to these tropical forest hunter-gatherers. In addition, field observations, excavations and sediment sampling were conducted in several other localities in the same forest area. Those localities were old sites, including open-air sites and a rock-shelter, abandoned ca. 20–30 years ago by the same group of people. These abandoned sites were located deeper in the forest and were recognized and introduced to us by the elder Nayaka people in the village where the ethnographic work took place.2) and open-air site 2 (OA2 = ca.90m2) were part of one settlement abandoned ca. 30 years ago (2) abandoned ca. 20 years ago  to provide quantitative data on the chemical elements. Representative bulk sediment samples (n = 77) were sent to ALS Laboratory Group in Seville (Spain), using method ME-41 ICP, where they were pre-treated with aqua-regia digestion for ICP-AES to measure the concentration of 35 main elements from Aluminum to zinc . Element-1, at 4cm-1 resolution using a Thermo Nicolet 380 spectrometer and interpreted using an internal library of infrared spectra of archaeological materials at the Kimmel Center for Archaeological Sciences at the Weizmann Institute of Science [All the bulk sediment samples (n = 110) were analyzed using Fourier-Transform Infrared (FTIR) spectroscopy in order to identify the mineral and organic components for each sample. The spectra were collected using the KBr method between 4000 and 250cm Science .Phytoliths were extracted from representative bulk sediment samples (n = 59) following the procedure of Katz et al. . PhytoliUndisturbed monolith sediment blocks (n = 5) were sampled using jackets made of Plaster of Paris. The blocks were dried in an oven at 30°C and then impregnated using a 9:1 mixture of polyester resin with acetone and 1% v:v MEKP. Pre-cut sample slices (2.6x5.5 inch) were ground to 30μm thickness thin sections following the method described by Murphy  and StooThe ethnographic observations conducted on contemporary site recorded how people’s activity resulted in the deposition of materials and how post-depositional behaviour influenced the preservation of these materials. Houses were mostly built from plant material such as bamboo, wood and grasses, but in some cases mud-bricks were also used . A typicThe processing and consumption of food resulted in the deposition of small fragments of bones and the remains of vegetables, fruits, oils and other liquids. Today plastic is part of the deposited waste, coming from food wrapping of items purchased in the markets. Maintenance of steel machetes and the production of timber tools resulted in the deposition of small chips of vegetal matter and metals. Hearths generally were of an ephemeral nature as they were often abandoned after a few days use, and a new fire started in another location on the terrace. Hearths used small amounts fuel, mostly wood, that, nevertheless produced sufficient amounts of ash and charcoal to be deposited on the terrace surface. Yet, the most significant activity in terms of material deposition patterns was the sweeping of the terraces, which was conducted on a daily basis. Sweeping resulted in the clearing and disposal of the waste materials that were normally re-deposited over the edge of the terrace. This process created small cumulative middens at the edge of the terraces on the hill-slope. The middens were actually the only designated areas where material were repeatedly deposited. Macroscopically, contemporary terrace surfaces look relatively clean of any activity remains except for some traces of ash in the location of recently abandoned hearths . FollowiThe abandoned sites, which were the main focus of this study, were covered with dense forest vegetation before excavations began . It woulDuring trenching of the sites some layering appeared in the excavation profiles but could not be interpreted as an activity surface as no artefacts were exposed during the excavation. The trench profiles revealed some variation. Open-air Site 1 had a lower brown-reddish layer, in some cases with rock fragments, and a brown to light brown sediment on top of it . Open-aiin situ of a hearth, which might have been used just before the abandonment of the site.The only macroscopic materials which could be associated with human activity were small fragments of charcoal found on the slope of open-air Site 1 . This fiGeochemical analysis of archaeological sediments is being broadly used for tracing residues of human activity , 33. RepThe entire set of results obtained from the geochemical analysis, including 35 elements, is presented in The concentrations of P, Sr, Ba, and Mg  were higThe concentrations of K, Na and Zn were higher in the contemporary site samples than in the rock-shelter samples, while the sediments of the two open-air sites exhibited even lower concentrations . To the Significant concentrations of Cu and Pb  were fou-1 and the bands in the O-H region of 3695-3445cm-1) and in most samples the main silicate absorbance band shifted to 1035cm-1 and it was wider .Black particles extracted from several samples presented absorbance bands indicative of charcoal . By usinHigh concentrations of phytoliths in sediments can be used as a reliable indicator of human activities associated with the exploitation of plants e.g., , 49–54).–54.49–54Two phytolith control samples were collected from a profile outside the range of human activities. The sample collected from the topsoil showed higher concentrations of phytoliths  than the sample collected below the topsoil  .The samples collected from the sites were divided into four groups according to their concentrations of phytoliths . The firThe second group of samples was attributed to sediments with phytolith concentrations ranging between 250,000 and 1M/g of sediment. This group includes the majority of the sediments sampled in the open-air and contemporary sites . The conin situ Nayaka activities. These samples could also represent specific locations of naturally occurring high phytolith concentrations. However, there are no differences in vegetation or taxa representation between the excavated sites and the spots sampled for control, and therefore we suggest that natural phytolith deposition is homogenous in all the study area. Thus, the accumulation of phytoliths with concentrations above 1M/g sediment should be regarded as in association to human activity.The third group of samples comprised sediments with phytolith concentrations higher than the control samples, ranging from 1M to 2M phytoliths/g sediment . This grThe last group, with the highest concentrations of phytoliths of above 2M /g includes only one sample from the contemporary site (CS-101) and several samples from the western part of the rock-shelter Figs  and 8. Tin situ hearths requires fast burial by colluvium [With five samples having more than 4M/g, the samples collected from the western part of the rock-shelter (in the upper and middle part of the profiles of sq. RS-C7—west profile only—and RS-B9) yielded the highest concentrations of phytoliths . Depositolluvium . The phyolluvium . Nayaka olluvium ). The spFurther study of the phytoliths assemblages including morphological analysis and identification of the taxonomy may in due course enhance the interpretation of the depositional processes of the phytoliths and their origins.Thin sections were produced from undisturbed sample blocks collected from open-air site 1 house, terrace and slope, open-air site 2 terrace and from the western part of the rock-shelter . The keyin situ buried soil that the house was built upon, or it may represent earthen construction material made from the local buried soil. In the latter case the structure of the fabric suggests that the material used as the floor make-up or for building the stage on which the house was built was not disturbed during the process. The similarity of the fabric to other fabrics found in the lowermost part of the profiles sampled elsewhere on the site strengthen its interpretation as the buried soil.The sample from open-air site 1 house (MM_OA1-House) was collected from the corner of the house near its wall, from the eastern profile of sq. OA1-K1 . Two thiAn horizon composed of two thin pure clay layers was found between the middle and low fabric no. 3 in . This feOne thin section was produced from a sample collected from the eastern profile of sq. OA1-F4, in the trench through the terrace of open-air site 1 . The samOne sample was collected from the slope of open-air site 1 from the eastern profile of sq. OA1-G11 . The sedOne sample was collected from open-air site 2 from the eastern profile of the trench in sq. OA2-D5 . AccordiOne block for micromorphological analysis was extracted from the southern profile of sq. RS-B9 in the rock-shelter . The samin situ hearth, coinciding with the site abandonment. Fabric 4 is interpreted as post-abandonment deposition of sediments and rock weathering, while the ‘turf-like’ fabric 3 represent the collapse of a lean-to thatched roof. This interpretation is further supported by the ethnographic data [Based on these observations, the lowermost fabric is interpreted as the surface on which activity took place, with the continuous deposition of gravels resulting from the rock weathering. The charcoal horizon represents an Nayaka architecture and their use of space reflects the importance of being-in-relation and living-together. In order to live together and maintain social relationships Nayaka people carry out most activities outdoors in full visibility of every person in the group, allowing a constant sharing of spaces, actions and things , 24. MobNevertheless, while field observations from the abandoned sites could not identify macroscopic artefacts (beside some remains of houses following the clearing of the dense forest vegetation), laboratory-based analyses identified occupation surfaces and deposits associated with hunter-gatherer activity .in situ, but enhance the chances for preserved horizons in secondary deposition on the slope beyond the living terrace used as the waste areas.Using micromorphology analysis, the house floor in open-air site 1 was identified but the sediment analysis showed that within the houses the floor is clean from activity remains. The terraces of the open-air sites yielded some spots with relatively high concentrations of phytoliths and heavy elements. Nonetheless, the overall pattern of occupation deposits associated with human activity does not support well defined activity areas for the designated use of space. Rather, the microscopic and chemical analyses support the ethnographic observation of hunter-gatherers’ tendency to act outside on the terrace, and the ephemeral nature of their activity areas. Site maintenance and cleaning practices observed during the ethnographic work were also evident at the micro- and sub-microscopic scale. The house floor in open-air site 1 showed indications of washing off and cleaning, and while no charcoal was found anywhere within the house or the terrace, it was found in high concentrations in the slope sediments. It is therefore concluded that routine cleaning practices reduce the chances for the preservation of primary occupation deposits The ethnographic data described the rock-shelter as a site of human habitation of several families, living under the rock and grass lean-to roof for at least several months. Yet, the only macroscopic remains that could be defiantly associated with past human activity were a few charcoal fragments buried under the topsoil. When examining the site’s sediments through microscopic and chemical analysis it was possible to trace the activity surface and collapsed thatch roof. Overall, the rock-shelter presented much better preservation of activity remains than the open-air sites.Archaeological site formation processes do not end with the deposition of materials due to human activity in the site. Post-depositional processes continue to affect the archaeological setting and may alter the archaeological record. The results of this study highlight the role of post-depositional and taphonomic processes in tropical forests and their implications for archaeological site formation processes in tropical environments .Samples collected from the contemporary site were used as control for occupation deposits prior to post-depositional alteration. When comparing the contemporary site samples to those from the open-air and the rock-shelter sites, several altering factors become evident. The tropical sediments appear to exhibit acidic conditions which result in complete dissolution of carbonates such as wood ash  and bone (in the form of carbonate hydroxylapatite) e.g., , 8, 10)., 10.8, 1The wet and warm conditions of tropical rainforests favor significant biological activity, which disrupts the sediment structure and enhances the degradation of organic matter. In addition, cycles of wet and dry conditions lead to impregnation with secondary iron , 8, 10. On the other hand, in acidic conditions charcoal and phytoliths do preserve and therefore can be used as markers for human activity in tropical forests. Being abandoned approximately at the same time, the rock-shelter presented a better state of preservation of organic matter as compared to the open-air sites. Yet, this could be the result of differences in the type of activity or due to the type of remains left behind during abandonment. Chemical analysis of elements associated with human activity  were relatively high in the contemporary site, as expected, and lower in the rock-shelter and much lower in the open-air sites. These lower values could be associated with post-depositional processes which indicate that chemical markers for anthropogenic activity are better preserved in rock-shelters as compared to open-air sites.The ethnoarchaeological approach of this study was designed to identifying the formation processes of the archaeological record of hunter-gatherer groups in tropical moist forest and to understand the anthropogenic signatures preserved in the archaeological record. In the light of this study, we argue that this is possible only through the integration of macro-, micro- and sub-microscopic approaches, which are apt to untangling the depositional and post-depositional processes that created the archaeological record. The ephemeral and moveable activities were carried out outside the houses on the external terrace. Social practices, site maintenance, cleaning practices and the impact of monsoon rains meant that primary deposition is minimal with little spatial differentiation (there is no direct correlation between activity area and deposition). The waste deposits (middens) at the edge of the activity areas (edge of platforms) are the areas where most of the sedimentation takes place and is preserved. The moist tropical environment further complicates the depositional and post-depositional dynamics due to significant biological activity, which disrupt the sediment structure and degrade the organic matter. In addition, the acidic conditions of the forest soil result in the dissolution of wood ash and bone, but charcoal and phytoliths all be preserved nonetheless.in situ activities. Although the identification and reconstruction of hunter-gatherer activity in archaeological sites within tropical forests is challenging, the use of several microscopic and chemical analyses allows the identification of anthropogenic signatures. In turn, this enables the reconstruction of the site formation processes including human activities and the use of space as well as post-depositional processes taking place that alter and shape the archaeological record.The current study has been able to identify the midden areas but also, and more importantly, highlighted the possibility of identifying the occupation surfaces and a few loci with primary deposition from S1 TableOA1 = open air 1; OA2 = open-air 2; RS = rock-shelter; CS = contemporary site.(XLSX)Click here for additional data file.S2 TableOA1 = open air 1; OA2 = open-air 2; RS = rock-shleter; CS = contemporary site.(XLSX)Click here for additional data file.


Pseudomonas aeruginosa, and present inhibition data for several ligands. These data should be useful for the production of homogeneous samples of phosphorylated or unphosphorylated proteins, which are essential for biophysical characterization of these enzymes.Most enzymes in the α-D-phosphohexomutase superfamily catalyze the reversible conversion of 1- to 6-phosphosugars. They play important roles in carbohydrate and sugar nucleotide metabolism, and participate in the biosynthesis of polysaccharides, glycolipids, and other exoproducts. Mutations in genes encoding these enzymes are associated with inherited metabolic diseases in humans, including glycogen storage disease and congenital disorders of glycosylation. Enzymes in the superfamily share a highly conserved active site serine that participates in the multi-step phosphoryl transfer reaction. Here we provide data on the effects of various phosphosugar ligands on the phosphorylation of this serine, as monitored by electrospray ionization mass spectrometry (ESI-MS) data on the intact proteins. We also show data on the longevity of the phospho-enzyme under various solution conditions in one member of the superfamily from  Specifications TableValue of the data•The catalytic phosphoserine of enzymes in the α-D-phosphohexomutase superfamily plays an integral role in enzyme mechanism.•We present data on the phosphorylation state of the serine under varying conditions.•These data will be useful information for preparation of homogeneous samples of phospho- or dephospho-enzyme for future biophysical and kinetic studies.1The data in this article show the effects of phosphosugar ligands and other variables on the phosphorylation state of the catalytic serine in several enzymes in the α-D-phosphohexomutase superfamily. Data were obtained by ESI-MS. Data on enzyme inhibition by several ligands is also presented.22.1Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase (G6PDH), and all bis- and monophosphorylated sugars except xylose 1-phosphate (X1P) were obtained from Sigma-Aldrich. X1P was kindly synthesized by Dr. Thomas Mawhinney (University of Missouri).2.2Pseduomonas aeruginosa PMM/PGM (PaPMM), Bacillus anthracis phosphoglucosamine mutase (BaPNGM), Salmonella typhimurium PGM (StPGM), Francisella tularensis PNGM (FtPNGM), and human phosphoglucosmutase 1 (hPGM1) were performed as described previously Expression and purification of 2.3Ligands tested for effects on phosphorylation included two bisphospho-sugars, and various monophosphosugars , which have been reported, in different instances, to either phosphorylate or dephosphorylate these enzymes 2.4Analysis of intact proteins by mass spectrometry was done as described previously The percentage phosphorylation was calculated by normalizing the sum of the dephosphorylated and phosphorylated peak heights to 1.0. As the proteins characterized herein are known to be phosphorylated on the conserved active site serine, and ESI-MS data confirmed a single phosphorylation site, no additional attempts were made to localize the site of phosphorylation. The exception to this was StPGM, which showed two phosphorylation sites via ESI-MS see .2.5Enzymatic activity of PaPMM was quantified by measuring the formation of glucose 6-phosphate (G6P) in a coupled assay with G6PDH. The conversion of NAD to NADH was monitored by UV–vis spectrophotometry on a CARY 100 spectrophotometer at 25 °C, as previously described Ki values for GlcN1P and the substrate analog X1P were determined as follows.For X1P, assays were performed with 0.1 µM enzyme, 1 µM G16P, and 10–500 µM G1P (substrate). For GlcN1P, assays were performed with 0.3 µM enzyme, 0.5 µM G16P, and 6.8–272 µM G1P. Data were fitted to the Michaelis–Menten equation. Apparent Km (for X1 P studies) or apparent kcat (for GlcN1P) values obtained at each inhibitor concentration were fitted using Eq. KI, the inhibitor dissociation constant. In Eq. Km and kcat are the Michaelis parameters of enzyme without the inhibitor.2.6A broad assessment of the effects of phosphosugar ligands on the phosphorylation of the catalytic serine was conducted on PaPMM, one of the best-characterized members of the superfamily Several other sequence-diverse proteins in the superfamily were examined for changes in phosphorylation: BaPNGM, FtPNGM, StPGM, and hPGM1. For all proteins, both G16P and F16P increase phosphorylation level of the active serine . For twoAs noted in 2.7Incubations of PaPMM with G1P and G6P and the related sugars GlcN1P and GlcN6P were performed . Under tSeveral concentrations of G1cN1P were tested at various time points in incubations with PaPMM. A time course of dephosphorylation  in the presence of increasing molar equivalents of GlcN1P is shown in A. The ef2.8Ki of 8.6±0.3 μM. GlcN1P shows evidence of noncompetitive inhibition with a Ki of 307±29 μM (To assess interactions between PaPMM and G1cN1P or GlcN6P, these two phosphosugars were tested as inhibitors for the PGM activity . GlcN6P 07±29 μM D.These data provide information on ligands and solution conditions that affect the phosphorylation state and lifetime of the active site serine of enzymes in the α-D-phosphohexomutase superfamily, and may assist with preparation of homogeneous protein samples for further studies.


Unc-51 Like Kinase 1 (ULK1) is a critical regulator of the biogenesis of autophagosomes, the central component of the catabolic macroautophagy pathway. Regulation of ULK1 activity is dependent upon several phosphorylation events acting to repress or activate the enzymatic function of this protein. Phosphorylation of Ser758 ULK1 has been linked to repression of autophagosome biogenesis and was thought to be exclusively dependent upon mTOR complex 1 kinase activity. In the present study, a novel regulation of Ser758 ULK1 phosphorylation is reported following prolonged inhibition of the Parkinson’s disease linked protein leucine rich repeat kinase 2 (LRRK2). Here, modulation of Ser758 ULK1 phosphorylation following LRRK2 inhibition is decoupled from the repression of autophagosome biogenesis and independent of mTOR complex 1 activity. LRRK2 locus have been identified in genome wide association studies (GWAs) that modestly increase the lifetime risk of PD [Macroautophagy is a critical eukaryotic catabolic pathway, conserved from yeast through to humans that allows the collection and degradation of cellular waste and contributes to maintenance of homoeostasis . The invsk of PD . The mossk of PD , leadingChemical compounds were purchased as follows: LRRK2in1  from theH4 cells (ATCC number HTB-148) were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% foetal calf serum (FCS). After 6 h from plating, medium was changed and H4 cells were treated with LRRK2 inhibitors LRRK2in1 or GSK2578215A, and/or with bafilomycin-A1 and/or torin-1 at the concentration and duration reported in each experiment. Vehicle  treated cells were used as controls. After incubation, cells were washed in Dulbecco’s phosphate buffered saline (DPBS) and collected in a lysis buffer containing: 0.5% Triton X-100, 2 mM ethylene di-amine tetra acetic acid (EDTA), 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate (SDS), protease inhibitors  and phosphatase inhibitors  in 50 mM Tris-HCl pH 7.5. m-TORC1 inhibition by starvation was achieved by overnight serum deprivation followed by exposure to Earle’s balanced salts solution for 2½ h before cell lysis.Primary astrocytes from cortex were isolated from 3 days old rats. The tissue was mechanically dissociated and trypsinized; the obtained cell pellet was plated in high glucose DMEM containing 20% FBS. At 7 days astrocytes were washed in PBS, trypsinized and seeded 1:2 in new flasks and kept in culture by splitting 1:2 when at confluency. Astrocytes were used for experiments after 14, 15 and 20 days in culture. They were seeded in six-well plates and treated with LRRK2-in1 as described for H4 cells. Purity was accessed by staining with anti-GFAP antibody D1F4Q, Cell Signalling.g for 5 min at 4°C. Protein concentrations were assessed by bicinchoninic acid assay  and 10–15 μg aliquots were separated by SDS polyacrylamide gel electrophoresis on Novex precast Bis-Tris 4–12% (Invitrogen), using MES running buffer (Invitrogen). After electrophoresis, gels were blotted onto 0.45 μm cut-off, PVDF membranes  and processed with antibodies diluted in Superblock solution (Pierce). Films were acquired as images in jpg format using an EPSON Perfection 4870 photo scanner and quantified using the ImageJ software (http://rsbweb.nih.gov/ij/).Cell lysates were frozen immediately upon collection. Lysates were clarified immediately before use by centrifugation at 10000 Statistical analyses were performed using Prism software (GraphPad). Details of the statistics are included in each figure legend. All the data are representative of results from at least three independent experiments. Quantification of each Western blot was done relative the β-actin loading control. In the case of phospho proteins, to avoid protein loss and degradation due to stripping procedures, all samples were run in two parallel gels. One gel was stained with the total antibody and one gel with the anti-phosphorylated protein antibody, each signal was normalized for the own β-actin loading control prior to normalization of phosphorylated protein over total. Comparison between different gels was performed after normalization against the internal, DMSO treated, control.As previously reported by our group, inhibition of the kinase activity of LRRK2 in the H4 astroglioma cell line results in mTORC1 independent induction of macroautophagy ,19. ConfTo assess a possible role for mTORC1/ULK1 in this increase in LC3-II following LRRK2 inhibition, we analysed Ser758 phosphorylation of ULK1 both as function of dose and time of treatment. No changes in ULK1 phosphorylation were recorded up to 6/10 h of LRRK2 kinase inhibition  co-treatment of cells with torin-1 and LRRK2in1 A or starde novo protein synthesis  and AMPK . During Previous studies have demonstrated that inhibition of mTOR induces a rapid de-phosphorylation of Ser758 ULK1 leading to the activation of ULK1 and to the formation of a protein complex able to initiate the macroautophagy cascade at the autophagosome assembly site . In the The data presented here confirm that LRRK2 has a role in the regulation of macroautophagy in astrocytes and supports a model where prolonged inhibition of LRRK2 kinase activity stimulates multiple feedback loops acting to repress canonical macroautophagy. Regulation of autophagy through feedback loops activated to maintain cellular homoeostasis is already documented ; the add


Substitution of the Ser/Thr-residues of this cluster by alanine residues enhances the transforming potential of B-Raf, indicating that these phosphorylation sites suppress its signaling output. Moreover, several B-Raf phosphorylation sites, including T401 and S419, are somatically mutated in tumors, further illustrating the importance of phosphorylation for the regulation of this kinase.B-Raf represents a critical physiological regulator of the Ras/RAF/MEK/ERK-pathway and a pharmacological target of growing clinical relevance, in particular in oncology. To understand how B-Raf itself is regulated, we combined mass spectrometry with genetic approaches to map its interactome in MCF-10A cells as well as in B-Raf deficient murine embryonic fibroblasts (MEFs) and B-Raf/Raf-1 double deficient DT40 lymphoma cells complemented with wildtype or mutant B-Raf expression vectors. Using a multi-protease digestion approach, we identified a novel ubiquitination site and provide a detailed B-Raf phospho-map. Importantly, we identify two evolutionary conserved phosphorylation clusters around T401 and S419 in the B-Raf hinge region. SILAC labelling and genetic/biochemical follow-up revealed that these clusters are phosphorylated in the contexts of oncogenic Ras, sorafenib induced Raf dimerization and in the background of the V600E mutation. We further show that the vemurafenib sensitive phosphorylation of the T401 cluster occurs  Drosophila and Caenorhabditis, respectively. Genetic approaches in mice and chicken DT40 B cells demonstrated that Raf-1 and B-Raf have unique, but also overlapping functions [BRAF mutations found in the neuro-cardio-facio-cutaneous syndromes or RASopathies [BRAFV600E/K mutations as the binding of these compounds to wildtype B-Raf provokes the paradoxical activation of the MEK/ERK pathway. This phenomenon, which causes therapy resistance and side effects including secondary neoplasms, involves the presence of active Ras and hetero-dimerization between a drug-bound and a drug-free Raf protomer [The Ras/Raf/mitogen-activated/extracellular-regulated kinase (MEK)/extracellular signal regulated kinase (ERK) pathway plays a pivotal role in controlling proliferation, survival and differentiation of metazoan cells. The Raf tier represents a particularly important node as these Ser/Thr-kinases are subject to a complex, still ill-defined activation process that integrates various protein-protein and –lipid interactions and positive as well as negative phosphorylation events -3. The Runctions . B-Raf, unctions . This isopathies , 10. Furopathies , 11. Theopathies , also taopathies , 14. Thuopathies . Howeverprotomer -19. Cons599VKS602-motif in its activation loop, a step mimicked by the most common oncogenic BRAF mutation, V600E [Our current knowledge of B-Raf regulation is best approached from a structural perspective . B-Raf an, V600E -24. The n, V600E -28 and cn, V600E , 30.D594A mutant, which behaves similarly to drug-bound B-Raf in that sense that it provokes paradoxical MEK/ERK phosphorylation by binding and transactivating Raf-1, also undergoes a dramatic EMS in cells with upregulated Ras activity [Although many details are still missing, the following model of the B-Raf activation cycle has emerged from studies conducted on B-Raf and Raf-1 over the last 20 years . In its activity , 16. In In this study, we report the detailed analysis of reconstituted and endogenous B-Raf complexes in various cellular model systems by quantitative and qualitative mass spectrometry. We provide a detailed catalog of interaction partners and map phosphorylation events induced by clinically relevant Raf inhibitors. Functional characterization of phosphorylation sites using combinatorial approaches of proteomics, biochemistry and genetics reveals molecular mechanisms underlying B-Raf regulation.Araf, this system provides the unique opportunity to study the function and protein-protein interactions of Raf mutants in vertebrate cells without the interference by endogenous Raf-proteins.To identify core and potentially accessory and cell-type specific components of B-Raf containing protein complexes as well as underlying regulatory mechanisms, we decided to analyze the composition of complexes and phosphorylation/ubiquitination sites on B-Raf itself across various cell lines and under different conditions. Therefore, we established four cell line models, allowing the highly efficient purification of hemagglutinin-(HA)-tagged B-Raf proteins. The first model system represents a genetic complementation system of B-Raf/Raf-1 double deficient DT40 cells  that werBraf deficient murine embryonic fibroblasts (MEFs) expressing a 4-hydroxytamoxifen- (4HT) controllable oncogenic H-RasG12V::ERTM fusion protein  to obtain quantitative insights into protein-protein interactions of B-Raf.Secondly, we used n Figure . In the tivation . Using tWT and B-RafCAAX, a constitutively activated Raf protein, which is tethered to the membrane via the polybasic region and CAAX-box of human K-Ras [WT and B-RafCAAX complexes  provide the unique opportunity to study the function, phosphorylation status and protein-protein interactions of B-Raf mutants without interference by the endogenous protein. Secondly, the complementation with a B-Raf cDNA allows the use of small epitope-tags such as the hemagglutinin (HA) tag for which highly specific antibody resins of high affinity are available. Since B-Raf is highly phosphorylated and entertains many protein-protein-interactions, antibodies raised against the endogenous protein might enrich or discriminate against certain subpopulations. Indeed, one commercially available antibody displays impaired binding to feedback-phosphorylated B-Raf . NeverthUsing label-free and SILAC-based quantitative mass spectrometry (MS), we aimed to obtain novel insights into the composition of B-Raf signalosomes Figure . TherefoIn addition to this B-Raf core interactome, we identified several other interaction partners, which have been reported as interaction partners of B-Raf or Raf-1 previously, further validating our approach. We detected peptides derived from the Casein kinase II holoenzyme and the peptidyl-prolyl-cis/trans-isomerase Pin1, which have been implicated in N-region phosphorylation and phosphorylation-dependent isomerization of Raf-kinases, respectively , 42. In Prohibitin has been implicated in the regulation of Raf-1 signaling . Here wePasteurella multocida toxin (PMT). This highly mitogenic bacterial toxin promotes the deamidation of a glutamine residue essential for GTP hydrolysis in the Gα subunits of heterotrimeric G-proteins, which in turn abolishes their intrinsic GTPase activity [Braf proficient MEFs with EGF or HRG promoted the interaction between B-Raf and Gα subunits, while stimulation with PMT caused an even stronger interaction between B-Raf and Gα subunits  and only eight have been subject to functional characterization so far , respectively. As a proof-of-principle, we noticed that the 14-3-3 binding site S365 and the adjacent S364 are replaced by leucine residues in tumor samples. Furthermore, P367, which represents an integral part of the 14-3-3 binding motif surrounding S365 , is sub65XP367; , 25, 27.in Raf-1 -62.http://www.phosphosite.org/siteAction.do?id=74357), but whose function is controversially described in the literature. In agreement with two other studies using mammalian cells [Xenopus oocytes suggests that phosphorylation of the S151 equivalent is required for activity of Xenopus B-Raf . In facXeB-Raf; , 27, 30.T Figure . Howevereraction , 63. Howd before . Similard before .BRAFP152S mutation  upon H-RasG12V::ERTM release indicating a similar pattern of PTMs. In contrast, MEFs expressing B-RafS465E showed lower pMEK/pERK levels compared to B-RafWT and B-RafS465A/D expressing cells and only a small and very discrete EMS upon H-RasG12V::ERTM release. This suggests that the glutamate substitution of S465 impairs the signaling potential of B-Raf and its activation dependent PTMs contributing to a maximum EMS. The strong negative charge introduced by the glutamate residue (or phosphate group) could either impair ATP coordination or block recognition of B-Raf by the HSP90 co-chaperone Cdc37, which binds to Raf kinases via its consensus motif GS465GSFG [Next, we focused on S465, which is located in the glycine-rich or P-loop and has been recently reported as an auto-phosphorylation site , althougas G469A , or RASoas G469A , 71. It as G469A , or MEK as G469A . We now d et al.  by showiS465GSFG . Both me599VKS602-motif and is required for maximum B-Raf activity [http://www.cancer.sanger.ac.uk/cancergenome/projects/cosmic/), albeit at very low frequency compared to other activation segment mutations such as V600E/K. In order to analyze the role of this phosphorylation site and the so far unknown relevance of its mutation for the signaling potential of B-Raf, we replaced S605 by an alanine residue to prevent phosphorylation, by a phospho-mimetic glutamate residue or by a glycine residue, as the latter substitution represents the most frequently observed S605 alteration in the COSMIC database. Surprisingly, however, these substitutions had little to no effect on basal or oncogenic H-Ras stimulated B-Raf activation  and immunoprecipitates. In contrast, alanine substitutions of the phosphorylation sites in the S419 cluster affected the EMS of B-RafD594A to a lesser but discernible extent. Similar findings were made for both clusters in the B-RafWT background in sorafenib treated MEFs with activated Ras signaling  assays involving B-RafV600E that this hyperactive oncoprotein also undergoes a pronounced EMS, which is reminiscent to that displayed by B-RafD594A in cells with active Ras signaling, we reasoned that the HR clusters could be targeted by an auto-phosphorylation in cis or in trans  to the IVK reaction prior to the addition of ATP  could phosphorylate these clusters. As we previously observed in ans Ref. . This asrodimers . To furts Figure . Plat-E s Figure , we can P Figure . This inhibitors . This inactivity . ConsequS Figure . Taken tV600E oncoprotein , a potent negative regulator of Ras proteins and tumor suppressor gene product [WT moderately increased the number and diameter of transformed foci compared to MEFs infected with the empty vector control construct, while expression of B-RafWT and NF1 knockdown cooperated in a more efficient focus formation. Focus formation was even more pronounced in MEFs transduced with B-RafT401cluster and, as expected from the well-established cooperation of kinase-inactive B-Raf mutants with active Ras [D594A proteins. Commensurate with its lack of an effect on MEK/ERK phosphorylation (compared to B-RafWT expressing MEFs), alanine substitution of the phosphorylation sites in the S419 cluster had no discernible impact on the transformation potential of B-RafWT. In summary, our data suggest that the hyper-phosphorylation of the T401 cluster represents a negative signaling event limiting the activation of B-RafWT.As conservation of the T401 cluster during vertebrate evolution suggests an important function, we next addressed its biological relevance. As mutation of both clusters had no discernible impact on the MEK/ERK phosphorylation potential of the highly active B-Rafn Figure , we deci product , 83  or Raf-1 or A-Raf is strongly enhanced by 4-HT treatment. These findings validate our approach and the identification of novel or less-established dynamic interaction partners such as Kidins220/ARMS  signaling systems. Indeed, while trans-activation of RTKs by GPCRs is a relatively well-established phenomenon , 88, othproteins . Secondlnd GNAO1 , 91. Furlue nevi , 93. Impmelanoma . Howevermelanoma -96. AlthV600E, of kinase-dead B-RafD594A or drug-bound B-RafWT , of B-RafD594A and even that of B-RafWT in the presence of oncogenic Ras and sorafenib [in trans and could be even mediated by another Raf-isoform such as Raf-1 or A-Raf that are both highly enriched in B-RafD594A complexes and should be strongly activated due to the paradoxical action of kinase-dead B-Raf [D594A mutant (B-RafD594A/AVKA) with alanine substitutions of the activation loop phosphorylation sites T599 and S602 exhibits an impaired EMS in the context of H-RasG12V::ERTM release and recruits significantly less Raf-1 than B-RafD594A proper [D594A and B-RafD594A/AVKA is reminiscent of that between B-RafD594A and B-RafD594A/T401cluster, it is tempting to speculate that the contrasting EMS of B-RafD594A and B-RafD594A/AVKA are caused by the less efficacious recruitment and transactivation of Raf-1 . Thgnaling; , 48, 78.orafenib . This suad B-Raf , 16. ThiA proper . As the see Ref.  for furtsee Ref. , 16; Fig B-RafWT , 23, it WT. As the structure of the HR is unknown and is probably highly disordered, we can only speculate about the precise molecular mechanisms by which phosphorylation of the T401 cluster contributes to B-Raf downregulation. For example, it is conceivable that HR phosphorylation counteracts the attachment to the negatively charged inner leaflet of the plasma-membrane by bulk changes in the electrostatic landscape of this B-Raf region [But what is the biological meaning of HR phosphorylation? While the precise role of the S419 cluster, which precedes one of the three established inhibitory AKT phosphorylation site S429 , remainsf region . Such a f region . Alternaf region , 30, 66.f region , 44, 66.in trans thereby contributing to the downregulation of B-Raf activity or expression . Indeeds [V600E . Moreoves [V600E . As we fs [V600E  and our 599VKS602-motif and S729. Thus, in line with its role as a potent proto-oncogene product, B-Raf is subject to multiple layers of negative regulation, allowing its fine-tuning in a spatio-temporal manner. Our study further highlights the usefulness to combine phospho-proteomic with sequencing data of tumor genomes, as both data sets can complement each other in the elucidation of phosphorylation site function.Finally, our study shows that B-Raf is a heavily phosphorylated protein. Although many of the sites identified or confirmed in broader settings by our study await their functional characterization, it is becoming clear that negative regulatory sites such as S151, S365, the T401 cluster, S429, S465 and the SPKTP-motif outnumber the few sites with a clear positive role such as S445, the TRaf-B , Raf-B , Raf-1 , β-actin , α-Tubulin , and NF1 (sc-67)were fromSanta Cruz Biotechnology; HA (3F10) monoclonal rat from Roche Bioscience. Antibodies against (p)ERK, (p)MEK were purchased from Cell Signaling Technologies. The Gα switch antibody and the source of PMT were described recently . DUSP6 wraf, pMIG/HAchB-raf, pAloxP-puro, pMIG/HAhBRAF and pMIG/BRAF-His/Myc expression vectors were described previously [BRAF vector was a kind gift from Dr. Niklas Engels (Göttingen) and contains the HA-tagged chicken c-Rmil/B-Raf cDNA [The pFLU/HAchB-eviously , 29, 107Braf−/− MEFs expressing the H-RasG12V::ERTM fusion protein has been described [BraffloxAVKA mice were used without previous Cre activation [Braf deficient mouse embryos  were described previously [The DT40 subline allowing the 4-hydroxy-tamoxifen (4-HT) inducible deletion of the chicken c-mil/raf-1 and c-Rmil/B-raf genes, DT40MCM/raf-1flE3/B-rafflE6 (DT40floxRaf in short) has been described [+ cells were complemented with pMIG/HAchB-raf as described previously [floxRaf cells (subclone DK37) were first converted to DT40Raf-less cells by 4-HT exposure for 24 h as described previously [AhdI-linearised pCDNA3.1loxPuro-HAchBRAF and selected with 0.5 μg/ml puromycine. PC12 cells were transfected by using Gene Juice (Novagen), kept in differentiation medium and scored for differentiation 6 days later as described previously [The propagation of DT40, PC12, MCF-10AecoR, Plat-E cells and escribed , 29, 108tivation . The geneviously . The DT4escribed . DK37+ ceviously . For indeviously  and subseviously , 29. Celeviously ) were ineviously . MEFs weeviously , 24, 74.These procedures were conducted as described in detail previously . In brieBRAF and V5-RasG12V expression vectors in a 2:1 ratio as described previously [HEK293T cells were co-transfected with pMIG/HAheviously . Cells weviously . AlphaScDetailed information on mass spectrometry procedures is provided in the supplement. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium  via the


Intrinsically disordered (ID) regions of the transcription factor proteins have much larger frequency of phosphorylation sites than ordered regions, suggesting an important role in their regulatory capacity. Consistent with this phenomenon, most of the functionally known phosphorylation sites in the steroid receptor family of transcription factors are located in the ID N-terminal domain that contains a powerful activation function (AF1) region. In this study, we determined the structural and functional consequences of functionally known phosphorylation residues  located in the human glucocorticoid receptor’s (GR’s) ID AF1 domain. We report the relative importance of each phosphorylation site in inducing a functionally active ordered conformation in GR’s ID AF1 domain. Our data demonstrate a mechanism through which ID domain of the steroid receptors and other similar transcription factors may adopt a functionally active conformation under physiological conditions. Like many other transcription factors, the activities of nuclear hormone receptors including glucocorticoid receptor (GR) are regulated by multiple phosphorylation sites4. The GR is phosphorylated in the absence of glucocorticoid, with enhanced phosphorylation occurring when it is bound to agonist but not antagonist steroids5. In the human GR, functionally important phosphorylated residues  are located within AF1, and are conserved among species6. In spite of discovery that AF1 is quantitatively the major transactivation domain of the GR, we are only beginning to understand its structure-function relationship. Lack of this information has undermined our understanding of how GR transmits the transcriptional signal from ligand to specific gene(s). There are also reports suggesting that phosphorylation of Ser211 may affect GR conformation and thus alter receptor activity, and AF1 appears to be a main player in this process9. However, it is not yet known whether other phosphorylation sites (Ser203 and/or Ser226) also influence the structure and functions of the GR AF1. To understand how the GR transmits the transcriptional signal from ligand to specific gene(s), it is essential to gain the information regarding relative phosphorylation of each site on the structure and functions of the GR AF1.Post-translational modifications such as phosphorylation may regulate protein functions of transcription factors by affecting their conformational dynamics leading to altered transcriptional activities13. It has been reported that ID AF1 must undergo a disorder-order conformational transition for receptor’s optimal transcriptional activity16. Several studies have suggested that ID regions of the transcription factors have much higher frequency of known phosphorylation sites than ordered regions19. This is particularly true for the ID AF1 domain of steroid hormone receptors16. It has earlier been suggested that when phosphorylated at Ser211 site, GR AF1 may be structurally more stable and functionally more active20. However, the role of each phosphorylation site in regulating the conformation and functions of AF1 is not known. In this study, we report that GR’s ID AF1 domain undergoes conformational rearrangements leading to significantly enhanced AF1-mediated GR’s transcriptional activity in a site-dependent manner.The structure of AF1 of the steroid receptors has been difficult to determine because in solution it exists as an intrinsically disordered (ID) domain, frequently found in many transcription factors including steroid hormone receptorsC peptide (an AF1 core sub-domain), bearing phosphorylated vs. non-phosphorylated residues  together are in the phosphorylated state, unique structural rearrangements are observed  showed that when both Ser203 and Ser211 are phosphorylated, Ser203 becomes solvent exposed whereas Ser211 interacts with Trp213 Figure . Similar8. On the other hand AF1 mutants Ser203A or Ser226A showed significant structural changes, though slightly less than WT AF1  reporter assay in GR-deficient CV-1 cells as describedons Fig. S. Lackinone Fig. . When GRone Fig. . Experimone Fig. . It is iTo further understand the effect of phosphorylation on the GR-dependent transcriptional activity, we measured the effects of phosphorylation on the AF1-mediated transcriptional activity of the GR using various combinations of site-specific mutants. Cells transfected with double mutant GR500-S203A/S226A showed similar modest effects as in case of individual GR500-S203A or GR500-S226A Fig. . On the 16. The GR protein is subject to multiple phosphorylation sites that regulate the function of the receptor16. These sites located in the ID NTD/AF1 domain contribute to multiple functions and the net effect of an individual phosphorylation depends on the activating kinase22. It has been suggested that ID AF1 domains of the GR and other steroid receptors with flexible structural dynamics play a critical role in dictating final outcome responsible for specific gene regulation. Post translational modifications such as phosphorylation elicit diverse effects on the biological functions of ID proteins by altering the energetics of their conformational landscape and by modulating interactions with other cellular components23 by stabilizing and/or inducing secondary structural elements26. Thus, signaling cascades that induce phosphorylation of the GR are important factors in determining the physiological actions of the GR. It has earlier been reported that p38-mediated phosphorylation of Ser211 plays an important role in regulating GR AF1’s conformation and subsequent glucocorticoid-induced apoptosis in leukemic cells9. However, the underlying mechanism that governs this important and yet complex phenomenon involving all major phosphorylation sites is not well understood. Our results provide a mechanism for how each phosphorylation site within its activation domain contributes to GR-mediated signaling through conformational rearrangements in otherwise ID AF1 domain.The mechanisms by which GR and other steroid receptors control gene expression pose a central problem in molecular biology and the role of their transcriptional activation domains in this complex process is of immense importance42−), which alters the steric, chemical, and electrostatic properties and thereby providing novel possibilities for intra- and inter- molecular interactions, including hydrogen-bonding networks. Under physiological conditions, the phosphoryl moiety is chemically different from the acidic amino acids (Asp and Glu) often used as phospho-mimics27. Although ID regions lack the ability to fold into stable 3-D structures on their own under physiological conditions, they undergo disorder-to-order conformational transitions under specific cellular environment28. Thus, changes in the surrounding physico-chemical environment can determine the ability of ID regions to fold into native-like species. In this context, site-specific phosphorylation-induced conformational changes due to altered physical/chemical properties of ID regions can be exploited for regulating biology. Although there are multiple phosphorylation sites critical for GR transcriptional activity , their respective role in driving GR functions is highly cell specific. Because p38 mitogen-activated protein kinase (MAPK) directly phosphorylates GR in different cell types in a stimulus- and cell-dependent manner, we used p38 MAPK for in vitro GR phosphorylation. Based on our results from the present studies combined with the results from earlier studies8, we propose that phosphorylation-induced conformational changes in the ID AF1 region of the GR are dependent upon the phosphorylation of individual site(s). It is interesting to note that phosphorylated Ser203 though appears to bind to nearby amino acids both when phosphorylation takes place alone or in combination with Ser211 or Ser226, yet fails to result into any significant structural changes unlike phosphorylation of Ser211 as reported earlier8. Further, functional data support that the moderate increases in AF1-mediated reporter activities of the GR due to site-specific phosphorylation are mostly reversed by Glu substitution in case of Ser203 and Ser226 but not with Ser211.Phosphorylation of Ser residue replaces a neutral hydroxyl (OH) group with a negatively phosphoryl group , phosphorylated Ser211 makes contacts with Trp213 depending upon the status of phosphorylation of either Ser203 or Ser226 but not alone. We infer from the models that the interactions between Ser211 and Trp213 may result in hindering interactions between Ser211 and Lys206, as the Trp213 and Lys206 are predicted to be on the opposite sides of Ser211. Interestingly, mutation of Trp213 is known to significantly reduce receptor transcriptional activation29. We’ve also reported that the environment of the tryptophan (fluorescence steady state spectra) change upon Ser211 phosphorylation8. We therefore propose that the regions surrounding S211 are of critical importance to GR transcriptional activation perhaps because they contribute to the formation of the putative protein-protein interaction surfaces as reported earlier30. The structural and subsequent functional effects of phosphorylated Ser211 may thus be influenced by the relative phosphorylation of other two Ser residues. These findings support our previous studies where we found that p38-mediated phosphorylation of GR is involved in GR-mediated apoptotic events31 as well as p38 MAPK can in vitro phosphorylate all three sites8. These findings further support other reports, which have shown that the relative effects of Ser211 phosphorylation on GR-mediated target gene expression is dependent upon the phosphorylation status of other sites32. Mutational studies using various point mutations within the AF1C have clearly established the role of important hydrophobic amino acids including Trp213 among others in the interaction between the AF1C and coregulatory proteins and subsequent AF1-mediated GR’s transcriptional activity35.It is important to note that phosphorylation of Ser211 induces a more structured conformation in AF1ts Figs   similar 8 may provide critical information on how different surfaces within the AF1 domain may be created and used to manipulate GR target gene expressions. Tissue-specific splice variants of the steroid receptors are located within the N-terminal domain, which can modulate AF1 conformation, giving rise to multivalent interactions and thereby regulating diverse cellular processes. Previous studies have illustrated the importance of synergistic effects of both AF1 and AF2 for target gene expression and our results from present studies provide a starting point for evaluating mechanisms for this selectivity, which are likely to involve relative effects of each phosphorylation site and of course other post-translational modifications36. Together, these results extend our previous studies8 by providing a detailed mechanism for cell/tissue specific effects of gene regulation by GR, and the role of each phosphorylation site in this process. We contend that understanding how each phosphorylation site of the receptor influenced by cell/tissue-specific kinases and coregulatory proteins combine to influence the structural dynamics of the receptor in general, and AF1 in particular will dramatically improve our ability to predictably perturb steroid receptor signaling and reduce undesired target activation. These strategies are general and can be employed within all ID segments of transcription factors and other signaling proteins in which ID sequences are found in abundance.Cell/tissue-specific effects of steroid receptors are tightly regulated through specific kinase(s)/phosphatase(s), and site-specific phosphorylation-induced conformational changes in AF1 and its subsequent effects on transactivation activities emerging from our studies combined with previously published studiesEscherichia coli BL21(DE3) using recombinant vector pGEX-4T1-AF1 . AF1 mutants were generated by using a QuikChange site-directed mutagenesis kit , and appropriate primers were designed. Recombinant proteins expressed in E. coli were induced with 1 mM IPTG  for 4 h, lysed, and extracted. The bacterial extracts were loaded onto a glutathione-Sepharose column at 4 °C as described previously13. His-tagged AF1C (residues 187–244) and mutants were expressed in Escherichia coli BL21(DE3), and purified on the Ni-NTA column  using imidazole step-gradient as described38. AF1 and AF1C proteins were further purified on a Resource Q column . Final protein purity of each protein was ≥ 95% as verified by presence of a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis.Recombinant AF1 protein was expressed in 39. The GR500 variants were generated via PCR by using phCMV2-GR500 as the starting template and inserting the PCR fragments into pECFP-C1 (BD Biosciences) using XhoI/SmaI cloning sites. DNA sequencing was performed on all clones to confirm correct sequence.The pGRE-SEAP vector  contains three copies of a GRE consensus sequence in tandem, fused to a TATA-like promoter (pTAL) upstream from the reporter gene for secreted alkaline phosphatase (SEAP). GR500 encodes amino acids 1 to 500 of the human GR, plus a five-residue nonspecific extensionC were generated using the ICM-Pro software . Eight different models were generated: 1) non-phosphorylated AF1C; 2) phosphorylated Ser203; 3) phosphorylated Ser211; 4) phosphorylated Ser226; 5) phosphorylated Ser203 & 211; 6) phosphorylated Ser203 & 226; 7) phosphorylated Ser211&226; and 8) phosphorylated Ser203, 211 & 226. The initial structure of the AF1C was generated in the non-phosphorylated form using the I-TASSER server with default parameters40. Each model was subjected to 200 global optimization moves using biased probability Monte Carlo41 and 100 local minimization calls. That was followed by an additional 800 global optimization moves and 100 local minimization calls.Models of the GR AF1In vitro phosphorylation of recombinant AF1 or AF1C was carried out by using a p38α/SAPKa assay kit . In each case, AF1 or AF1C was incubated with active p38 MAPK for 2 h at 37 °C in a shaking water bath. The level of Ser203, Ser211 or Ser226 phosphorylation was determined by using immunoblot analysis with specific antibodies against phospho- Ser203, Ser211 or Ser226 and GR  as described8.13. Each spectrum is representative of at least three independent experiments, corrected for the contribution of the buffer and smoothed.CD spectra of proteins (at 200 µg/ml with or without p38 MAPK in 5 mM Tris [pH 7.9] and 25 mM NaCl) were recorded on a Jasco-815 spectropolarimeter using a 0.1-cm quartz cell, with a bandwidth of 1.0 nm and a scan step of 0.5 nm, as described previously2 and 95% air for the duration of the experiment (24 h).CV-1 cells (American Type Culture Collection) were grown at 37 °C in minimal essential medium with Earle’s salts (Invitrogen) supplemented with 10% (vol/vol) fetal bovine serum . Cells were sub-cultured every 2 to 3 days. CV-1 cells were plated on a 24-well plate (1000 µl/well) 1 day before the transfection and transfected by using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. Transfected cells were maintained at 37 °C in 5% CO32. The total amount of DNA added was kept fixed at 0.3 µg by the addition of empty pECFP vector as and when needed. Medium (25 µl) was collected 24 h later and tested for the presence of SEAP  according to the manufacturer’s protocol. Experiments were performed at least three times, in triplicate. The data from different experiments were normalized to GR500 activity.CV-1 cells were cotransfected as described above with 0.15 µg of pGRE_SEAP reporter vector and 0.15 µg of pECFP-GR500 or mutant as describedSupplementary File


In addition, MS1 was found to co-precipitate with JNK, and inspection of the amino acid sequence upstream of the phosphorylation site, at Thr-62, revealed a putative Mitogen-Activated Protein Kinase (MAPK) binding site. Taken together, these data identify MS1 as a likely transcriptional and post-translational target for the JNK pathway in cardiac myocytes subjected to metabolic stress.Myocyte Stress Protein 1 (MS1) is a muscle-specific, stress-responsive, regulator of gene expression. It was originally identified in embryonic mouse heart which showed increased expression in a rat model of left ventricular hypertrophy. To determine if MS1 was responsive to other stresses relevant to cardiac myocyte function, we tested if it could be induced by the metabolic stresses associated with ischaemia/reperfusion injury in cardiac myocytes. We found that metabolic stress increased MS1 expression, both at the mRNA and protein level, concurrent with activation of the c-Jun N-terminal Kinase (JNK) signalling pathway. MS1 induction by metabolic stress was blocked by both the transcription inhibitor actinomycin D and a JNK inhibitor, suggesting that activation of the JNK pathway during metabolic stress in cardiac myocytes leads to transcriptional induction of MS1. MS1 was also found to be an efficient JNK substrate  Myocyte stress protein 1 (MS1) is a myofibrillar protein involved in the regulation of gene expression in both cardiac and skeletal muscle. It is also known as striated muscle activator of Rho Signalling and Serum Response Factor dependent transcription (STARS). MS1 was13More recent evidence suggests that MS1 likely plays a key role in regulating many aspects of myocyte cell physiology during development, in regeneration after injury and also in the adaptive responses to metabolic and mechanical stresses experienced by both cardiac and skeletal muscle 67. MS1 a61149Many of the adaptive responses of cardiac myocytes to the pathophysiological processes associated with cardiovascular disease involve activation of Mitogen-Activated Protein Kinase (MAPK) pathways 12. These13141515161712171920211111323242526Anti-phospho JNK (Cat. No. 9251S), anti-JNK (Cat. No. 9252S) and phospho-c-Jun (Cat. No. 9146) were purchased from Cell Signalling Technologies . Anti-a-tubulin (Cat. No. T5168) and anti-Flag M2 (Cat. No. F-3165) were purchased from Sigma . Anti-HA (Cat. No. Sc805) was from Santa Cruz Biotechnology . A rabbit polyclonal antiserum against full-length rat MS1 (1–375) was produced at Cambridge Research Bioscience . Anti-rabbit and anti-mouse horseradish peroxidase (HRP) conjugates were purchased from GE Healthcare Life Sciences . Dulbecco’s Modified Eagle Medium (DMEM), penicillin, streptomycin, collagenase, heat-inactivated fetal calf serum (FCS), new-born calf serum (NCS) and optiMEM were purchased from Invitrogen . Pancreatin (Cat. No. P3292) and gelatin (Cat. No. G2500) were purchased from Sigma . The inhibitors SP600125 and actinomycin D were from CN Biosciences Inc .E. coli DH5α and plasmid DNA for transfection prepared using the QIAprep Maxiprep Kit according to the manufacturer’s instructions .pETM-11-MS1 expression plasmids and pcDNA3-HA-JNK1 were generous gifts from Dr. Mark Pfuhl  and Dr. Raj Patel (University of Leicester) respectively. The pFlag-CMV2-MS1 expression vector was constructed by cloning the full-length rat MS1 cDNA between the HindIII and EcoRI sites of the pFlag-CMV2 polylinker (Sigma). A threonine to alanine mutation at residue 62 (T62A) of MS1 was made using the Quikchange site directed mutagenesis kit . Plasmid vectors were maintained in E.coli BL21-DE3 using pGEX vectors and purified by glutathione agarose affinity chromatography as described previously  dCTP-labelled probes generated by random priming of cDNAs . Membranes were then washed twice for 20 minutes with wash solution I  SDS), and 3 times for 20 minutes with wash solution II  SDS). Levels of mRNA were quantified by PhosporImager analysis of the dried membranes   dCTP-labelled probes generated by random priming of cDNAs using the Prime-IT II kit (Stratagene). Bands were quantified by PhosphorImager analysis and statistical significance determined by a global one-way analysis of variance (ANOVA) at a significance level of 95% (α = 0.05)  ATP (2000 cpm/pmol) and 2 µg of purified protein substrate: either GST, GST-Jun (1–79) or MS1 (1–118). Kinase assays were incubated for 30 min at 30°C and terminated by the addition of SDS-PAGE sample buffer prior to electrophoresis on 10% SDS-PAGE gels. Incorporation of 32P into substrates was quantified by PhosporImager analysis of the dried gels. [Transfected HEK-293 cells were washed once in ice-cold PBS and lysed in ice-cold lysis buffer. Cell lysates were clarified by centrifugation at 20,000 × g for 15 min at 4°C. Cell lysates  were immunoprecipitated by tumbling for 3 h at 4°C with 5 mg Protein-A Sepharose (Sigma) and 5 µl of anti-Flag, anti-HA or control antibody in a final volume of 500 µl. Immune complexes were isolated by brief low-speed centrifugation at 4°C and washed three times with 1-ml of ice-cold lysis buffer. Immunoprecipitates were either resuspended in sample buffer before SDS-PAGE and western blotting or used for JNK immune-complex kinase assays. For kinase assays the immunoprecipitates were washed once with 1ml kinase assay buffer , was immunoprecipitated from transfected HEK-293 cells and used in a kinase assay with an N-terminal fragment of MS1, GST or GST-Jun (1–79) as substrates  at the University of Leicester Proteomics Facility. Analysis of the spectrum and fragmentation pattern indicated phosphorylation at Thr-62  was phosphorylated -62 Fig. . To confein Fig. . JNK efftro Fig. .Skeletal and cardiac muscle are plastic tissues that adapt to both the physiological stresses associated with normal contractile function and the pathophysiological stresses associated with disease. Such adaptive responses involve integration of signals arising from many sources, including mechanical loading of the muscle, metabolic perturbations and associated redox stress. Stress-activated signalling pathways couple these signals to downstream responses such as changes in gene expression 161739. T161756102740412233in vivo or in the isolated, perfused heart, and further work will be required to determine this. However, MS1 is involved in the physiological adaptations that occur in skeletal muscle during exercise. Both endurance and resistance training in skeletal muscle bring about an increase MS1 expression via intracellular signals generated in response to the mechanical stresses of contraction, suggesting that MS1 expression may be regulated by a wide variety of cellular stresses in vivo [274344MS1 was originally identified as a gene induced by pressure overload in a rat model of left ventricular hypertrophy 1. Since h in vivo 27434445.The JNK pathway is a major stress-responsive pathway activated by ischaemia/reperfusion injury in the heart 1217. Our12The kinetics of JNK activation during metabolic stress were strikingly similar to those of MS1 induction, with both occurring only upon recovery from metabolic inhibition Fig. .Pharmacological inhibition of JNK was found to completely abolish the transcriptional induction of MS1 Fig. , suggestThe MS1 promoter has been characterised in some detail with binding sites for several transcription factors having been identified and some of these may be JNK targets 404146. O40411314484950in vitro substrate for JNK. This protein was an excellent substrate for JNK, appearing comparable with its canonical substrate c-Jun, with a major phosphorylation site identified at Thr-62 , close to the N-terminus of the protein. We therefore tested a purified N-terminal fragment of MS1 (residues 1–118) as an 62 Figs.  and 5.The substrate selectivity of MAPKs usually involves interaction with their substrates via a MAPK docking motif 53. JNK i2–3 – X2–6 – ФA – X – ФB) that mediates interactions between MAPKs and their substrates and upstream activators [The JNK phosphorylation site we identified in MS1 at Thr-62 also lies just downstream of a putative MAPK docking motif (residues 19–27: KVRRATLVI; Fig. tivators . We wereMS1 is generally thought to be a cytoplasmic protein localised at the myofibril by well-characterised actin binding domains at its C-terminus 1. However2The presence of a DNA binding domain, a putative MAPK docking site and a MAPK phosphorylation site within MS1 suggests that MS1 may function as a transcription factor regulated by MAPK signalling pathways to regulate gene expression in muscle cells. Further work using phosphorylation site mutants of MS1 coupled with more in-depth MS1 transcription reporter assays will be required to test if JNK targets MS1 in the nucleus to regulate its transcriptional activity and hence hypertrophic gene expression in response to cardiac myocyte cell stress.in vitro substrate for JNK with a major phosphorylation site at Thr-62.In summary, our results identify MS1 as a transcriptional target for the JNK pathway in cardiac myocytes subjected to metabolic stress. We further demonstrate the physical interaction of JNK and MS1, possibly mediated by a putative MAPK docking site in MS1, and the identification of MS1 as an


The S205D mutant enzyme (mimicking the phosphorylated form) exhibited a significant decrease in activity but similar affinity toward substrates. Immunodetection and activity assays showed that NAD-GAPDH is phosphorylated in vivo, the enzyme depicting different activity, abundance and phosphorylation profiles during development of seeds that mainly accumulate starch (wheat) or lipids (castor oil seed). NAD-GAPDH activity gradually increases along wheat seed development, but protein levels and phosphorylation status exhibited slight changes. Conversely, in castor oil seed, the activity slightly increased and total protein levels do not significantly change in the first half of seed development but both abruptly decreased in the second part of development, when triacylglycerol synthesis and storage begin. Interestingly, phospho-NAD-GAPDH levels reached a maximum when the seed switch their metabolism to mainly support synthesis and accumulation of carbon reserves. After this point the castor oil seed NAD-GAPDH protein levels and activity highly decreased, and the protein stability assays showed that the protein would be degraded by the proteasome. The results presented herein suggest that phosphorylation of NAD-GAPDH during seed development would have impact on the partitioning of triose-phosphate between different metabolic pathways and cell compartments to support the specific carbon, energy and reducing equivalent demands during synthesis of storage products.Cytosolic glyceraldehyde-3-phosphate dehydrogenase (NAD-GAPDH) is involved in a critical energetic step of glycolysis and also has many important functions besides its enzymatic activity. The recombinant wheat NAD-GAPDH was phosphorylated  Then, the high energy intermediate 1,3-bisP-glycerate is metabolized by P-glycerate kinase  to produce ATP and 3P-glycerate (3PGA): 1,3-bisP-glycerate + ADP ←→ 3PGA + ATP  is a homotetrameric glycolytic enzyme that catalyzes the phosphorylating-coupled oxidation of Ga3P: Ga3P + Pi + NADGA + ATP . These tGA + ATP . NAD-GAPGA + ATP ,b,c. ItsGA + ATP .+ → 3PGA + NADPH + 2 H+, as an alternative to the steps mediated by NAD-GAPDH and PGK  allowing an adequate metabolic adaptation to the changing environmental conditions that they face owing their sessile life style . The Ga3 and PGK . The sim and PGK , 2003; a and PGK . It was 2 sensor .Triticum aestivum) heterotrophic tissues np-NADP-GAPDH is phosphorylated at Ser404 by a SnRK1 (SNF1 related) protein kinase  tissues of plants growing under natural conditions. Also, we describe that phosphorylation of NAD-GAPDH occurs in vivo with specific profiles of NAD-GAPDH activity, protein level and phosphorylation degree during seed development that, interestingly, were different in seeds accumulating starch (wheat) or lipids (castor oil seed).In this work, we report that phosphorylation of NAD-GAPDH occurs in wheat and castor oil seeds. Rabbit muscle aldolase, ATP, NAD, Fru1,6bisP, Rib5P, and 3PGA were purchased from Sigma Aldrich . All other reagents were of the highest quality available.Escherichia coli BL21Codon Plus®(DE3) RIL cells transformed with [pRSETB/TagapC], and the enzyme was purified to near homogeneity by metal affinity chromatography . Enzyme activity assay (50 μl) contained (unless otherwise specified): 50 mM Tricine-NaOH pH 8.5, 1 mM NAD+, 10 mM sodium arsenate, 0.4 units aldolase, 1.2 mM Fru1,6bisP, and an adequate quantity of protein. NADH generation was monitored spectrophotometrically at 30°C and 340 nm. One unit (U) is defined as the amount of enzyme that catalyzes the formation of 1 μmol NADH per minute under the specified assay conditions.Recombinant wheat NAD-GAPDH production, purification and assay for activity were performed as previously described . Briefly+ and D-Ga3P were obtained fitting the experimental data to the generalized Hill equation by a non-linear least-square regression kinetics computer program , Santa Fe, Argentina] from June 13th (sowing date) until December 20th in 2011 and 2013 [It should be noted that the experiment was done in the South hemisphere]. The wheat density was 4 × 106 seedlings per hectare and plot dimensions were 10 × 30 m. Anthesis date was recorded when the first anther appears above the glumes in the central region of the spike. Samples were harvested at 3, 6, 10, 14, 17, and 27 days post-anthesis (DPA); and spikes frozen immediately in liquid nitrogen. Seed samples contained grains from the central part of the frozen spike between the fifth and tenth spikelet that were store at −80°C until analysis.Ricinus communis) seeds were harvested from the field around the University Campus  in 2011 and 2013. Castor seeds were reaping at various stages of development as previously described  determinations.2, 5 mM thiourea, 2 mM dithiothreitol (DTT), 2 mM phenylmethylsulfonyl fluoride (PMSF), 5 mM malate, and 1% (w/v) polyvinyl polypyrrolidone] was used to extract total proteins  was incubated under activity conditions determined for different plant protein kinases in previous works, essentially as already detailed (2+-independent SNF1-related protein kinase (WPK4): 20 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.1 mM PMSF, 20 mM MgCl2, and 100 mM ATP at 25°C : 20 mM Tris-HCl, pH 7.2, 5 mM MgCl2, 0.5 mM CaCl2, 2 mM DTT, and 10 mM ATP at 30°C  and were initiated by adding wheat endosperm or leaves extract as kinase resource, or partially purified SnRK1 from wheat endosperm. After reaction, the protein mixtures were resolved by electrophoresis under denatured conditions on discontinuous polyacrylamide gels (SDS-PAGE) according to TM system .Unless otherwise specified, reactions were performed in 20 μl with 2 μCi of  with a standard starch solution treated identically to the sample.For starch quantification in seeds, the protocols described by 2O and 3.8 ml of chloroform/methanol: 2/1 (v/v) solution. Tubes were hermetically closed and incubated 2 h at room temperature with gently mixing every 30 min. Samples were filtered on oil free filter paper previously washed with the extraction solution. Samples transferred to previously weighed tubes were vigorously mixed with 0.7 ml of 0.02 % CaCl2 solution in chloroform/methanol/H2O: 3/48/47 (v/v) and centrifuged 5 min at 3,000 ×g until phase separation. The upper phase was discarded and 0.7 ml of chloroform/methanol/H2O: 3/48/47 (v/v) were added. Samples were vigorously mixed, centrifuged as before until phase separation and the upper phase discarded by suction. After these washes, chloroform was evaporated at 45–50°C to obtain the lipids. After weighing, lipids were dissolved in isopropanol and TAGs quantified by an enzymatic colorimetric assay. TAGs were digested by lipase and the resulting glycerol phosphorylated to glycerol-3P by a specific kinase and then oxidized by a glycerol-3P oxidase producing H2O2. The last product was quantified through a peroxidase coupled to a colorimetric compound. The reaction mixture (505 μl) consisted of 500 μl of the commercial reactive  and 5 μl of sample conveniently diluted. The reaction was incubated 10 min at 37°C and color quantified at 492 nm.Lipid quantification was based on the method described by in vitro, following the methodology previously employed for similar studies performed with np-NADP-GAPDH (TaeNAD-GAPDH) was expressed and purified as described Figure 1 (gray columns), NAD-GAPDH was phosphorylated by the seed extract in more than one condition, with the highest signal observed for that promoting WPK4 activity. We carried out experiments in parallel and under identical conditions, except that the extract was obtained from leaves instead of seeds. In this case, almost no phosphorylation of the recombinant enzyme was observed . These results suggest that the seed protein kinase that was able to phosphorylate NAD-GAPDH was either absent or inactive in the photosynthetic tissues analyzed. The absence or lack of activity of this kinase was not due to a general lack of protein kinase activity in our leaf extracts. Indeed, in control experiments performed with myelin basic protein, a general substrate for kinases, this substrate was equally phosphorylated in all incubation media performed with leaves or seeds extracts (data not shown). The latter suggests that under natural and normal growth conditions the phosphorylation of NAD-GAPDH in photosynthetic tissues would be negligible. It should be considered that in our studies we only determined phosphorylation in leaves harvested during day-time. Thus, the possibility of find differences in activity of the kinase in night-time leaves should not be discarded and is a subject for future research. For further studies, we partially purified a SnRK1 kinase (SNF1-related protein kinase) from wheat seeds strictly following the protocol assayed before . The partially purified protein kinase  was inhibited by Rib5P and, to a lesser extent, by Fru1,6bisP and 3PGA. These results are similar to those previously reported for the characterization of the SnRK1 phosphorylating np-NADP-GAPDH  two SnRK1 recognition motifs , surrounding the residues Ser205 and Ser66, the former having a better context. To study if these residues are involved in the phosphorylation of the enzyme, we performed site-directed mutagenesis to change these two Ser residues to Ala. We also produced an Ala mutant for the Ser124, as it has been previously reported as the probable site for the interaction of NAD-GAPDH with 14-3-3 regulatory proteins . Additionally, we also phosphorylated in vitro all the NAD-GAPDH versions with the SnRK1 purified from wheat seeds . We also tested the phosphorylation of the reduced and oxidized forms of the NAD-GAPDH versions, as redox regulation is also a post-translational modification affecting the enzyme activity and switching the protein to its non-glycolytic role  mutant version was not phosphorylated by the SnRK1 purified from wheat seeds. The experimental data indicate that Ser205 is an in vitro phosphorylation site for NAD-GAPDH and also that SnRK1 would be the kinase involved in the phosphorylation reaction, thus supporting the in silico analysis showing the domain containing a Ser residue matching better with a SnRK1 phosphorylation motif. Moreover, results suggest that the different redox states undergone by NAD-GAPDH  with no significant changes in Km values for substrates, but a 350-fold decrease in Vmax when compared with the wild type. These results support that phosphorylation of NAD-GAPDH at Ser205 would impair enzyme catalysis.To further explore the kinetic consequences of the post-translational modification on the rylation . The S20in vitro phosphorylation of NAD-GAPDH could be catalyzed by SnRK1 protein kinases extracted from heterotrophic wheat tissues. Because this post-translational modification would negatively impact enzyme catalysis, we decided to explore activity, protein and phosphorylation profiles of NAD-GAPDH along seed development. The complete growth of wheat seeds is reached at 45 DPA2, being the respective phases of development: cell proliferation 0–10 DPA, accumulation of reserves 11–30 DPA, and seed maturation and desiccation among 30–45 DPA. For the analysis we collected wheat seeds from the experimental field at 6 different times: 3, 6, 10, 14, 17, and 27 DPA; and determined the fresh weight and the content of starch immediately after harvesting . The profiles determined were similar to those established for wheat seed fresh weight . When the protein was detected with specific antibodies in the whole protein extracts , more than one polypeptide (∼34–47 kDa) were recognized, with minor changes in the protein abundance along development. When NAD-GAPDH was specifically detected in extracts enriched in phospho-proteins after IMAC-Fe3+ affinity chromatography, two major protein bands were detected in all samples through the end of the seed development . To confirm that the phospho-protein enrichment was related to the specific post-translational modification of the NAD-GAPDH, we assayed a de-phosphorylation treatment of the whole protein extract obtained from seeds at 27 DPA with alkaline phosphatase (AlkPase). After treatment, the extract was purified by IMAC-Fe3+ chromatography and the NAD-GAPDH specifically detected in the fractions loaded and eluted from the column . As expected, after the treatment with AlkPase the IMAC-Fe3+ column retained significantly less amount of the enzyme, even though it is still being detected within the proteins loaded onto the column. This result confirms that purification of the phosphorylated NAD-GAPDH form in all the wheat seed samples analyzed is not due to an unspecific interaction with the IMAC-Fe3+ matrix.in vivo along development of wheat seeds, we specifically detected the kinase in the whole protein extracts using monoclonal antibodies against the αAMPK phospho-Thr172. This antibody recognizes α subunits of AMPK and it proved useful to detect SnRK1 in plants  also store carbon in the endosperm, but in the form of TAGs instead of starch . The fresh weight profile agreed with the above referenced report, while the synthesis of TAGs initiated at 25 DPP and highly increased (∼40-fold) through 40 DPP, representing at this point ∼28% of the seed fresh weight. NAD-GAPDH profiles along castor seed development showed that the enzyme activity  increased by 2-fold from 5 to 20 DPP, decreasing abruptly afterward (∼4-fold) at the point where the TAGs start to being accumulated and becoming almost undetectable at the end of the process. Regarding the protein profile, the specific antibodies recognized one polypeptide (around 34–47 kDa) remarkably diminishing in the last two seed sample analyzed . The immunodetection after phospho-protein enrichment by IMAC-Fe3+  revealed that NAD-GAPDH also undergoes phosphorylation in castor seeds, with levels of the phospho-form exhibiting large variations along development. NAD-GAPDH phosphorylation seems to increase from 10 to 25 DPP and then decreased in agreement with the whole protein. Immunodetection of SnRK1 kinase with αAMPK phospho-Thr172 antibodies also shown that SnRK1 kinases are present from 5 to 25 DPP in castor developing seeds ; concurring with the profile detected for the phospho-NAD-GAPDH. To confirm that the phosphoprotein enrichment of NAD-GAPDH was specifically related with the phosphorylation status of the enzyme, we used AlkPase for de-phosphorylation and protein immunodetection before and after IMAC-Fe3+ purification in protein extracts from castor oil seeds at 25 DPP . As a result, NAD-GAPDH was surprisingly neither detected in the IMAC-Fe3+ elution nor in the sample loaded in the column after AlkPase treatment, suggesting that the protein stability was affected. It is worth to note that controls determining levels of actin as a signal of same amount of protein loaded on the electrophoresis gels also exhibited a huge reduction of protein in samples of castor oil seeds at 40 DPP. This suggests that in this late stage of development, the seed may contain high levels of proteolytic enzymes which, together with the accumulation of oil could constitute an environment detrimental for the general stability of proteins. These instability conditions could start with more specificity (involving NAD-GAPDH) at earlier developmental periods and then could generalize to favor a tissue that finally mainly accumulates oils.Long-term storage of carbon in wheat seeds takes place in the endosperm and mainly as starch. Seeds like those of castor bean  under phosphorylation conditions determined for SnRK, CKII, Tsl, and CDPK in previous works. For further studies, we partially purified a SnRK1 kinase from wheat seeds that exhibited the ability to phosphorylate in vitro the recombinant TaeNAD-GAPDH. An analysis of the NAD-GAPDH amino acidic sequence identified the Ser205 residue with the better chances to be the target of SnRK1 action, which was supported by the fact that the TaeNAD-GAPDH S205A mutant proved recalcitrant to phosphorylation. It is worth noting that NAD-GAPDH was found phosphorylated by AMPK in animal cells on a Ser residue equivalent to the Ser124 of the wheat enzyme  residue, while Ser205  residue with an Arg at –3 only occurs in the plant enzymes. As it has been proposed that phosphorylation of Ser124 would trigger NAD-GAPDH interaction with 14-3-3 proteins , which supports that the phosphorylation site identified herein would be specific for cytosolic plant NAD-GAPDHs. It should be noted that the equivalent of wheat NAD-GAPDH Ser205 in both NAD-GAPDHs from Arabidopsis is the main phosphorylation site found in vivo according to PhosPhAT database3. Although this NAD-GAPDH phosphorylation site seems to represent a target that could have a physiological role in plants, the consequences of such a post-translational modification on the enzyme activity or stability were never studied before. In our work, we found that the S205D mutant (mimicking phosphorylation) exhibited a significant reduction in enzyme activity, thus suggesting that phosphorylation would have an inhibitory role on the enzyme. Previous reports point those enzymes like 3hydroxy-3-methylglutaryl-coenzyme A reductase  decreases by 4-fold as seed develop  the proportion of GAPDH phosphorylated specifically at Ser205 could be low in vivo in wheat; (ii) phosphorylation status could modulate the enzyme’s interaction with other components  to be fully active/inactive; and/or (iii) the measurements of activity we performed could be also detecting the plastidic NAD-GAPDH that was shown to play a specific role in glycolytic energy production in non-green plastids  will differ within plant species.Seeds are typically heterotrophic. Their supplies for carbon and energy mostly depend on the breakdown and oxidation of sucrose imported from photosynthetic tissues. In these cells, glycolysis is the main pathway using cleaved sucrose to provide the energy and building blocks for metabolism in different cell compartments. Glucose-6P, Ga3P and phosphoirements . At the The analysis of NAD-GAPDH activity, protein and phosphorylation profiles in wheat and castor developing seeds, respectively, accumulating mainly starch or TAGs highlight some interesting clues. Even though these seeds are from different species and accumulate carbon in different ways, at the beginning (during the pre-storage phase) the carbon fate and partitioning would be similar, as both seeds need to supply the cell proliferation process. In fact, the profiles observed for NAD-GAPDH protein and activity during that phase (from 3 to 10 DPA in wheat and from 5 to 20 DPP in castor seed) looked similar in both plants; the NAD-GAPDH activity increases and the protein level remains with slight variations in both cases. Then, when the seed perform a metabolic adaptation to redirect carbon sources into reserves, the NAD-GAPDH profiles showed significant differences. In wheat, the NAD-GAPDH activity slightly increases but abruptly decreases in castor seed. Going ahead in development, NAD-GAPDH remains active and unchanged in wheat, but is almost undetectable in castor seed that are using carbon sources to supply the storage of TAGs. Precursors for fatty acids and TAGs synthesis are acetyl-CoA and glycerol-3P, both being synthesized from two glycolytic intermediates. Our results suggest that phosphorylation of NAD-GAPDH would switch the protein from its enzymatic role to other moonlighting non-glycolytic functions, as it was shown for the enzyme in animal cells . Phosphoin vivo NAD-GAPDH phosphorylation at Ser205 in wheat and castor oil seed constitute the subject of future research. However, this work highlights that the post-translational modification process would play critical roles for determining carbon, energy and reducing power flux in plants during seed development.Oils and carbohydrates stored in seeds are not only essential reserves to support germination and early growth of plants, but they are also critical to humans and animals as major source of food . Hence, CVP and AAI conceived and designed the project. CVP and DMLF generated experimental data and together with ID and AV collected and classified the seeds samples. CVP and AAI analyzed data and wrote the article.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.