Development of kinomic analyses to identify dysregulated signaling pathways in cells expressing cytoplasmic PrP
© Shott et al.; licensee BioMed Central Ltd. 2014
Received: 5 June 2014
Accepted: 26 September 2014
Published: 3 October 2014
Dysregulated protein kinase signaling is involved in the pathogenesis of many chronic diseases. However, the dysregulated signaling pathways critical to prion pathogenesis remain incompletely characterized. Global analyses of signaling pathways may be useful to better characterize these pathways. We therefore set out to develop such global assays. To this end, we used as a model cytoplasmic mutants of the cellular prion protein (PrPC), which are toxic to N2a neuroblastoma cells. We tested the global assays for their sensitivity to detect changes in signaling pathways in cells expressing cytoplasmic PrP mutants.
We developed a targeted proteomics (kinomics) approach using multiplex Western blots to identify signaling pathways dysregulated in chronic neurological pathologies. We tested the approach for its potential ability to detect signaling changes in N2a cells expressing cytoplasmic PrP mutants.
Multiplex Western blots were designed to quantitate the expression levels of 137 protein kinases in a single membrane and using only 1.2 mg of sample. The response of the blots was sensitive and linear to changes of 6% in protein levels. Hierarchical and functional clustering of the relative expression levels identified an mTOR signaling pathway as potentially dysregulated in N2a cells expressing cytoplasmic PrP. The mTOR signaling pathway regulates global protein synthesis, which is inhibited in cells expressing cytoplasmic PrP. The levels of proteins involved in the Akt1/p70S6K branch of mTOR signaling changed in synchrony with time of cytoplasmic PrP expression. Three kinases in this pathway, Akt, p70S6K, and eIF4B were in their inactive states, as evaluated by phosphorylation of their regulatory sites.
The results presented are consistent with the previously reported inhibition of Akt/p70S6K/eIF4B signaling as mediating pathogenesis of cytoplasmic PrP. We conclude that the kinomic analyses are sensitive and specific to detect signaling pathways dysregulated in a simple in vitro model of PrP pathogenesis.
KeywordsPrion disease Cytoplasmic PrP Neurotoxicity Kinomics Protein kinase Multiplex Western blots Akt p70S6K eIF4B
Dysregulation of protein kinase signaling is implicated in the pathogenesis of many chronic diseases, including neurodegenerative diseases such as Alzheimer’s and Parkinson’s [1–3]. Not surprisingly, inhibitors of protein kinases are the largest group of new cancer therapeutics . Thirty-one such inhibitors are in clinical use, over 500 are involved in approximately 2,700 clinical trials, and thousands more are in various stages of pre-clinical development ([4–6] and summary of ). Up to 30% of the research and development budget of the pharmaceutical industry is estimated to be invested towards protein kinase inhibitors [8, 9]. Considering the importance of protein kinases in chronic disease, it would be desirable to have approaches to identify protein kinase signaling pathways that are dysregulated in chronic diseases.
Transmissible spongiform encephalopathies (TSEs), or prion diseases, are a family of chronic neurodegenerative diseases against which there are no preventative or therapeutic treatments . Prion diseases are invariably lethal to humans (kuru; Creutzfeldt-Jakob disease, CJD; Gerstmann-Sträussler-Scheinker disease, GSS; fatal familial insomnia, FFI), and other species such as cattle (bovine spongiform encephalopathy, BSE), goat, sheep (scrapie), deer, elk and moose (chronic wasting disease, CWD) [11, 12]. The characteristic neuropathology of prion diseases includes gliosis, spongiform degeneration, and neuronal death. The conversion of the cellular prion protein (PrPC) to an abnormal conformation (PrPSc) is widely accepted to be essential for pathogenesis. However, the molecular mechanisms whereby such conversion eventually mediates the consequent neurodegeneration are not yet fully understood.
Considering the critical roles that protein kinases play in the pathogenesis of other chronic neurodegenerative diseases, it is not surprising that they also participate in that of prion diseases. For example, feline Gardner-Rasheed sarcoma virus oncogene cellular homolog/Yamaguchi 73 and Esh avian sarcoma virus oncogene cellular homolog-related novel protein kinase (Fyn) knockout mice died faster than wild-type mice after scrapie infection . Conversely, inhibition of protein kinase R-like endoplasmic reticulum kinase (PERK) by the overexpression of growth arrest and DNA damage-inducible protein 34 (GADD34) prolonged survival of scrapie-infected mice . The activation of vascular endothelial growth factor receptor (VEGFR) inhibited death of cultured neurons treated with the neurotoxic prion peptide PrP106-126 . The Abelson leukemia oncogene cellular homolog (c-Abl)/Hardy-Zuckerman 4 feline sarcoma virus oncogene cellular homolog (c-Kit)/platelet-derived growth factor receptor (PDGFR) inhibitor STI571 impaired scrapie neuroinvasion and prolonged survival of mice after intraperitoneal infection . Several other inhibitors of protein kinases regulated PrPSc accumulation in scrapie-infected cultured cells [17–22]. Unfortunately, the signaling pathways most critical to prion disease pathogenesis have yet to be fully identified.
Although prion diseases are characterized by the accumulation of PrPSc, endogenous PrPC is also required for pathogenesis [23–26]. PrPC is physiologically attached to the outside of the plasma membrane via a glycosylphosphatinositol (GPI) anchor [27, 28]. PrPC may accumulate in the cytoplasm as a result of inefficient endoplasmic reticulum (ER)-targeting [29–31], ER-associated degradation [32–34], alternative translation initiation , or persistent pre-emptive quality control . Low levels of cytoplasmic PrP have been observed in certain subpopulations of neurons without overt neurodegeneration [37–39], and the roles of cytoplasmic PrP in prion infection are disputed . Nonetheless, the accumulation of PrPC in the cytoplasm is often neurotoxic and has also been considered as a possible neurodegeneration mechanism . Mice expressing a truncated mutant of PrPC lacking its N-terminal ER-targeting and C-terminal GPI-membrane-anchoring signals (named cytoplasmic PrP, or CyPrP) suffered from ataxia with gliosis and cerebellar degeneration . The molecular mechanisms of such neurodegeneration can be studied in culture because CyPrP is also toxic to mouse N2a neuroblastoma cells .
The expression of CyPrP inhibits heat shock protein 70 (Hsp70) synthesis in stressed N2a cells . Hsp70 overexpression inhibits CyPrP-mediated toxicity, suggesting that the inhibition of Hsp70 synthesis may contribute to cell death [44, 45]. Hsp70 promotes assembly and activation of the mammalian target of rapamycin complex 2 (mTORC2; consisting of mammalian target of rapamycin [mTOR], rapamycin-insensitive companion of mTOR [rictor], mammalian lethal with SEC13 protein 8 [mLST8] and stress-activated protein kinase-interacting protein 1 [SIN1]), which then activates AKT8 virus oncogene cellular homolog (Akt)/ribosomal protein S6 kinase, 70 kilodalton, polypeptide 1 (p70S6K)/eukaryotic initiation factor 4B (eIF4B) signaling [46, 47]. Active eIF4B promotes protein synthesis, which is otherwise inhibited in cells expressing CyPrP [43, 48].
Here, we describe the development of a multiplex Western blot-based kinomics approach. Before embarking on the analysis of prion infected animals, we used a simple in vitro model to test the sensitivity of the approach to identify dysregulated signaling pathways, accumulation of enhanced green fluorescent protein-tagged cytoplasmic PrP (CyPrPEGFP) in N2a cells. The approach identified the Hsp70-regulated Akt/p70S6K/eIF4B signaling pathway to be inhibited in cells expressing CyPrPEGFP, consistently with previously known consequences of CyPrPEGFP expression . The results support the ability of the kinomics approach to detect signaling pathways dysregulated in an in vitro model of prion pathogenesis. As described in the companion manuscript, we have applied this approach to an in vivo model, infection of mice with mouse-adapted scrapie, to discover two signaling pathways dysregulated during prion disease pathogenesis.
Multiplex Western blots quantitate the expression levels of 137 protein kinases or regulatory subunits in only 1.2 mg of sample
We developed multiplex Western blots to analyze the expression levels of protein kinases potentially involved in prion pathogenesis. In these assays, protein extracts are run in SDS-PAGE in a single well, transferred to a membrane and probed with several pools of antibodies in a multiplex Western blot apparatus.
Mice and human kinomes are well conserved, allowing the use of mice to identify and analyze protein kinases of potential importance in human disease. We performed an extensive literature search for human protein kinases that may be involved in prion or other neurodegenerative diseases (Alzheimer’s, Parkinson’s, Huntington’s, multiple sclerosis, or amyotrophic lateral sclerosis). We also included protein kinases involved in cellular pathologies associated with prion disease (neuronal apoptosis, gliosis, glial activation, neuronal degeneration, or neuronal survival). The search was restricted to protein kinases the mouse orthologs of which were detected by antibodies commercially available at the time. Following these criteria, we selected 145 protein kinases, almost 30% of the 540 or 518 protein kinases in the mouse or human kinomes, respectively (Additional file 1: Figure S1) [49, 50]. The selected protein kinases are distributed among the eight groups of protein kinases (AGC, CAMK, CMGC, CK1, STE, TK, TKL, and atypical) . The most under-represented kinases in the selection are involved in muscle contraction [myosin light chain kinases, MLCK], spermatogenesis [testis specific serine/threonine kinases, TSSK], or developmental processes [transforming growth factor-beta receptor kinases, TGF-β]), which are not expected to be critical in prion disease. We also included the eleven cyclins or cyclin-like proteins (p25/p35, p39), which are the activating subunits required for the activity of the catalytic cyclin-dependent kinase (CDK) moiety of the active CDK/cyclin heterodimers.
Our long-term objective was to analyze the kinomic changes in brains of scrapie-infected mice (see companion paper). We therefore optimized the antibodies in multiplex Western blots with mouse brain homogenate. Antibodies specific for 122 protein kinases or regulatory subunits (Additional file 2: Table S1) recognized their cognate proteins in 1.2 mg of mouse brain homogenate (200 μg loaded per linear cm). We selected the dilution of each antibody that resulted in maximum signal intensity and minimum background with no antibody saturation (i.e., signal did not increase with increasing antibody concentrations). Antibodies specific for calcium/calmodulin-dependent kinase 4 (CaMK4), mitogen-activated protein kinase/extracellular signal-regulated kinase 5 (MEK5), and Jun N-terminal kinase 2 (JNK2) detected two isoforms each. Fifteen antibodies specific for proteins not recognized in mouse brain homogenate were optimized in Western blots using lysate from cultured 3T3 mouse fibroblasts (200 μg loaded per linear cm). The remaining 19 antibodies did not detect their cognate protein in mouse brain homogenate or 3T3 cell lysates.
The multiplex Western blots were tested for reproducibility. Mouse brain homogenate resolved throughout a single-well gel (1.2 mg; 200 μg per linear cm) was transferred, and 16 lanes were isolated in the membrane with a multi-screen apparatus. The extracellular signal-regulated kinases (Erk) 1 and 2 were probed in each of the 16 lanes. The standard deviation between all 16 lanes was only 2.2% or 1.0% of the average for Erk1 or Erk2, respectively, and the range was 8% of the average for Erk1 or 4% of Erk2.
Importantly, the multiplex Western blots allowed for the identification and resolution of: (i) kinases yielding high and low intensity signal (for example, Set 1; Akt3 in lane 11 [green band at 60 kDa] vs. Akt2 in lane 16 [green band at 60 kDa]); (ii) kinases of similar molecular weight in the same lanes (for example, Set 1, lane 3; GRK2 [green band at 80 kDa] and p70S6K [red band at 75 kDa]); and (iii) as many as 5 protein kinases in a single lane (for example, Set 2 lane 9: EphA3 [green band at 140 kDa], CASK [red band at 104 kDa], GSK3β [red band at 46 kDa], p38γ [green band at 43 kDa], CDK5 [red band at 30 kDa]; Set 2, lane 14; SLK [red band at 220 kDa], HGK [green band at 140 kDa], PKCα [red band at 82 kDa], LIMK1 [green band at 70 kDa], Erk1 [green band at 44 kDa]) (Figure 1).
In summary, we developed multiplex Western blots to quantitate 137 protein kinases or regulatory subunits involved in neurological diseases or pathologies using only 1.2 mg of sample on a single membrane stripped only once.
Multiplex Western blots are sensitive and linear, detecting incremental 6 (or 3)% changes in protein levels
Primary kinomic screens of N2a cells expressing cytoplasmic PrP mutants identified the mTOR signaling pathway as potentially dysregulated
The neurotoxicity of CyPrP expression requires residues 116–156 . We transfected N2a neuroblastoma cells with CyPrP or two mutants truncated within this region, CyPrP124stop and CyPrP124-230. The expression of CyPrP124stop is not toxic to N2a cells , and cells expressing CyPrP124-230 also are healthy, although toxicity has not been quantitatively assessed. We expected cells expressing CyPrP to affect different subsets of signaling pathways than, or to differentially affect the same signaling pathways as, those expressing CyPrP124stop or CyPrP124-230. Such differentially affected signaling pathways might be involved in CyPrP-mediated neurotoxicity.
We first performed hierarchical clustering of the three PrP mutants to evaluate any potential (unexpected) similarities in changes in protein kinase expression. If two different cytoplasmic PrP mutants resulted in identical changes in protein kinase expression, the correlation would be 1; a correlation of 0 indicates no relationship. The city-block distance metric correlation between CyPrPEGFP124stop and CyPrPEGFP124-230 was 0.088, and between them and CyPrPEGFP was 0. The lack of the correlation indicated that, as expected, the different cytoplasmic PrP mutants dysregulated different signaling pathways.
The levels of proteins in the Akt1/p70S6K branch of the mTOR signaling pathway decreased synchronously with time of CyPrPEGFP expression
If the changes in the levels of the proteins involved in mTOR signaling were the result of CyPrPEGFP expression, then their levels would be expected to change in synchrony with time of expression. We analyzed three sets of N2a cell lysates prepared 12, 24 and 48 h after transfection with the CyPrPEGFP-expressing construct in three independent biological repeats. Cells expressing EGFP were used as control.
Inhibition of Hsp70-regulated Akt/p70S6K/eIF4B signaling in cells expressing CyPrPEGFP
To test whether Akt1/p70S6K signaling was dysregulated in cells expressing CyPrPEGFP, we characterized the activation states of the ten proteins previously tested. We optimized phosphorylation-specific antibodies for the sites directly phosphorylated by the relevant upstream protein kinases. Akt is activated by phosphorylation on S473 by mTORC2 and on T308 by 3-phosphoinositide-dependent protein kinase 1 (PDK1) [58, 59]. The synthesis of Hsp70, which activates mTORC2, is inhibited in cells expressing CyPrP [43, 46]. We focused on Hsp70-regulated phosphorylation of Akt1. No available antibody was specific for the phosphorylation at the activation-specific site S473 on Akt1 only. We used an antibody that detects S473 phosphorylation on all Akt isoforms (Akt1, 2, and 3). Although mTOR phosphorylation is not required for mTORC1 activation, active mTORC1 typically contains S2448 phosphorylated mTOR . We included an antibody specific for this phosphorylation (P-S2448). We also included antibodies specific for the phosphorylation at activation-specific sites (activating phosphorylation) on AMPKα (P-T172), Mnk1 (P-T197/202), PKCα (P-S657), p70S6K (P-T389), S6 (P-S235/236; P-S240/244), eIF4B (P-S422) and eIF4E (P-S209), or the inhibition-specific site (inhibitory phosphorylation) on eEF2 (P-T56) [61–67]. The phosphorylation level of Akt (P-S473) at 12 h could be tested in only two of the three independent biological replicates due to limiting sample.
Here we describe the development of kinomic analyses aimed at identifying signaling pathways dysregulated during chronic pathologies. We designed and optimized multiplex Western blots to quantitate the expression of 137 protein kinases (including regulatory subunits) in a single membrane, and using only 1.2 mg of sample. These multiplex Western blots were reproducible, sensitive and linear, detecting 6% incremental changes in protein level. We tested the multiplex Western blots in a kinomic screen of an in vitro model of prion pathogenesis, N2a neuroblastoma cells expressing cytoplasmic PrP mutants. The mTOR signaling pathway was identified in the primary screen. The levels of proteins involved in the Akt1/p70S6K branch of the mTOR signaling pathway changed synchronously and were phosphorylated or unphosphorylated to their inhibited states in CyPrPEGFP expressing cells.
Hsp70 overexpression inhibits CyPrP-mediated toxicity and the synthesis of Hsp70 is inhibited in cells expressing CyPrPEGFP[43–45]. The inhibition of Hsp70-regulated Akt/p70S6K/eIF4B signaling in cells expressing CyPrPEGFP is fully consistent with those previous data [43–45], supporting the ability of the approach to detect kinomic changes. Inhibition of Hsp70-activated Akt/p70S6K/eIF4B signaling may also be important in CyPrP pathogenesis. Depletion of eIF4B by RNA interference promotes cell death . Hsp70 overexpression protects against this cell death in part by promoting the expression of the anti-apoptotic protein B-cell CLL/lymphoma 2 (Bcl-2) [68, 69]. Active eIF4B is also required for the translation of Bcl-2 (and other proteins translated from mRNAs with highly structured 5’ untranslated regions). CyPrP may promote cell death by inhibiting Bcl-2 synthesis through the Akt/p70S6K/eIF4B pathway. Overexpression of Akt1, mTOR, and p70S6K in cells expressing CyPrPEGFP for 12 h may well be an ultimately fruitless early attempt to overcome neurotoxic inhibition of Akt/p70S6K/eIF4B signaling.
Active eIF4B promotes translation initiation by stimulating the helicase activity of eukaryotic initiation factor 4A (eIF4A) and promoting ribosome binding [70–73]. Global protein synthesis is inhibited in cells expressing CyPrPEGFP. Previous studies have indicated that eukaryotic initiation factor 2 alpha (eIF2α) was inhibited in cells expressing CyPrPEGFP in a protein kinase R (PKR)-dependent manner . The inhibition of eIF4B also inhibits protein synthesis , suggesting that the inhibition of Akt/p70S6K/eIF4B signaling may too contribute to the inhibition of protein synthesis in cells expressing CyPrPEGFP.
The kinomics approach we describe here is dependent on the primary screens to first identify signaling pathways of potential interest. The protein kinases for the primary screens were selected based on their potential roles in prion or other neurodegenerative diseases, or in pathologies associated with prion disease. However, the screens are extensive, evaluating almost one-third of the protein kinases in the mouse (or human) genome. Other kinomic analyses  used peptide arrays to identify different signaling pathways induced by PrPC stimulation with a PrP antibody (6H4) or a small peptide (PrP106-126). These assays are limited by the availability of the peptide arrays and the different activities of several kinases on small peptide or protein substrates. Other previous studies have only analyzed a limited number of protein kinases (c-Abl, Src, Fyn, Yes, Lck, Lyn, Syk, Akt, mTOR, p70S6K, CaMK2α, CDK5, PYK2, PKA, PKC, PKR, PERK, MEK1/2 and MAPKs), based mostly on their hypothesized involvement in prion disease pathogenesis [13, 14, 17–20, 22, 74–88]. Such analyses provide information about the selected signaling pathways only. High-throughput reverse-phase protein arrays  can also be used to analyze changes in protein kinase levels. This approach requires highly specific antibodies, however, limiting the number of protein kinases that can be analyzed. The antibodies included in the experiments described here are also specific. Nonetheless, multiplex Western blots discriminate specific from non-specific binding (by molecular weight), tolerating some level of cross-reactivity. More proteins can therefore be analyzed by the multiplex Western blot approach than by protein arrays.
High-throughput gene arrays have identified hundreds of genes expressed differentially during, and a number of cellular processes affected by, prion diseases [90–102]. However, only 518 or 540 of the approximately 30,000 genes in the human or mouse genomes encode for protein kinases. Hierarchical clustering of gene array data thus results in very few clusters with multiple protein kinases, making it difficult to identify any particular signaling pathway. Moreover, protein kinases are extensively regulated post-transcriptionally and therefore their mRNA and protein levels often do not correspond . Global analyses of changes in microRNA (miRNA) which may post-transcriptionally regulate as many as 60% of human genes , have also been performed during prion disease pathogenesis, and have identified potentially dysregulated signaling pathways [100, 105, 106]. However, the biological roles of most miRNAs have yet to be characterized, which makes it difficult to interpret the effects mediated by many of the identified miRNAs.
The kinomics approach described here of course has limitations, too. First, the primary screens identify signaling pathways of potential interest using hierarchical clustering. The identification of potentially dysregulated pathways requires multiple kinases in the pathway to be included in the primary screen. The 137 protein kinases selected for the screens are involved in most of the best characterized signaling pathways. The approach is thus unable to detect signaling pathways that are less well characterized, or for which only one or very few kinases are included in the primary screen. Protein kinases are also extensively regulated post-translationally. Dysregulation of signaling pathways during chronic conditions often result in, or is the result of, changes in expression levels of involved proteins. We therefore screened for expression levels in the primary analysis. Consequently, these screens cannot detect pathways that are only affected post-translationally (unless the posttranslational regulation is at the level of degradation). The described approach is also unable to differentiate signaling pathways dysregulated as a result of disease from those critical in the pathogenesis. Previous studies have screened protein kinase inhibitors and identified STI571 (an inhibitor of c-Abl, c-Kit, and PDGFR kinases) as promoting PrPSc degradation . The signaling pathway identified by the kinomics approach can be tested with siRNA, knockouts, or specific inhibitors, to identify whether it is involved in CyPrP pathogenesis, or dysregulated as a result of it.
We used N2a cells expressing cytoplasmic PrP mutants to test the sensitivity and specificity of kinomics analyses developed to detect the dysregulation of specific signaling pathways. The assays identified a dysregulated signaling pathway which is fully consistent with previous data. We conclude that the kinomics analyses are sensitive and specific enough to detect signaling pathways dysregulated in a simple in vitro model of prion pathogenesis. We have now used these analyses to test critical signaling pathways dysregulated in brains of prion-infected animals (see companion paper).
Materials and methods
Cloning, cell culture and transfections
Cloning of CyPrPEGFP, CyPrPEGFP124stop and CyPrPEGFP124-230 in pCEP4β (Life Technologies Inc., Carlsbad, California, USA) was previously described . Mouse N2a neuroblastoma cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (Wisent, St. Bruno, Quebec, Canada). Transfections were carried out using Exgen (MBI Fermentas, Burlington, Ontario, Canada) or GeneCellin (BioCellChallenge, Toulon, France), according to the manufacturer’s protocol. To evaluate transfection efficiency, cells were harvested with trypsin and EDTA and centrifuged for 5 min at 500 × g. Following a wash with PBS, cells were fixed with 4% paraformaldehyde in 4% sucrose for 20 min at room temperature, washed with PBS and analyzed on a cytometer for GFP expression.
Mouse 3T3 cells were propagated in DMEM (Life Technologies Inc.) supplemented with 5% fetal bovine serum (FBS; PAA Laboratories GmbH, Pasching, Austria), 50 U/mL penicillin and 50 U/mL streptomycin (Life Technologies Inc.), at 37°C in 5% CO2.
Cell lysis and brain homogenization
Mouse 3T3 and transfected N2a cells were cultured to approximately 85% confluency on 8 × 10 cm tissue culture dishes. All subsequent procedures were performed on ice or at 4°C using reagents pre-chilled to 4°C. Each dish was washed twice with 2 mL of phosphate-buffered saline (PBS; 150 mM NaCl, 1 mM KH2PO4, 3 mM Na2HPO4, pH 7.4). Cells were collected by scraping into freshly prepared lysis buffer (0.2 mL per dish) (20 mM MOPS [pH 7.0)], 2 mM EGTA, 5 mM EDTA, 1% Nonidet P-40, 0.001% phosphatase inhibitor cocktail [Pierce, Rockford, Illinois, USA], 0.002% protease inhibitor cocktail [Sigma-Aldrich, St. Louis, Missouri, USA], 1 mM DTT, pH 7.2). The lysates were passed twice through a 20 gauge needle, sonicated five times for 20 s intervals at 88 W output (XL-2020; Misonix, Farmingdale, New York, USA), and pre-cleared at 14,000 × g for 30 min (JA.14 rotor, Avanti J-E centrifuge; Beckman/Coulter, Brea, California, USA). Approximately 1 mL volumes of supernatant were aliquoted, snap frozen in liquid nitrogen, and immediately stored at −80°C (3T3 lysates) or shipped on dry ice and then stored at −80°C (N2a lysates).
Brain homogenates were prepared using snap-frozen mouse brains stored at −80°C. Weighed brains were homogenized in 3 mL freshly prepared lysis buffer per 250 mg of brain, using a tissue homogenizer (TH; OMNI International, Kennesaw, Georgia, USA) with disposable tips (hard tissue OMNI tip; OMNI International). Homogenates were then sonicated, centrifuged, and stored as described for cell lysates.
Protein concentration was determined by Bradford’s assay (Bio-Rad Laboratories, Hercules, California, USA). Protein concentration and equal sample loading were then verified by preliminary sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Brain homogenates or cell lysates were mixed with equal volumes of 2X SDS loading buffer (125 mM Tris-Cl [pH 6.8], 20% glycerol, 4% SDS, 0.005% bromophenol blue, 260 mM DTT), denatured at 100°C for 10 min, and loaded onto 10- or 15-well 8% SDS-PAGE gels (Mini-PROTEAN; Bio-Rad Laboratories) (running buffer; 190 mM glycine, 24.8 mM Tris, 0.1% SDS, pH 8.3). Proteins were run through the stacking gel at 50 V, and then resolved for 90 min at 100 V, always at room temperature. Proteins were stained with Coomassie blue G-250 (Bio-Safe Coomassie; Bio-Rad Laboratories) according to the manufacturer’s instructions. Signal from Coomassie-stained protein was detected using an Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, Nebraska, USA) in the 700 nm channel and quantitated using Odyssey 3.0 software (LI-COR Biosciences). Protein amounts were calculated relative to a pre-quantitated standard brain homogenate.
All procedures were performed at room temperature and all washes used gentle rocking. Proteins were denatured by incubating for 10 min at 100°C with an equal volume of 2X, or one-fifth volume of 6X (375 mM Tris-Cl [pH 6.8], 60% glycerol, 12% SDS, 0.015% bromophenol blue, 780 mM DTT), SDS-PAGE loading buffer.
For Western blots of PrP and EGFP, lysates from N2a cells transfected with empty vector (100 μg), or vector encoding EGFP (50 μg), CyPrPEGFP (200 μg), CyPrPEGFP124stop (100 μg), or CyPrPEGFP124-230 (100 μg) were loaded onto 15-well 10% SDS-PAGE gels (Mini-PROTEAN; Bio-Rad Laboratories). Proteins were resolved as described for protein quantitation and the gels were then equilibrated in transfer buffer (384 mM glycine, 49.6 mM Tris, 20% methanol, 0.01% SDS)  for 30 min. Meanwhile, polyvinylidene fluoride (PVDF) membranes (Immuno-Blot, 0.2 μm; Bio-Rad Laboratories) were soaked in methanol for 2 min, and equilibrated in transfer buffer for 20 min. For each membrane, four sheets of filter paper were equilibrated in transfer buffer for 5 min. Transfer cassettes were loaded into a transfer tank (TE22; Hoefer, Holliston, Massachusetts, USA) filled with transfer buffer at 4°C. Proteins were transferred for 23 h at 4°C; 1 h at 54 mA, 4 h at 189 mA, 8 h at 270 mA and finally 10 h at 378 mA. The temperature was maintained at 4°C by heat exchange (Isotemp 1016D; Thermo Fisher Scientific, Waltham, Massachusetts, USA) and gentle stirring of the transfer buffer. After transfer, membranes were dried, soaked in methanol for 2 min and then washed twice for 10 min each in Tris-buffered saline (TBS; 140 mM NaCl, 3 mM KCl, 25 mM Tris, pH 7.6). Membranes were blocked for 1 h in 10% blocking buffer (Sigma-Aldrich) then simultaneously probed for 18 h at 4°C with primary antibodies specific for GFP (a kind gift from Dr. Luc Berthiaume, University of Alberta) and PrP (clone 3F4; a kind gift from Dr. Deborah McKenzie, University of Alberta) diluted to 1:10,000 and 1:2,500, respectively, in 10% blocking buffer with 0.1% Tween-20. Afterward, membranes were washed in TBS with 0.1% Tween-20 (TBST) once for 5 min and thrice for 10 min each. Membranes were incubated with donkey anti-mouse IRDye 680- and donkey anti-rabbit IRDye 800-labeled secondary antibodies (LI-COR Biosciences), diluted to 1:20,000 in 10% blocking buffer with 0.1% Tween-20 and 0.01% SDS for 1 h. Membranes were washed thrice in TBST for 10 min each and once in TBS for 5 min. Signal from pre-stained protein standards and IRDye 680-labeled secondary antibody was detected at 700 nm, and from IRDye 800-labeled secondary antibody at 800 nm, using an Odyssey infrared imaging system. Signal was quantitated using Odyssey 3.0 software. To evaluate the amounts of protein transferred to the membranes, the membranes were stained with Coomassie blue R-250 (Bio-Rad Laboratories) for 10 min and destained with 40% methanol in 10% glacial acetic acid thrice for 10 min each, or until excess stain was removed. Signal from Coomassie-stained protein was detected at 700 nm using the Odyssey and quantitated using Odyssey 3.0 software.
For multiplex Western blot spiking experiments, 85 μg (for p39 and TrkB) or 200 μg (for PKCγ, CaMK4, CDK1, CHK1, RSK2, CDK4, PDGFRβ, BTK and PKCθ) of total protein was loaded per linear cm of four- or five-well 8% SDS-PAGE gels. For primary multiplex Western blots of N2a cells expressing cytoplasmic PrP mutants, 150 μg of protein was loaded per linear cm of single-well 8% SDS-PAGE gels; 8 or 10% gels, as appropriate for the target MW, loaded with 150, 300, or 450 μg of protein per linear cm were used for targeted analyses. Similar signal intensities were reached using 150 μg of N2a or 200 μg of brain lysates (Additional file 4: Figure S3). Proteins were resolved and transferred to PVDF membranes as described for Western blots of EGFP or CyPrPEGFP. Membranes were blocked for 1 h in 10% blocking buffer (Sigma-Aldrich) for protein-specific antibodies, or in 3% BSA (Rockland, Gilbertsville, Pennsylvania, USA) for phosphorylation-specific antibodies. Membranes were rinsed briefly with TBS and positioned within a 20-lane multi-screen apparatus (Bio-Rad Laboratories, Hercules, California, USA). Meanwhile, combinations of primary antibodies were diluted in 2.6 mL (primary blots) or 3.9 mL (spiked, secondary and tertiary blots) of 10% blocking buffer or 3% BSA with 0.1% Tween-20. Six hundred microliters of antibody solutions were loaded in each lane of the multi-screen apparatus and incubated for 18 h at 4°C. Membranes were then briefly washed once with TBST within the multi-screen apparatus, removed from the apparatus and further washed in TBST, once for 5 min and four times for 15 min each. Membranes were incubated with secondary antibody diluted to 1:20,000 in 10% blocking buffer or 3% BSA with 0.1% Tween-20 and 0.01% SDS for 1 h. Mouse monoclonal primary antibodies were detected with goat anti-mouse Alexa Fluor (Molecular Probes, Eugene, Oregon, USA) or IRDye (LI-COR Biosciences) 680-labeled secondary antibody. Rabbit or goat polyclonal primary antibodies were detected with goat anti-rabbit or donkey anti-goat (LI-COR Biosciences or Rockland) IRDye 800-labeled secondary antibodies, respectively. Signal from pre-stained protein standards and Alexa Fluor or IRDye 680-labeled secondary antibody was detected at 700 nm, and from IRDye 800-labeled secondary antibody at 800 nm, using the Odyssey system. Signal was quantitated using Odyssey 3.0 software.
Membranes with protein from cells expressing cytoplasmic PrP mutants were always stripped (only once) in parallel with the membranes from the control cells, under conditions that minimize protein loss . Membranes were washed with a mild stripping buffer (25 mM glycine, 1% SDS, pH 2.0) once for 5 min and twice for 15 min each. Membranes were then washed with TBST once and TBS once for 5 min each. If necessary, they were further washed with mild stripping buffer for a maximum of six times of 15 min each. If still necessary, membranes were then incubated with harsh stripping buffer (50 mM Tris-Cl [pH 7.0], 2% SDS, 50 mM DTT)  for 15 min at 65°C with gentle rocking. Membranes were then washed with TBST once for 5 min and TBS twice for 10 min each. Stripped membranes were blocked and reprobed with another set of primary antibodies as described. No membrane was stripped more than once.
Hierarchical cluster analysis
The integrated intensity levels (pixel volume) of proteins in cells expressing cytoplasmic PrP mutants were normalized to levels in cells expressing empty vector, and then log2 transformed to avoid bias by proteins with extreme changes in expression. Hierarchical clustering was performed with Gene Cluster 3.0  using city-block distance (similarity metric) and complete linkage (clustering method). Java Treeview was used to present the resulting clusters .
Time-course data normalization
Gi: pixel volume in lysates from cells expressing EGFP in replicate “i”,
Pi: pixel volume in lysates from cells expressing CyPrPEGFP in replicate “i”, and
TE: transfection efficiency.
The differences in the integrated intensity levels (pixel volume) in the populations of cells transfected with the vectors expressing CyPrPEGFP or EGFP from the same biological replicate were corrected by the fraction of cells actually expressing the recombinant proteins (transfection efficiency, 47, 45 and 34% for replicates 1, 2 and 3, respectively), which are the cells in which the expressed proteins directly induce changes in signaling, and added to the basal levels (those in the population of cells transfected with the vector expressing EGFP). The corrected levels in cells expressing CyPrPEGFP were then normalized to the levels in cells expressing EGFP in the same biological replicate.
All data was analyzed using Prism (Version 5.0f; GraphPad Software Inc., La Jolla, California, USA). For nonlinear regression analyses, curves of the normalized phosphorylation levels were compared to a straight line (y-intercept = 1; slope = 0), representing the levels in cells expressing EGFP, using a replicates test for lack-of-fit.
This work was supported by PrioNet Canada Grants to LMS and XR, an Alberta Prion Research Institute grant to LMS, a Burroughs Wellcome Fund Award to LMS, and a Canadian Institutes for Health Research grant to XR (MOP-89881). LMS is a Burroughs Wellcome Fund Investigator in the Pathogenesis of Infectious Disease. RHS was supported by NSERC, 75th Anniversary Graduate Student Award (Faculty of Medicine and Dentistry, University of Alberta) and Queen Elizabeth II Doctoral Scholarship (Government of the Province of Alberta).
- Martin L, Latypova X, Wilson C, Magnaudeix A, Perrin M, Yardin C, Terro F: Tau protein kinases: involvement in Alzheimer’s disease. Ageing Res Rev 2013, 12: 289-309. 10.1016/j.arr.2012.06.003PubMedGoogle Scholar
- Wood-Kaczmar A, Gandhi S, Wood N: Understanding the molecular causes of Parkinson’s disease. Trends Mol Med 2006, 12: 521-528. 10.1016/j.molmed.2006.09.007PubMedGoogle Scholar
- Kinase-Disease association. [http://www.cellsignal.com/common/content/content.jsp?id=science-tables-kinase-disease] 
- Knight Z, Lin H, Shokat K: Targeting the cancer kinome through polypharmacology. Nat Rev Cancer 2010, 10: 130-137. 10.1038/nrc2787PubMedPubMed CentralGoogle Scholar
- Cohen P, Alessi D: Kinase drug discovery–what’s next in the field? ACS Chem Biol 2013, 8: 96-104. 10.1021/cb300610sPubMedPubMed CentralGoogle Scholar
- Clinical Trials of Protein Kinase Inhibitors. [http://www.researchandmarkets.com/reports/1196697/protein_kinase_inhibitors_in_oncology_drug] 
- Protein Kinase Inhibitors in Oncology Drug Pipeline Update 2014. 2014. [http://www.researchandmarkets.com/reports/1196697/protein_kinase_inhibitors_in_oncology_drug] 
- Fabbro D, Cowan-Jacob S, Mobitz H, Martiny-Baron G: Targeting cancer with small-molecular-weight kinase inhibitors. Methods Mol Biol 2012, 795: 1-34. 10.1007/978-1-61779-337-0_1PubMedGoogle Scholar
- Bamborough P: System-based drug discovery within the human kinome. Expert Opin Drug Discov 2012, 7: 1053-1070.PubMedGoogle Scholar
- Colby D, Prusiner S: Prions. Cold Spring Harb Perspect Biol 2011, 3: a006833.PubMedPubMed CentralGoogle Scholar
- Prusiner S: Prions. Proc Natl Acad Sci U S A 1998, 95: 13363-13383. 10.1073/pnas.95.23.13363PubMedPubMed CentralGoogle Scholar
- Baeten L, Powers B, Jewell J, Spraker T, Miller M: A natural case of chronic wasting disease in a free-ranging moose (Alces alces shirasi). J Wildl Dis 2007, 43: 309-314. 10.7589/0090-3558-43.2.309PubMedGoogle Scholar
- Schwarz A, Burwinkel M, Riemer C, Schultz J, Baier M: Unchanged scrapie pathology in brain tissue of tyrosine kinase Fyn-deficient mice. Neurodegener Dis 2004, 1: 266-268. 10.1159/000085065PubMedGoogle Scholar
- Moreno J, Radford H, Peretti D, Steinert J, Verity N, Martin M, Halliday M, Morgan J, Dinsdale D, Ortori C, Barrett DA, Tsaytler P, Bertolotti A, Willis AE, Bushell M, Mallucci GR: Sustained translational repression by eIF2alpha-P mediates prion neurodegeneration. Nature 2012, 485: 507-511.PubMedPubMed CentralGoogle Scholar
- Arsenault R, Li Y, Potter A, Griebel P, Kusalik A, Napper S: Induction of ligand-specific PrP (C) signaling in human neuronal cells. Prion 2012, 6: 477-488. 10.4161/pri.21914PubMedPubMed CentralGoogle Scholar
- Yun S, Ertmer A, Flechsig E, Gilch S, Riederer P, Gerlach M, Schatzl H, Klein M: The tyrosine kinase inhibitor imatinib mesylate delays prion neuroinvasion by inhibiting prion propagation in the periphery. J Neurovirol 2007, 13: 328-337. 10.1080/13550280701361516PubMedGoogle Scholar
- Nordstrom E, Fisone G, Kristensson K: Opposing effects of ERK and p38-JNK MAP kinase pathways on formation of prions in GT1-1 cells. FASEB J 2009, 23: 613-622.PubMedGoogle Scholar
- Aguib Y, Heiseke A, Gilch S, Riemer C, Baier M, Schätzl H, Ertmer A: Autophagy induction by trehalose counteracts cellular prion infection. Autophagy 2009, 5: 361-369. 10.4161/auto.5.3.7662PubMedGoogle Scholar
- Nordstrom E, Luhr K, Ibanez C, Kristensson K: Inhibitors of the mitogen-activated protein kinase kinase 1/2 signaling pathway clear prion-infected cells from PrPSc. J Neurosci 2005, 25: 8451-8456. 10.1523/JNEUROSCI.2349-05.2005PubMedGoogle Scholar
- Ertmer A, Gilch S, Yun S, Flechsig E, Klebl B, Stein-Gerlach M, Klein M, Schatzl H: The tyrosine kinase inhibitor STI571 induces cellular clearance of PrPSc in prion-infected cells. J Biol Chem 2004, 279: 41918-41927. 10.1074/jbc.M405652200PubMedGoogle Scholar
- Heiseke A, Aguib Y, Riemer C, Baier M, Schatzl H: Lithium induces clearance of protease resistant prion protein in prion-infected cells by induction of autophagy. J Neurochem 2009, 109: 25-34. 10.1111/j.1471-4159.2009.05906.xPubMedGoogle Scholar
- Allard E, Grujic M, Fisone G, Kristensson K: Prion formation correlates with activation of translation-regulating protein 4E-BP and neuronal transcription factor Elk1. Neurobiol Dis 2013, 58: 116-122.PubMedGoogle Scholar
- Collinge J, Palmer M, Sidle K, Gowland I, Medori R, Ironside J, Lantos P: Transmission of fatal familial insomnia to laboratory animals. Lancet 1995, 346: 569-570. 10.1016/S0140-6736(95)91405-6PubMedGoogle Scholar
- Medori R, Montagna P, Tritschler H, LeBlanc A, Cortelli P, Tinuper P, Lugaresi E, Gambetti P: Fatal familial insomnia: a second kindred with mutation of prion protein gene at codon 178. Neurology 1992, 42: 669-670. 10.1212/WNL.42.3.669PubMedGoogle Scholar
- Brandner S, Isenmann S, Raeber A, Fischer M, Sailer A, Kobayashi Y, Marino S, Weissmann C, Aguzzi A: Normal host prion protein necessary for scrapie-induced neurotoxicity. Nature 1996, 379: 339-343. 10.1038/379339a0PubMedGoogle Scholar
- Mallucci G, Dickinson A, Linehan J, Klohn P, Brandner S, Collinge J: Depleting neuronal PrP in prion infection prevents disease and reverses spongiosis. Science 2003, 302: 871-874. 10.1126/science.1090187PubMedGoogle Scholar
- Stahl N, Borchelt D, Hsiao K, Prusiner S: Scrapie prion protein contains a phosphatidylinositol glycolipid. Cell 1987, 51: 229-240. 10.1016/0092-8674(87)90150-4PubMedGoogle Scholar
- Baldwin M, Burlingame A, Prusiner S: Mass spectrometric analysis of a GPI-anchored protein: the scrapie prion protein. Trends Anal Chem 1993, 12: 239-248. 10.1016/0165-9936(93)87063-4Google Scholar
- Drisaldi B, Stewart R, Adles C, Stewart L, Quaglio E, Biasini E, Fioriti L, Chiesa R, Harris D: Mutant PrP is delayed in its exit from the endoplasmic reticulum, but neither wild-type nor mutant PrP undergoes retrotranslocation prior to proteasomal degradation. J Biol Chem 2003, 278: 21732-21743. 10.1074/jbc.M213247200PubMedGoogle Scholar
- Rane N, Yonkovich J, Hegde R: Protection from cytosolic prion protein toxicity by modulation of protein translocation. EMBO J 2004, 23: 4550-4559. 10.1038/sj.emboj.7600462PubMedPubMed CentralGoogle Scholar
- Levine C, Mitra D, Sharma A, Smith C, Hegde R: The efficiency of protein compartmentalization into the secretory pathway. Mol Biol Cell 2005, 16: 279-291.PubMedPubMed CentralGoogle Scholar
- Ma J, Lindquist S: Wild-type PrP and a mutant associated with prion disease are subject to retrograde transport and proteasome degradation. Proc Natl Acad Sci U S A 2001, 98: 14955-14960. 10.1073/pnas.011578098PubMedPubMed CentralGoogle Scholar
- Ma J, Lindquist S: Conversion of PrP to a self-perpetuating PrPSc-like conformation in the cytosol. Science 2002, 298: 1785-1788. 10.1126/science.1073619PubMedGoogle Scholar
- Yedidia Y, Horonchik L, Tzaban S, Yanai A, Taraboulos A: Proteasomes and ubiquitin are involved in the turnover of the wild-type prion protein. EMBO J 2001, 20: 5383-5391. 10.1093/emboj/20.19.5383PubMedPubMed CentralGoogle Scholar
- Lund C, Olsen C, Skogtvedt S, Tveit H, Prydz K, Tranulis M: Alternative translation initiation generates cytoplasmic sheep prion protein. J Biol Chem 2009, 284: 19668-19678. 10.1074/jbc.M109.003566PubMedPubMed CentralGoogle Scholar
- Rane N, Kang S, Chakrabarti O, Feigenbaum L, Hegde R: Reduced translocation of nascent prion protein during ER stress contributes to neurodegeneration. Dev Cell 2008, 15: 359-370. 10.1016/j.devcel.2008.06.015PubMedPubMed CentralGoogle Scholar
- Bailly Y, Haeberle A, Blanquet-Grossard F, Chasserot-Golaz S, Grant N, Schulze T, Bombarde G, Grassi J, Cesbron J, Lemaire-Vieille C: Prion protein (PrPc) immunocytochemistry and expression of the green fluorescent protein reporter gene under control of the bovine PrP gene promoter in the mouse brain. J Comp Neurol 2004, 473: 244-269. 10.1002/cne.20117PubMedGoogle Scholar
- Barmada S, Piccardo P, Yamaguchi K, Ghetti B, Harris D: GFP-tagged prion protein is correctly localized and functionally active in the brains of transgenic mice. Neurobiol Dis 2004, 16: 527-537. 10.1016/j.nbd.2004.05.005PubMedGoogle Scholar
- Mironov AJ, Latawiec D, Wille H, Bouzamondo-Bernstein E, Legname G, Williamson R, Burton D, DeArmond S, Prusiner S, Peters P: Cytosolic prion protein in neurons. J Neurosci 2003, 23: 7183-7193.PubMedGoogle Scholar
- Norstrom EM, Ciaccio MF, Rassbach B, Wollmann R, Mastrianni JA: Cytosolic prion protein toxicity is independent of cellular prion protein expression and prion propagation. J Virol 2007, 81: 2831-2837. 10.1128/JVI.02157-06PubMedPubMed CentralGoogle Scholar
- Hegde R, Rane N: Prion protein trafficking and the development of neurodegeneration. Trends Neurosci 2003, 26: 337-339. 10.1016/S0166-2236(03)00143-7PubMedGoogle Scholar
- Ma J, Wollmann R, Lindquist S: Neurotoxicity and neurodegeneration when PrP accumulates in the cytosol. Science 2002, 298: 1781-1785. 10.1126/science.1073725PubMedGoogle Scholar
- Goggin K, Beaudoin S, Grenier C, Brown A, Roucou X: Prion protein aggresomes are poly(A) + ribonucleoprotein complexes that induce a PKR-mediated deficient cell stress response. Biochim Biophys Acta 2008, 1783: 479-491. 10.1016/j.bbamcr.2007.10.008PubMedGoogle Scholar
- Rambold A, Miesbauer M, Rapaport D, Bartke T, Baier M, Winklhofer K, Tatzelt J: Association of Bcl-2 with misfolded prion protein is linked to the toxic potential of cytosolic PrP. Mol Biol Cell 2006, 17: 3356-3368. 10.1091/mbc.E06-01-0083PubMedPubMed CentralGoogle Scholar
- Zhang J, Wang K, Guo Y, Shi Q, Tian C, Chen C, Gao C, Zhang B, Dong X: Heat shock protein 70 selectively mediates the degradation of cytosolic PrPs and restores the cytosolic PrP-induced cytotoxicity via a molecular interaction. Virol J 2012, 9: 303. 10.1186/1743-422X-9-303PubMedPubMed CentralGoogle Scholar
- Martin J, Masri J, Bernath A, Nishimura R, Gera J: Hsp70 associates with Rictor and is required for mTORC2 formation and activity. Biochem Biophys Res Commun 2008, 372: 578-583. 10.1016/j.bbrc.2008.05.086PubMedPubMed CentralGoogle Scholar
- Rosner M, Fuchs C, Siegel N, Valli A, Hengstschlager M: Functional interaction of mammalian target of rapamycin complexes in regulating mammalian cell size and cell cycle. Hum Mol Genet 2009, 18: 3298-3310. 10.1093/hmg/ddp271PubMedPubMed CentralGoogle Scholar
- Shahbazian D, Parsyan A, Petroulakis E, Topisirovic I, Martineau Y, Gibbs B, Svitkin Y, Sonenberg N: Control of cell survival and proliferation by mammalian eukaryotic initiation factor 4B. Mol Cell Biol 2010, 30: 1478-1485. 10.1128/MCB.01218-09PubMedPubMed CentralGoogle Scholar
- Caenepeel S, Charydczak G, Sudarsanam S, Hunter T, Manning G: The mouse kinome: discovery and comparative genomics of all mouse protein kinases. Proc Natl Acad Sci U S A 2004, 101: 11707-11712. 10.1073/pnas.0306880101PubMedPubMed CentralGoogle Scholar
- Manning G, Whyte D, Martinez R, Hunter T, Sudarsanam S: The protein kinase complement of the human genome. Science 2002, 298: 1912-1934. 10.1126/science.1075762PubMedGoogle Scholar
- Valin A, Cook J, Ross S, Saklad C, Gill G: Sp1 and Sp3 regulate transcription of the cyclin-dependent kinase 5 regulatory subunit 2 (p39) promoter in neuronal cells. Biochim Biophys Acta 2009, 1789: 204-211. 10.1016/j.bbagrm.2009.01.007PubMedPubMed CentralGoogle Scholar
- Soppet D, Escandon E, Maragos J, Middlemas D, Reid S, Blair J, Burton L, Stanton B, Kaplan D, Hunter T, Nikolics K, Parada LF: The neurotrophic factors brain-derived neurotrophic factor and neurotrophin-3 are ligands for the trkB tyrosine kinase receptor. Cell 1991, 65: 895-903. 10.1016/0092-8674(91)90396-GPubMedGoogle Scholar
- Klein R, Nanduri V, Jing S, Lamballe F, Tapley P, Bryant S, Cordon-Cardo C, Jones K, Reichardt L, Barbacid M: The trkB tyrosine protein kinase is a receptor for brain-derived neurotrophic factor and neurotrophin-3. Cell 1991, 66: 395-403. 10.1016/0092-8674(91)90628-CPubMedPubMed CentralGoogle Scholar
- Alevizopoulos A, Dusserre Y, Ruegg U, Mermod N: Regulation of the transforming growth factor beta-responsive transcription factor CTF-1 by calcineurin and calcium/calmodulin-dependent protein kinase IV. J Biol Chem 1997, 272: 23597-23605. 10.1074/jbc.272.38.23597PubMedGoogle Scholar
- Cuadrado A, Molloy C, Pech M: Expression of protein kinase CI in NIH 3T3 cells increases its growth response to specific activators. FEBS Lett 1990, 260: 281-284. 10.1016/0014-5793(90)80123-ZPubMedGoogle Scholar
- Grenier C, Bissonnette C, Volkov L, Roucou X: Molecular morphology and toxicity of cytoplasmic prion protein aggregates in neuronal and non-neuronal cells. J Neurochem 2006, 97: 1456-1466. 10.1111/j.1471-4159.2006.03837.xPubMedPubMed CentralGoogle Scholar
- Foster K, Fingar D: Mammalian target of rapamycin (mTOR): conducting the cellular signaling symphony. J Biol Chem 2010, 285: 14071-14077. 10.1074/jbc.R109.094003PubMedPubMed CentralGoogle Scholar
- Sarbassov D, Guertin D, Ali S, Sabatini D: Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex. Science 2005, 307: 1098-1101. 10.1126/science.1106148PubMedGoogle Scholar
- Alessi D, James S, Downes C, Holmes A, Gaffney P, Reese C, Cohen P: Characterization of a 3-phosphoinositide-dependent protein kinase which phosphorylates and activates protein kinase Balpha. Curr Biol 1997, 7: 261-269. 10.1016/S0960-9822(06)00122-9PubMedGoogle Scholar
- Copp J, Manning G, Hunter T: TORC-specific phosphorylation of mammalian target of rapamycin (mTOR): phospho-Ser2481 is a marker for intact mTOR signaling complex 2. Cancer Res 2009, 69: 1821-1827. 10.1158/0008-5472.CAN-08-3014PubMedPubMed CentralGoogle Scholar
- Hawley S, Davison M, Woods A, Davies S, Beri R, Carling D, Hardie D: Characterization of the AMP-activated protein kinase kinase from rat liver and identification of threonine 172 as the major site at which it phosphorylates AMP-activated protein kinase. J Biol Chem 1996, 271: 27879-27887. 10.1074/jbc.271.44.27879PubMedGoogle Scholar
- Sarbassov D, Ali S, Kim D, Guertin D, Latek R, Erdjument-Bromage H, Tempst P, Sabatini D: Rictor, a novel binding partner of mTOR, defines a rapamycin-insensitive and raptor-independent pathway that regulates the cytoskeleton. Curr Biol 2004, 14: 1296-1302. 10.1016/j.cub.2004.06.054PubMedGoogle Scholar
- Burnett P, Barrow R, Cohen N, Snyder S, Sabatini D: RAFT1 phosphorylation of the translational regulators p70 S6 kinase and 4E-BP1. Proc Natl Acad Sci U S A 1998, 95: 1432-1437. 10.1073/pnas.95.4.1432PubMedPubMed CentralGoogle Scholar
- Ferrari S, Bandi H, Hofsteenge J, Bussian B, Thomas G: Mitogen-activated 70 K S6 kinase. Identification of in vitro 40 S ribosomal S6 phosphorylation sites. J Biol Chem 1991, 266: 22770-22775.PubMedGoogle Scholar
- Raught B, Peiretti F, Gingras A, Livingstone M, Shahbazian D, Mayeur G, Polakiewicz R, Sonenberg N, Hershey J: Phosphorylation of eucaryotic translation initiation factor 4B Ser422 is modulated by S6 kinases. EMBO J 2004, 23: 1761-1769. 10.1038/sj.emboj.7600193PubMedPubMed CentralGoogle Scholar
- Waskiewicz A, Johnson J, Penn B, Mahalingam M, Kimball S, Cooper J: Phosphorylation of the cap-binding protein eukaryotic translation initiation factor 4E by protein kinase Mnk1 in vivo. Mol Cell Biol 1999, 19: 1871-1880.PubMedPubMed CentralGoogle Scholar
- Redpath N, Price N, Severinov K, Proud C: Regulation of elongation factor-2 by multisite phosphorylation. Eur J Biochem 1993, 213: 689-699. 10.1111/j.1432-1033.1993.tb17809.xPubMedGoogle Scholar
- Kelly S, Zhang Z, Zhao H, Xu L, Giffard R, Sapolsky R, Yenari M, Steinberg G: Gene transfer of HSP72 protects cornu ammonis 1 region of the hippocampus neurons from global ischemia: influence of Bcl-2. Ann Neurol 2002, 52: 160-167. 10.1002/ana.10264PubMedGoogle Scholar
- Jiang B, Liang P, Deng G, Tu Z, Liu M, Xiao X: Increased stability of Bcl-2 in HSP70-mediated protection against apoptosis induced by oxidative stress. Cell Stress Chaperones 2011, 16: 143-152. 10.1007/s12192-010-0226-6PubMedPubMed CentralGoogle Scholar
- Rozen F, Edery I, Meerovitch K, Dever T, Merrick W, Sonenberg N: Bidirectional RNA helicase activity of eucaryotic translation initiation factors 4A and 4F. Mol Cell Biol 1990, 10: 1134-1144.PubMedPubMed CentralGoogle Scholar
- Rozovsky N, Butterworth A, Moore M: Interactions between eIF4AI and its accessory factors eIF4B and eIF4H. RNA 2008, 14: 2136-2148. 10.1261/rna.1049608PubMedPubMed CentralGoogle Scholar
- Methot N, Pickett G, Keene J, Sonenberg N: In vitro RNA selection identifies RNA ligands that specifically bind to eukaryotic translation initiation factor 4B: the role of the RNA remotif. RNA 1996, 2: 38-50.PubMedPubMed CentralGoogle Scholar
- Methot N, Song M, Sonenberg N: A region rich in aspartic acid, arginine, tyrosine, and glycine (DRYG) mediates eukaryotic initiation factor 4B (eIF4B) self-association and interaction with eIF3. Mol Cell Biol 1996, 16: 5328-5334.PubMedPubMed CentralGoogle Scholar
- Combs C, Johnson D, Cannady S, Lehman T, Landreth G: Identification of microglial signal transduction pathways mediating a neurotoxic response to amyloidogenic fragments of beta-amyloid and prion proteins. J Neurosci 1999, 19: 928-939.PubMedGoogle Scholar
- Jin J, Choi J, Lee H, Kim Y, Carp R, Choi E: Increased expression of CaM kinase II alpha in the brains of scrapie-infected mice. Neurosci Lett 1999, 273: 37-40. 10.1016/S0304-3940(99)00622-9PubMedGoogle Scholar
- Mouillet-Richard S, Ermonval M, Chebassier C, Laplanche J, Lehmann S, Launay J, Kellermann O: Signal transduction through prion protein. Science 2000, 289: 1925-1928. 10.1126/science.289.5486.1925PubMedGoogle Scholar
- Chiarini L, Freitas A, Zanata S, Brentani R, Martins V, Linden R: Cellular prion protein transduces neuroprotective signals. EMBO J 2002, 21: 3317-3326. 10.1093/emboj/cdf324PubMedPubMed CentralGoogle Scholar
- Lopes J, Oliveira C, Agostinho P: Role of cyclin-dependent kinase 5 in the neurodegenerative process triggered by amyloid-Beta and prion peptides: implications for Alzheimer’s disease and prion-related encephalopathies. Cell Mol Neurobiol 2007, 27: 943-957. 10.1007/s10571-007-9224-3PubMedGoogle Scholar
- Lopes M, Hajj G, Muras A, Mancini G, Castro R, Ribeiro K, Brentani R, Linden R, Martins V: Interaction of cellular prion and stress-inducible protein 1 promotes neuritogenesis and neuroprotection by distinct signaling pathways. J Neurosci 2005, 25: 11330-11339. 10.1523/JNEUROSCI.2313-05.2005PubMedGoogle Scholar
- Kanaani J, Prusiner S, Diacovo J, Baekkeskov S, Legname G: Recombinant prion protein induces rapid polarization and development of synapses in embryonic rat hippocampal neurons in vitro. J Neurochem 2005, 95: 1373-1386. 10.1111/j.1471-4159.2005.03469.xPubMedGoogle Scholar
- Weise J, Sandau R, Schwarting S, Crome O, Wrede A, Schulz-Schaeffer W, Zerr I, Bahr M: Deletion of cellular prion protein results in reduced Akt activation, enhanced postischemic caspase-3 activation, and exacerbation of ischemic brain injury. Stroke 2006, 37: 1296-1300. 10.1161/01.STR.0000217262.03192.d4PubMedGoogle Scholar
- Krebs B, Dorner-Ciossek C, Schmalzbauer R, Vassallo N, Herms J, Kretzschmar H: Prion protein induced signaling cascades in monocytes. Biochem Biophys Res Commun 2006, 340: 13-22. 10.1016/j.bbrc.2005.11.158PubMedGoogle Scholar
- Schneider B, Mutel V, Pietri M, Ermonval M, Mouillet-Richard S, Kellermann O: NADPH oxidase and extracellular regulated kinases 1/2 are targets of prion protein signaling in neuronal and nonneuronal cells. Proc Natl Acad Sci U S A 2003, 100: 13326-13331. 10.1073/pnas.2235648100PubMedPubMed CentralGoogle Scholar
- Monnet C, Gavard J, Mege R, Sobel A: Clustering of cellular prion protein induces ERK1/2 and stathmin phosphorylation in GT1-7 neuronal cells. FEBS Lett 2004, 576: 114-118. 10.1016/j.febslet.2004.08.076PubMedGoogle Scholar
- Lee H, Jun Y, Choi J, Kim J, Carp R, Kim Y: Activation of mitogen-activated protein kinases in hamster brains infected with 263 K scrapie agent. J Neurochem 2005, 95: 584-593. 10.1111/j.1471-4159.2005.03429.xPubMedGoogle Scholar
- Nixon R: Prion-associated increases in Src-family kinases. J Biol Chem 2005, 280: 2455-2462. 10.1074/jbc.M410883200PubMedGoogle Scholar
- Caetano F, Lopes M, Hajj G, Machado C, Pinto Arantes C, Magalhaes A, Vieira Mde P, Americo T, Massensini A, Priola S, Vorberg I, Gomez MV, Linden R, Prado VF, Martins VR, Prado MA: Endocytosis of prion protein is required for ERK1/2 signaling induced by stress-inducible protein 1. J Neurosci 2008, 28: 6691-6702. 10.1523/JNEUROSCI.1701-08.2008PubMedPubMed CentralGoogle Scholar
- Roffe M, Beraldo F, Bester R, Nunziante M, Bach C, Mancini G, Gilch S, Vorberg I, Castilho B, Martins V, Hajj G: Prion protein interaction with stress-inducible protein 1 enhances neuronal protein synthesis via mTOR. Proc Natl Acad Sci U S A 2010, 107: 13147-13152. 10.1073/pnas.1000784107PubMedPubMed CentralGoogle Scholar
- Spurrier B, Ramalingam S, Nishizuka S: Reverse-phase protein lysate microarrays for cell signaling analysis. Nat Protoc 2008, 3: 1796-1808. 10.1038/nprot.2008.179PubMedGoogle Scholar
- Booth S, Bowman C, Baumgartner R, Dolenko B, Sorensen G, Robertson C, Coulthart M, Phillipson C, Somorjai R: Molecular classification of scrapie strains in mice using gene expression profiling. Biochem Biophys Res Commun 2004, 325: 1339-1345. 10.1016/j.bbrc.2004.10.150PubMedGoogle Scholar
- Booth S, Bowman C, Baumgartner R, Sorensen G, Robertson C, Coulthart M, Phillipson C, Somorjai R: Identification of central nervous system genes involved in the host response to the scrapie agent during preclinical and clinical infection. J Gen Virol 2004, 85: 3459-3471. 10.1099/vir.0.80110-0PubMedGoogle Scholar
- Brown A, Rebus S, McKimmie C, Robertson K, Williams A, Fazakerley J: Gene expression profiling of the preclinical scrapie-infected hippocampus. Biochem Biophys Res Commun 2005, 334: 86-95. 10.1016/j.bbrc.2005.06.060PubMedGoogle Scholar
- Riemer C, Neidhold S, Burwinkel M, Schwarz A, Schultz J, Kratzschmar J, Monning U, Baier M: Gene expression profiling of scrapie-infected brain tissue. Biochem Biophys Res Commun 2004, 323: 556-564. 10.1016/j.bbrc.2004.08.124PubMedGoogle Scholar
- Sawiris G, Becker K, Elliott E, Moulden R, Rohwer R: Molecular analysis of bovine spongiform encephalopathy infection by cDNA arrays. J Gen Virol 2007, 88: 1356-1362. 10.1099/vir.0.82387-0PubMedGoogle Scholar
- Skinner P, Abbassi H, Chesebro B, Race R, Reilly C, Haase A: Gene expression alterations in brains of mice infected with three strains of scrapie. BMC Genomics 2006, 7: 114. 10.1186/1471-2164-7-114PubMedPubMed CentralGoogle Scholar
- Xiang W, Windl O, Wunsch G, Dugas M, Kohlmann A, Dierkes N, Westner I, Kretzschmar H: Identification of differentially expressed genes in scrapie-infected mouse brains by using global gene expression technology. J Virol 2004, 78: 11051-11060. 10.1128/JVI.78.20.11051-11060.2004PubMedPubMed CentralGoogle Scholar
- Xiang W, Hummel M, Mitteregger G, Pace C, Windl O, Mansmann U, Kretzschmar H: Transcriptome analysis reveals altered cholesterol metabolism during the neurodegeneration in mouse scrapie model. J Neurochem 2007, 102: 834-847. 10.1111/j.1471-4159.2007.04566.xPubMedGoogle Scholar
- Sorensen G, Medina S, Parchaliuk D, Phillipson C, Robertson C, Booth S: Comprehensive transcriptional profiling of prion infection in mouse models reveals networks of responsive genes. BMC Genomics 2008, 9: 114. 10.1186/1471-2164-9-114PubMedPubMed CentralGoogle Scholar
- Filali H, Martin-Burriel I, Harders F, Varona L, Lyahyai J, Zaragoza P, Pumarola M, Badiola J, Bossers A, Bolea R: Gene expression profiling and association with prion-related lesions in the medulla oblongata of symptomatic natural scrapie animals. PLoS One 2011, 6: e19909. 10.1371/journal.pone.0019909PubMedPubMed CentralGoogle Scholar
- Majer A, Medina S, Niu Y, Abrenica B, Manguiat K, Frost K, Philipson C, Sorensen D, Booth S: Early mechanisms of pathobiology are revealed by transcriptional temporal dynamics in hippocampal CA1 neurons of prion infected mice. PLoS Pathog 2012, 8: e1003002. 10.1371/journal.ppat.1003002PubMedPubMed CentralGoogle Scholar
- Hwang D, Lee I, Yoo H, Gehlenborg N, Cho J, Petritis B, Baxter D, Pitstick R, Young R, Spicer D, Price ND, Hohmann JG, Dearmond SJ, Carlson GA, Hood LE: A systems approach to prion disease. Mol Syst Biol 2009, 5: 252.PubMedPubMed CentralGoogle Scholar
- Kim H, Snyder G, Blazey T, Race R, Chesebro B, Skinner P: Prion disease induced alterations in gene expression in spleen and brain prior to clinical symptoms. Adv Appl Bioinform Chem 2008, 1: 29-50.PubMedPubMed CentralGoogle Scholar
- Gygi S, Rochon Y, Franza B, Aebersold R: Correlation between protein and mRNA abundance in yeast. Mol Cell Biol 1999, 19: 1720-1730.PubMedPubMed CentralGoogle Scholar
- Friedman R, Farh K, Burge C, Bartel D: Most mammalian mRNAs are conserved targets of microRNAs. Genome Res 2009, 19: 92-105.PubMedPubMed CentralGoogle Scholar
- Saba R, Goodman C, Huzarewich R, Robertson C, Booth S: A miRNA signature of prion induced neurodegeneration. PLoS One 2008, 3: e3652. 10.1371/journal.pone.0003652PubMedPubMed CentralGoogle Scholar
- Montag J, Hitt R, Opitz L, Schulz-Schaeffer W, Hunsmann G, Motzkus D: Upregulation of miRNA hsa-miR-342-3p in experimental and idiopathic prion disease. Mol Neurodegener 2009, 4: 36. 10.1186/1750-1326-4-36PubMedPubMed CentralGoogle Scholar
- Otter T, King S, Witman G: A two-step procedure for efficient electrotransfer of both high-molecular-weight (greater than 400,000) and low-molecular-weight (less than 20,000) proteins. Anal Biochem 1987, 162: 370-377. 10.1016/0003-2697(87)90406-4PubMedGoogle Scholar
- Yeung Y, Stanley E: A solution for stripping antibodies from polyvinylidene fluoride immunoblots for multiple reprobing. Anal Biochem 2009, 389: 89-91. 10.1016/j.ab.2009.03.017PubMedPubMed CentralGoogle Scholar
- Harlow E, Lane D: Using Antibodies: A Laboratory Manual. New York: Cold Spring Harbor Laboratory Press; 1999.Google Scholar
- de Hoon M, Imoto S, Nolan J, Miyano S: Open source clustering software. Bioinformatics 2004, 20: 1453-1454. 10.1093/bioinformatics/bth078PubMedGoogle Scholar
- Saldanha A: Java Treeview–extensible visualization of microarray data. Bioinformatics 2004, 20: 3246-3248. 10.1093/bioinformatics/bth349PubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.