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A novel whole-cell lysate kinase assay identifies substrates of the p38 MAPK in differentiating myoblasts

A novel whole-cell lysate kinase assay identifies substrates of the p38 MAPK in differentiating... Background: The p38a mitogen-activated protein kinase (MAPK) is a critical mediator of myoblast differentiation, and does so in part through the phosphorylation and regulation of several transcription factors and chromatin remodelling proteins. However, whether p38a is involved in processes other than gene regulation during myogenesis is currently unknown, and why other p38 isoforms cannot compensate for its loss is unclear. Methods: To further characterise the involvement of p38a during myoblast differentiation, we developed and applied a simple technique for identifying relevant in vivo kinase substrates and their phosphorylation sites. In addition to identifying substrates for one kinase, the technique can be used in vitro to compare multiple kinases in the same experiment, and we made use of this to study the substrate specificities of the p38a and b isoforms. Results: Applying the technique to p38a resulted in the identification of seven in vivo phosphorylation sites on six proteins, four of which are cytoplasmic, in lysate derived from differentiating myoblasts. An in vitro comparison with p38b revealed that substrate specificity does not discriminate these two isoforms, but rather that their distinguishing characteristic appears to be cellular localisation. Conclusion: Our results suggest p38a has a novel cytoplasmic role during myogenesis and that its unique cellular localisation may be why p38b and other isoforms cannot compensate for its absence. The substrate-finding approach presented here also provides a necessary tool for studying the hundreds of protein kinases that exist and for uncovering the deeper mechanisms of phosphorylation-dependent cell signalling. Keywords: differentiation, FSBA, kinase assay, mitogen-activated protein kinase, myoblast, p38, phosphorylation, quantitative MS Background substrate-finding techniques exist, they are hindered by Protein kinases are well-known regulators of cell signal- problems that prevent them from being easily or readily ling and cellular behaviour that execute their function employed [1-4] and are generally limited to providing in through the covalent attachment of an ATP-derived vitro substrate identifications that may or may not be phosphate to protein substrates. To understand the relevant in vivo. In vivo approaches currently available, function of any protein kinase on a large and cell-wide such as that employed by Holt et al. [5], can associate a scale first requires the development of a substrate kinase with in vivo phosphorylation events, but direct screening technique that allows for the proteins phos- phosphorylation cannot be inferred without additional phorylated by a kinase of interest to be comprehensively experimentation. A simple technique that can identify identified, ideally in a single experiment. Although direct in vivo substrates is an obvious need for the field. The mitogen-activated protein kinase p38a is involved in several cellular processes, but its critical role during * Correspondence: rkothary@ohri.ca differentiation, and particularly the differentiation of Regenerative Medicine Program, Ottawa Hospital Research Institute, 501 Smyth Road, Ottawa, ON, K1H 8L6, Canada myoblasts, has been a major focus. At the initiation of Full list of author information is available at the end of the article © 2012 Knight et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Knight et al. Skeletal Muscle 2012, 2:5 Page 2 of 12 http://www.skeletalmusclejournal.com/content/2/1/5 myoblast differentiation, p38a is known to phosphory- and p38b were acquired from Addgene (Cambridge, late several transcription factors and chromatin remo- MA, USA) [10]. Constructs were transfected into C2C12 delling proteins, thereby inducing the expression of a myoblasts with Lipofectamine 2000 reagent (Invitrogen, myogenic gene program [6]. Although much is known Carlsbad, CA, USA) according to the manufacturer’s about p38a’s role in this process, it is likely very partial, instructions. To inhibit p38 activity, SB 202190 (Pro- and whether p38a plays an important role in other pro- mega, Madison, WI, USA) solubilised in dimethyl sulph- cesses during myoblast differentiation, such as cell oxide (DMSO) or DMSO as control was added to fusion or sarcomere formation, is unknown. At the differentiation media at 48 hours following the induction same time, there are questions regarding the other p38 of differentiation, and medium with inhibitor was chan- isoforms and their role, or lack thereof, in myogenesis. ged daily. p38b is also expressed in myoblasts and is activated in the same manner as p38a, but despite having a kinase Immunofluorescence domain 75% identical to that of p38a (72% sequence Cells were fixed with 4% formaldehyde and stained with identity overall), p38b is unable to compensate for the the following antibodies: Flag M2 (1:1,000 dilution; loss of p38a, even when overexpressed [7-9]. The Sigma-Aldrich, St Louis, MO, USA), myosin heavy chain obvious and suspected explanation is that there are criti- (MyHC) (1:20 dilution; Developmental Studies Hybri- cal myogenic phosphorylations specific to the a isoform, doma Bank, Department of Biology, University of Iowa, but these have yet to be discovered and whether this Iowa City, IA, USA), Alexa Fluor 488 goat anti-mouse assumption is correct is unknown. antibody (1:1,000 dilution; Molecular Probes/Invitrogen) Here we describe a simple approach for substrate find- and Alexa Fluor 555 goat anti-mouse antibody (1:1,000 ing that can be used to identify in vitro and in vivo sub- dilution; Molecular Probes/Invitrogen). The differentia- strates. The technique begins with treatment of cell tion index was calculated as the number of MyHC-posi- lysate to inactivate endogenous kinases, followed by an tive nuclei divided by the total number of nuclei. The in vitro assay using an exogenous kinase of interest, and fusion index was quantified as the number of nuclei per concludes with quantitative mass spectrometry (MS) to MyHC-positive cell. Five fields of view at ×20 magnifica- identify phosphorylation sites specific to the added tion were counted and averaged per replicate, with a kinase. By using lysate derived from vehicle- or inhibi- total of three replicate experiments. tor-treated cells, this in vitro approach can be simulta- neously coupled with biologically relevant information Statistical analysis to identify direct substrates regulated by the kinase of Statistical analyses were performed using StatPlus soft- interest in vivo. Applying this technique to p38a with ware (AnalystSoft Inc; http://www.analystsoft.com/en/ lysate from differentiating myoblasts resulted in the products/statplus/). The data shown are means with SD, identification of several new in vivo substrates that sug- and Student’s t-tests were performed to determine sig- gest novel functions for p38a during myogenesis. We nificance for the differentiation and fusion indices. did not identify a single phosphorylation specific to the p38a isoform compared with p38b,atleast in termsof Western blot analysis in vitro substrate specificity, but we did see a clear dif- For Western blot analysis, cells were lysed in radioim- ference in cellular localisation during myoblast differen- munoprecipitation assay (RIPA) buffer (50 mM tiation. This leads us to propose that although the Tris∙HCl, pH 7.5, 1% Nonidet P-40, 0.1% SDS, 150 mM kinase domains of p38a and b likely have the same NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 50 capacity to phosphorylate substrates, there are major mM NaF, 200 μMNa VO , 1 mM phenylmethanesul- 3 4 differences in actual substrate specificity in an in vivo phonylfluoride (PMSF), 10 μg/ml aprotinin, 10 μg/ml context. leupeptin and 10 μg/ml pepstatin), and 5× Laemmli buf- fer (300 mM Tris, pH 6.8, 0.01% bromophenol blue, Methods 10% SDS, 50% glycerol and 5% b-mercaptoethanol) was Cell culture added to 25 μg of protein per sample to a final concen- C2C12 cells were grown in Dulbecco’s modified Eagle’s tration of 1× for SDS-PAGE. For fractionation, cells medium (DMEM) supplemented with 10% (vol/vol) foe- were lysed in hypotonic buffer (10 mM 4-(2-hydro- tal bovine serum with 100 U/ml penicillin, 100 μg/ml xyethyl)-1-piperazineethanesulphonic acid (HEPES), pH streptomycin and 250 ng/ml amphotericin B. To induce 7.5, 1.5 mM MgCl ,10mMKCl,0.5 mM dithiothreitol differentiation, cells were grown to 85-90% confluence, (DTT),50 mMNaF,200 μMNa VO ,1mM PMSF,10 3 4 and the medium was changed to DMEM with 2% horse μg/ml aprotinin, 10 μg/ml leupeptin and 10 μg/ml pep- serum supplemented with penicillin, streptomycin and statin) and left on ice for 10 minutes. Cells were then amphotericin B as described above. FLAG-tagged p38a passedthrougha25-gaugeneedlethree timesand Knight et al. Skeletal Muscle 2012, 2:5 Page 3 of 12 http://www.skeletalmusclejournal.com/content/2/1/5 centrifuged at 500 g to pellet nuclei and unlysed cells. sample was electrophoresed. For two-dimensional elec- The supernatant was collected as whole cytoplasm. For trophoresis, 17-cm ReadyStrip immobilised pH gradient further fractionation of the cytoplasm, the supernatant (IPG) strips (Bio-Rad Laboratories, Hercules, CA, USA) was centrifuged again at 5,000 g to pellet mitochondria were directly rehydrated with labelled lysate diluted in and membrane fractions. The supernatant was then col- rehydration buffer (7 M urea, 2 M thiourea, 4% 3-[(3- lected and centrifuged at 100,000 g to pellet any remain- cholamidopropyl)dimethylammonio]-1-propanesulpho- ing cell particles, and the resulting supernatant was nate (CHAPS) and 1% DTT) following the manufac- collected as cytosol. RIPA buffer was added to all frac- turer’s directions. Isoelectric focusing was performed on tions to a final concentration of 1× for complete lysis. a PROTEAN IEF Cell (Bio-Rad Laboratories) under the Recombinant phosphorylated p38a (Millipore, Billerica, following conditions: 200 V for 1 hour, 500 V for 1 MA, USA) was dephosphorylated using l-protein phos- hour, 5,000 V ramp for 5 hours and 5,000 V for 80,000 phatase (New England Biolabs, Inc, Ipswich, MA, USA) VH. IPG strips were then equilibrated following the by adding 800 U of phosphatase to 200 ng of p38a manufacturer’sinstructionsand overlaid onto a12% diluted in l-phosphatase buffer, and the sample was SDS-PAGE gel. Following electrophoresis, gels were assayed at 30°C for 1 hour. Antibodies used for blotting dried and imaged. For one-dimensional electrophoresis, were as follows: a-actinin (1:125 dilution; Abcam, Cam- 100 μg of lysate was used per reaction. For two-dimen- bridge, MA, USA), COX IV (1:1,000 dilution; Abcam), sional electrophoresis, 300 μg of lysate was used. GRP78 (1:500 dilution; Cell Signaling Technology, Dan- For in vitro substrate identification, assays were per- vers,MA, USA),lamin A/C (1:500dilution;Abcam), formed as described above with the following modifica- MyHC (1:100 dilution; Developmental Studies Hybri- tions. A quantity of 1.5 mg of lysate was treated with doma Bank), MyoD (1:1,000 dilution; Santa Cruz Bio- FSBA, the sample was desalted and 2× kinase assay buf- technology, Santa Cruz, CA, USA), myogenin (1:100 fer was added (40 mM MOPS, pH 7.2, 50 mM b-glycer- dilution; Developmental Studies Hybridoma Bank), ophosphate,10mM EGTA, 2mMNa VO ,2mM 3 4 neural cell adhesion molecule (1:200 dilution; Abcam), DTT, 50 mM MgCl and 2 mM cold ATP). The sample p38a (1:500 dilution; Cell Signaling Technology), phos- was then split into three 500-μgaliquots, and5 μgof pho-p38 (1:500 dilution; Abcam) and b-tubulin (1:1,000 heat-inactivated p38a was added to the control, 5 μgof dilution; Developmental Studies Hybridoma Bank). An active p38a was added to the second aliquot and 5 μg Alpha Innotech HD2 imaging system (R&D Systems, of active p38b was added to the third aliquot. The sam- Minneapolis, MN, USA) was used to quantify phospho- ples were then assayed for 3 hours at 30°C. p38 and tubulin expression. For in vivo substrate identification, assays were per- formed as above with the following modifications. At 48 FSBA treatment and substrate labelling hours of differentiation, cells were treated with 5 μMof Cells were lysed in Nonidet P-40 buffer (50 mM SB 202190 or an equivalent amount of DMSO as vehi- Tris∙HCl, pH 7.8, 150 mM NaCl, 1% (vol/vol) Nonidet cle. Twenty-four hours later the cells were lysed in Non- P-40, 1 mM PMSF, 10 μg/ml aprotinin, 10 μg/ml leu- idet P-40 buffer. Lysate (1 mg) from DMSO-treated cells peptin and 10 μg/ml pepstatin). Lysate was treated at a or 2× 1 mg of lysate from SB 202190-treated cells was concentration of 2 mg/ml with 20 mM 5’-4-fluorosul- inhibited with FSBA, the samples were desalted and 2× phonylbenzoyladenosine (FSBA) solubilised in DMSO kinase assay buffer was added to each. A quantity of 5 and placed at 30°C for 1 hour. The sample was then μgofactivep38a was added to one of the lysate ali- diluted down to 1:5 with Nonidet P-40 buffer minus quots from SB-treated cells. The samples were then protease inhibitors and desalted using Millipore Amicon assayed for 3 hours at 30°C. ultrafiltration columns with a 10 kDa molecular weight cutoff. Following concentration, the sample was diluted Dimethyl labelling to 4 mg/ml with Nonidet P-40 buffer and diluted 1:2 After assaying the samples, they were precipitated by with 2× kinase assay buffer (40 mM 3-morpholinopro- methanol chloroform, then redissolved in 200 μlof 8 M pane-1-sulphonic acid (MOPS), pH 7.2, 50 mM b-gly- urea and 50 mM Tris∙HCl, pH 8.1, with sonication. The cerophosphate, 10 mM ethylene glycol tetraacetic acid samples were then reduced with 20 mM DTT for 1 (EGTA), 2 mM Na VO , 2 mM DTT, 50 mM MgCl , hour at 60°C and alkylated by 100 mM iodoacetamide 3 4 2 400 μMcoldATP and5 μCi [g- P]ATP). Recombinant for 30 minutes at room temperature in the dark. Subse- p38a or p38b (Millipore) was added to a final concen- quently, the samples were diluted to 2 M urea with 50 tration of 0.5% (wt/wt) total protein. Control and mM Tris∙HCl, pH 8.1, and digested with trypsin at a kinase-added samples were assayed at 30°C for 1.5 protein-to-trypsin ratio of 50:1 (wt/wt) for 16 hours at hours. For one-dimensional SDS-PAGE, 5× Laemmli 37°C. Next, the digested samples were acidified to pH 2 buffer was added following the assay to 1×, and the using 10% (vol/vol) formic acid. Dimethyl labelling of Knight et al. Skeletal Muscle 2012, 2:5 Page 4 of 12 http://www.skeletalmusclejournal.com/content/2/1/5 the samples was performed as reported previously [11] and MS/MS spectra were acquired in a data-dependent and is described briefly as follows. The acidified peptides mode, and one full MS scan was followed by ten MS/ were loaded onto C18 solid phase extraction (SPE) col- MS scans. The resolution was set at 60,000 at m/z 400 umns (50 mg of packing material). After brief washing after accumulation to a target value of 500,000. with 50 mM sodium phosphate buffer, pH 7.5, 3 ml of light, intermediate and heavy labelling reagents were Protein identification and quantification loaded onto C18 SPE columns trapped with control, All MS/MS spectra in one acquired raw file were con- p38a-and p38b-labelled samples, respectively. After verted to a single *.mgf file using DTASuperCharge ver- being washed with 0.1% (vol/vol) formic acid, the sion 2.0a7 (Matrix Science, Boston, MA, USA). The *. labelled samples were eluted with 80% acetonitrile mgf file was queried against the International Protein (ACN) (vol/vol) and 0.1% (vol/vol) formic acid, then Index mouse database version 3.52 (EMBL-EBI; http:// dried by vacuum centrifugation. www.ebi.ac.uk/IPI/IPIhelp.html) using Mascot version 2.1 (Matrix Science). To evaluate the false discovery rate Phosphopeptide enrichment (FDR), reversed sequences were appended to the data- Phosphopeptide enrichment by TiO was carried out as base. Cysteine residues were searched as a static modifi- reported previously [12] with modifications. The dried cation of +57.0215 Da; methionine residues were samples were redissolved with 65% ACN/2% trifluoroa- searched with a variable modification of +15.9949 Da; cetic acid (TFA)/saturated glutamic acid and combined. and serine, threonine and tyrosine residues were TiO beads suspended in 65% ACN/2% TFA/saturated searched with a variable modification of +79.9663 Da. glutamic acid were added into the above samples with a Light, intermediate and heavy dimethylation of peptide peptide to TiO bead ratio of 1:4 (wt/wt). After being amino termini and lysine residues were set as variable nutated for 40 minutes, the TiO beads were recovered modifications of +28.0313 Da, +32.0564 Da and by centrifugation and washed thoroughly with 65% +36.0757 Da, respectively. Peptides were queried using ACN/2% TFA. Finally, the enriched phosphopeptides full tryptic cleavage constraints with up to two missed were eluted with 10% (vol/vol) NH ·H Oand dried by cleavage sites. The mass tolerances were 7 ppm for par- 3 2 vacuum centrifugation. ent masses and 0.5 Da for fragment masses. Phospho- peptides with a Mascot score ≥ 30 (rank 1, P ≤ 0.05, Online liquid chromatography tandem mass spectrometry bold red required) were selected and quantified (FDR < analysis 0.01). Phosphorylation site localisation and phosphopep- Online liquid chromatography tandem mass spectrome- tide quantification were performed using a dimethyl- adapted version of MSQuant version 2.0a81. For each try (LC-MS/MS) analysis was performed as reported previously [13,14] with modifications. The dried sample peptide, the putative site of phosphorylation yielding the was redissolved with 0.1% formic acid and loaded onto a highest posttranslational modification (PTM) score was biphasic trap column (200 μmID×10 cm;5-cm accepted (PTM score > 13 required, as described pre- reversed phase column packed with ReproSil-Pur C18 viously [15]). Peptide ratios were obtained by calculating resin (5 μm at 200 Å; Dr.Maisch GmbH, Ammerbuch- the extracted ion chromatograms of the light, medium Entringen, Germany) and a 5-cm monolith strong cation and heavy forms of the peptide using the monoisotopic exchange (SCX) column). The trapped phosphopeptides peaks only, and protein ratios were calculated from the were eluted from the trap column onto a C18 tip col- average of all quantified peptides. All MSQuant outputs umn (75 μmID × 20 cm,3 μmat200 Å; Dr.Maisch of the same online multidimensional separation were GmbH) by a series of salt washes at increasing concen- then imported into StatQuant version 1.2.2, and the trations (0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, quantified phosphopeptides were sorted together and 70, 80, 90, 100, 150, 200 and 1,000 mM). Each fraction exported. was then separated by reversed phase-based gradient elution and detected using an LTQ Orbitrap XL Fourier Substrate validation transform mass spectrometer (Thermo Scientific, Wal- 1 μg recombinant HSP27 (Enzo Life Sciences, Farming- tham, MA, USA). The reversed phase gradient was set dale,NY, USA),recombinantRps27(Abnova,Taipei as follows: 0% to 5% ACN for 2 minutes, 5% to 30% City, Taiwan) or recombinant Smad9 (Abnova) were ACN for 90 minutes and 30% to 80% ACN for 5 min- diluted in 30 μl of 1× kinase assay buffer (20 mM utes. After flushing with 80% ACN for 10 minutes, the MOPS,pH7.2,25mM b-glycerophosphate, 5 mM column was equilibrated with 0.1% formic acid aqueous EGTA, 1 mM Na VO , 1 mM DTT, 25 mM MgCl , 200 3 4 2 solution for 13 minutes. The LTQ Orbitrap XL Fourier μMcoldATP and2.5 μCi [g- P]ATP). A quantity of transform mass spectrometer was operated in positive 500 ng of p38a or p38b was then added, and the sam- ionization mode. A voltage of 1.8 kV was applied. MS ples were assayed at 30°C for 1 hour. Laemmli buffer at Knight et al. Skeletal Muscle 2012, 2:5 Page 5 of 12 http://www.skeletalmusclejournal.com/content/2/1/5 5× concentration was added to 1× to terminate the assays, then the samples were electrophoresed and the gels were dried and imaged. Peptide assays were per- formed similarly with 5 μgofthe followingpeptides synthesised by Biomatik (Cambridge, ON, Canada): HSP27 S180-APLPKAVTQSAEITIPVTF, HSP27 A180- APLPKAVTQAAEITIPVTF, Rps27 S27-KHKKKRLVQ SPNSYFMDVK, Rps27 A27-KHKKKRLVQAPNSYF MDVK, Smad9 T136-NPYHYQRVETPVLPPVLVP and Smad9 A136-NPYHYQRVEAPVLPPVLVP. The fre- quency logo was generated using WebLogo [16]. Results FSBA inhibits endogenous protein kinases A substrate-finding approach that works with cell lysate must overcome the obstacle of endogenous pro- tein kinase activity. Lysate contains tens if not hun- dreds of active kinases, making it difficult to attribute individual phosphorylations that occur during a lysate- based assay to a particular kinase. 5’-4-fluorosulphonyl- benzoyladenosine (FSBA) offers a simple solution. FSBA is an ATP analogue that inhibits protein kinases by occupying the ATP binding site and covalently attaching to an invariant lysine [17-20], the fully func- tionally conserved and so-called catalytic lysine [21]. As FSBA irreversibly occupies the ATP binding site, a bound kinase will permanently lose activity. Treatment of whole-cell C2C12 myoblast lysate with this com- pound can completely eliminate the endogenous kinase signal present (Figure 1A). Kinase-specific substrate labelling Cell lysate treated with FSBA can be desalted to remove any unbound inhibitor and a pool of protein with no inherent kinase activity is generated. A kinase of interest can then be added with a kinase assay buf- fer, and any labelling that subsequently occurs is due to the added kinase as opposed to an endogenous one. To specifically label substrates of p38a,akinase assay buffer containing [g- P]ATP was added to FSBA-trea- ted C2C12 lysate, along with recombinant p38a,and the sample was assayed at 30°C. Substrates labelled by Figure 1 FSBA is a pan-kinase inhibitor that allows for kinase- p38a appeared as bands following one-dimensional gel specific substrate labelling of cell lysate. (A) C2C12 cell lysate was treated with either nothing (lane 1), dimethyl sulphoxide electrophoresis or as spots in two-dimensional gel elec- (DMSO) or 5’-4-fluorosulphonylbenzoyladenosine (FSBA) solubilised trophoresis with no contaminating signal from endo- in DMSO. Subsequently, samples were desalted, kinase assay buffer genous kinases (Figure 1B and Additional file 1 Figure 32 containing P-g-ATP was added and the samples were assayed for S1). Although this type of approach is excellent for 1.5 hours. Treatment with FSBA abolished the labelling of visualising phosphorylation, it is very difficult to iden- endogenous protein kinase substrates. (B) After pretreatment of C2C12 cell lysate with FSBA, purified p38a was added with a kinase tify phosphorylated proteins through spot-picking and assay buffer to specifically label its substrates and visualised using MS. We therefore sought an alternative gel- and radio- one-dimensional SDS-PAGE or two-dimensional gel electrophoresis active-free approach for identifying phosphorylated (Additional file 1 Figure S1). proteins. Knight et al. Skeletal Muscle 2012, 2:5 Page 6 of 12 http://www.skeletalmusclejournal.com/content/2/1/5 dimethyl labels. As a final step before MS, the samples are combined and an enrichment for phosphopeptides is performed using either TiO for serine/threonine phos- phorylation or a phosphotyrosine antibody for tyrosine phosphorylation. Phosphopeptides are then identified by performing LC-MS/MS, and their relative abundance between samples quantified by the differential dimethyl labelling of peptides. Phosphopeptides that are more abundant in the kinase-added sample are from proteins labelled by the added kinase during the in vitro assay, and by this means substrates can be identified. This approach results not only in substrate identification but also in the identification of the site of phosphorylation. Because up to three samples can be compared using dimethyl labelling, the phosphorylation profile of two kinases (plus a control) can be compared in a single experiment. We began our initial in vitro substrate-finding proce- dure by treating 1.5 mg of C2C12 lysate with FSBA. For the assay, the sample was split into three equal parts, with active p38a and b added to the second and third samples, respectively. The rationale behind comparing p38a with p38b was to identify specific p38a phosphor- ylations that might explain why p38b cannot compen- sate for the loss of p38a in differentiating myoblasts. In total, 387 unique serine/threonine phosphopeptides were identified (Additional file 1 Table S1). A histogram of their relative abundance ratios (p38a/control) is shown in Additional file 1 Figure S2. A threefold increase in the abundance ratio was selected as a cutoff for high-confidence in vitro substrates as this ratio is beyond the range of inherent variability, and 158 phos- phorylation sites from 94 different proteins showed at least a threefold increase in the p38a-assayed sample relative to the control. The list of p38a phosphorylation Figure 2 Methodology used to identify in vitro kinase substrates using FSBA and quantitative mass spectrometry. The sites is presented in Additional file 1 Table S2. We iden- differential tagging of peptides with dimethyl labels allows for the tified five previously known substrates (caldesmon, his- relative quantification and identification of phosphopeptides tone H2B, Psmd1, SAKS1 and Smad3) and eighty-nine simultaneously in a high-mass accuracy mass spectrometer. An that were previously unknown. Three of these previously example/theoretical substrate identification for the added kinase (green) is shown and is based on the higher intensity of the unknown targets were validated to determine if the phosphopeptide in MS. technique was discovering true in vitro p38a substrates. Recombinant forms of these three proteins could be phosphorylated in vitro by p38a (Figure 3A), and a vali- dation of peptides confirmed the site of phosphorylation Quantitative MS coupled with a phosphopeptide (Figure 3B). After aligning substrates on their phosphor- enrichment to identify substrates ylation sites, we found that a consensus phosphorylation The approach we devised to identify substrates is out- motif was present in many substrates, although it was lined in Figure 2. Cell lysate is treated with FSBA and not an absolute requirement (Figure 3C). The motif desalted as described in the previous section. This is fol- contains a proline immediately downstream from the lowed by the addition of a nonradioactive kinase assay target serine or threonine and an aliphatic residue two buffer, and the sample is split in two, to one of which is residues upstream. This motif is in agreement with that added active kinase (kinase-added). After assaying at 30° previously described for p38 [22], providing further sup- C, the two samples are digested, and peptides from each port for our substrate-finding approach. sample are differentially tagged using isotopomeric Knight et al. Skeletal Muscle 2012, 2:5 Page 7 of 12 http://www.skeletalmusclejournal.com/content/2/1/5 Figure 3 Validation of newly discovered p38a substrates. (A) Validation of three newly identified targets as in vitro p38a substrates was performed using full-length substrate incubated with a kinase assay buffer and p38a. (B) Wild-type peptides containing the phosphorylation site or peptides harbouring an S/T-to-A mutation of the phosphorylation site were incubated with p38a and a kinase assay buffer to confirm the location of the phosphorylation site. (C) The 158 phosphorylation sites identified were aligned, and a frequency logo was generated. p38a has a consensus phosphorylation motif of aXS/TP, where “a” is an aliphatic residue and “X” is any amino acid. Substrate specificity does not distinguish p38a and p38b We first assayed p38a and p38b on cell lysate using the radioactive approach and were surprised to see no obvious differences in the substrate banding patterns Figure 4 Localisation, not substrate specificity, distinguishes produced by the two isoforms (Figure 4A). Consistent p38a from p38b. (A) FSBA-treated C2C12 cell lysate was incubated with this observation, of the 158 p38a phosphorylations with a kinase assay buffer and p38a, p38b or no kinase as a control. identified using the quantitative approach, none Both isoforms produced similar banding patterns. The prominent appeared to be specific to this isoform. The 158 quanti- bands marked with the asterisks in each of the p38a and p38b lanes represent autophosphorylation of the added recombinant tative values for the p38a phosphorylations are plotted kinase. (B) The quantitative values for all 158 identified p38a from highest to lowest in Figure 4B (blue line for p38a). phosphorylations are plotted in blue on a log scale, with the The red line represents the corresponding p38b values. corresponding values for p38b shown in red. The dashed line Although p38a and p38b showed apparent differences represents the fold cutoff for accepted substrates. Four p38b in preference, they had very similar overall profiles. phosphorylations fell appreciably below the fold cutoff (point 2 corresponds to serine 27 of the 40S ribosomal protein S27-Rps27). There were only four phosphorylations that fell above (C) Incubation of p38a or p38b with purified Rps27 and a kinase the threefold cutoff for p38a,whereas for p38b they assay buffer. (D) Incubation of p38a or p38b with a peptide from were well below. However, the values were still positive Rps27 containing serine 27 (or mutation of serine 27 to alanine) and for p38b, suggesting that these could be real phosphory- a kinase assay buffer. (C) and (D) demonstrate that serine 27 of lations but there is simply less confidence in them. To Rps27 is not a specific p38a phosphorylation site. (E) FLAG-tagged p38a or p38b were transfected into C2C12 cells, and at 48 hours of determine if these might represent specific phosphoryla- differentiation immunofluorescence staining for FLAG was tions, we performed in vitro assays with either p38a or performed. p38a has a ubiquitous distribution, whereas p38b is p38b using purified substrate for one candidate. The found solely at the cell periphery. Cells were imaged using a Zeiss purified substrate was the 40S ribosomal protein S27 LSM 510 META confocal microscope (Carl Zeiss MicroImaging (Rps27), and both p38 isoforms were able to phosphory- GmbH, Jena, Germany). Scale bar = 20 μm. late this protein at the same site (Figure 4C and 4D). Knight et al. Skeletal Muscle 2012, 2:5 Page 8 of 12 http://www.skeletalmusclejournal.com/content/2/1/5 Therefore, none of the 158 phosphorylations found for p38a appear to be specific to this isoform. If substrate specificity does not distinguish the two p38 isoforms, and yet p38b is unable to compensate for the loss of p38a, there must be an alternative character- istic that discriminates them. An obvious possibility is cellular localisation. If p38a and p38b localise differently within the cell, then p38b wouldsimplybe unableto fulfil p38a’s role, even though it might have the catalytic potential to do so. To study this, we overexpressed FLAG-tagged p38a and p38b in C2C12 cells and assessed their localisation during differentiation (Figure 4E). Whereas p38a has a ubiquitous localisation pattern, p38b is found only at the periphery of the cell. p38a therefore has access to a substrate pool that p38b does not, highlighting a major reason why p38b cannot com- pensate for the loss of p38a. Identification of novel in vivo p38a substrates Although the approach we have outlined can identify in vitro kinase substrates, such in vitro approaches will also turn up irrelevant substrates that are phosphorylated only because of the absence of the appropriate cellular context. For example, we identified just over 400 phos- phorylation sites in the screen described above, 158 of which are in vitro p38 targets. If these numbers are representative, it suggests that p38 could be responsible for approximately 40% of all cellular phosphorylations in differentiating myoblasts, a number that seems unlikely, Figure 5 Methodology used to identify in vivo kinase given that there are about 500 mouse and human substrates using FSBA and quantitative MS. In vivo substrates are kinases [23]. identified based on the higher phosphopeptide intensity in MS for To overcome the drawbacks associated with in vitro both the vehicle-treated (blue) and kinase-added (green) samples. A substrate identification, we modified the approach to higher phosphopeptide abundance in MS for the vehicle-treated (blue) sample relative to the inhibitor-treated (red) sample indicates allow for the identification of relevant, direct in vivo a phosphorylation site regulated by the kinase of interest in vivo.A substrates (outlined in Figure 5). For our purposes, dif- higher phosphopeptide abundance for the kinase-added (green) ferentiating C2C12 cells were treated either with the sample relative to the inhibitor-treated (red) sample indicates a p38 inhibitor SB 202190 or with DMSO as vehicle prior phosphorylation site directly targeted by the kinase in vitro. to lysis. SB inhibitor or vehicle treatment began at 48 hours of differentiation, and the cells were lysed 24 hours later. The 48-hour time point was chosen because aliquots were taken from the lysate of inhibitor-treated of our interest in identifying novel functions for p38a cells. All three aliquots were treated with FSBA and during the middle stages of myoblast differentiation. desalted to remove unbound FSBA and SB inhibitor, Myogenic gene activation occurs within the first 48 and a kinase assay buffer was added to each. Active hours of differentiation [24] and is followed by cell p38a was added to one of the inhibitor-treated aliquots, fusion, sarcomere formation and other processes. If dif- and the samples were assayed at 30°C. Following the ferentiating C2C12 myoblasts are treated with a p38 assay, the samples were digested, dimethyl-labelled and inhibitor at 48 hours, there is a reduction in cell fusion phosphopeptides enriched as described above. What we and overall differentiation (Additional file 1 Figure S3), sought to identify were phosphorylation sites that indicating that there is a requirement for long-term p38 decreased on inhibitor treatment and could be elevated activity for efficient myogenesis beyond the initial stage back up by direct in vitro p38a labelling of SB inhibitor- of myogenic gene activation, possibly both for maintain- treated lysate. These would be direct phosphorylation ing such gene activation and for fusion-related pro- sites regulated by p38a in vivo. cesses. Following cell lysis, a 1-mg aliquot was taken By using this approach, we identified a total of 717 from the lysate of vehicle-treated cells and two 1-mg phosphorylation sites (Additional file 1 Table S3), 73 of Knight et al. Skeletal Muscle 2012, 2:5 Page 9 of 12 http://www.skeletalmusclejournal.com/content/2/1/5 Table 1 Newly discovered in vivo p38a substrates Genes Protein names Sites Ahnak Neuroblast differentiation-associated protein AHNAK S4906 Iws1 Protein IWS1 homolog S183, S185 Grp78 78 kDa glucose-regulated protein T649 Pgrmc Membrane-associated progesterone receptor S181 component 1 Prdx6 Peroxiredoxin 6 T93 Ranbp2 E3 SUMO protein ligase RanBP2 S2505 SUMO, small ubiquitin-like modifier. which showed a threefold decrease following SB inhibi- tor treatment, and seven of these sites were direct p38a phosphorylation sites (that is, showed at least a threefold increase in the p38a-labelled sample relative to the lysate from inhibitor-treated cells). In contrast to the pure in vitro approach, which suggested that approxi- mately 40% of all cellular phosphorylations might be p38 target sites, the numbers derived from the in vivo study suggest that the actual level is closer to about 1%. The seven in vivo sites (on six substrates) that we iden- tified are listed in Table 1. The substrate Iws1 is a largely nuclear protein involved in several processes, such as chromatin remo- Figure 6 Cytoplasmic characterization of p38a during C2C12 delling and mRNA export [25,26], functions with which cell differentiation. (A) C2C12 cells were fractionated over a p38 has previously been associated. Ranbp2 localises differentiation time course. C, control whole-cell lysate; P, predominantly to the nuclear pore complex and is criti- proliferating lysate. Western blotting for phospho-p38 shows a clear cal for myotube formation [27]. The other four sub- increase in the cytoplasm as differentiation proceeds. Nuclei were removed in the initial step of fractionation, and lamin A/C was used strates are cytoplasmic proteins without known or to demonstrate that there were no contaminating nuclei in suggested nuclear functions (Ahnak, Grp78, Pgrmc and cytoplasmic extracts. Quantification of phospho-p38 expression is Prdx6). On the basis of fractionations and Western blot shown in Additional file 1 Figure S4. (B) After 48 hours of analysis, we found that p38a was indeed present in the differentiation, C2C12 cells were lysed and the cytoplasmic fraction cytoplasm during differentiation (Figure 6A), which is in was collected and further fractionated into cytosolic (including the cytoskeleton) and noncytosolic fractions. Nuclear marker: lamin A/C; agreement with the immunofluorescence staining of cytoskeletal marker: a-actinin; mitochondrial marker: COX IV; FLAG-tagged p38a. The levels of phosphorylated p38, membrane marker: neural cell adhesion molecule (NCAM); the active form, increased in the cytoplasm with differ- endoplasmic reticulum marker: GRP78. (C) Interconnections entiation (Figure 6A and Additional file 1 Figure S4), between the p38a activation pathway and newly discovered in vivo substrates. Proteins previously associated with p38a in its activation suggesting that p38a might play an important role pathway are indicated as blue nodes. New substrates are indicated there. Validation of the phospho-p38 antibody is shown as red nodes. Edge colouring indicates the type of association in Additional file 1 Figure S5. Further fractionation of between nodes. the cytoplasm revealed that p38a and phospho-p38 were present only in the cytosolic fraction (Figure 6B), meaning that all four of the in vivo cytoplasmic phos- 6C), providing additional support that these targets are phorylation sites we identified would be accessible to relevant and that their phosphorylation could be critical p38a. Using Scansite [28], we found that five of the six for pathway function. substrates contained one or more predicted D domains, MAPK docking domains found on many in vivo sub- Discussion strates (D domain sites and scores are listed in Addi- We have described here a simple technique for kinase tional file 1 Table S4). We next used Genemania [29] to substrate finding that uses whole-cell lysate, can identify identify previously known associations between these sites of phosphorylation in addition to the protein phos- substrates and the p38 activation pathway we recently phorylated, has the ability to identify both in vivo and in reviewed and outlined [30]. Four of the substrates have vitro substrates, and can be used to compare the in vitro previously known associations with this pathway (Figure substrate specificity of two kinases in the same Knight et al. Skeletal Muscle 2012, 2:5 Page 10 of 12 http://www.skeletalmusclejournal.com/content/2/1/5 experiment. Performing the assay with p38a and p38b the in vivo approach on unfractionated lysate, when revealed that, although differences in preference are combined with fractionation prior to LC-MS/MS, the apparent, there are no obvious phosphorylation sites number of substrates identified could be much greater specific to p38a. However, a major characteristic that than what we have demonstrated in principle here. distinguishes p38a from p38b during myoblast differen- As mentioned, a major advantage of the technique tiation is localisation, with p38a located ubiquitously presented here is that it can be used to compare the substrate profiles of two or more kinases, and we used throughout the cell while p38b is found only at the cell this to study the substrate specificity of the p38a and b periphery. We also have shown that p38a can phos- isoforms. However, we were unable to identify any phos- phorylate several cytosolic phosphorylation sites in vivo and that this may be a critical but previously unknown phorylations that were specific to p38a.There are function for it. These results demonstrate the utility of apparent differences in preference for substrates the substrate screening technique we have developed between p38a and p38b, which could be functionally and how its use not only can find proteins phosphory- relevant. Assuming that p38b had the same ubiquitous lated by a kinase of interest but also can answer particu- localisation pattern as p38a, a low preference for certain lar questions and propose novel avenues of research. critical myogenic phosphorylations could result in a dif- The technique we have developed has several advan- ference in cell behaviour if p38b were theonlyisoform tages over existing substrate finding approaches. It can present. However, it seems likely that differentiation provide direct substrate identifications and information would still occur, but possibly at a reduced rate or in a on in vivo relevance in a single experiment. Whole-cell compromised way. This is not the case, as p38b-con- lysate can be used as a source of candidate substrates, taining myoblasts that lack p38a fail to differentiate at allowing screens to be performed on samples of specific all [7-9,34]. Rather, we believe what truly distinguishes relevance to the area of interest instead of on protein or the two isoforms is their localisation. p38a has a known peptide arrays. Our approach for eliminating endogen- critical role in the nucleus, and our results suggest that ous kinase activity in lysate is nondenaturing, decreasing the same may be true for the cytoplasm; therefore, p38b the likelihood of false-positive and false-negative sub- would be unable to compensate because it is found strate phosphorylations that would result from the heat- solely at the periphery of the cell. A similar distinction inactivating approach that others have employed [31,32]. exists for two other closely related kinases, focal adhe- At the same time, the recombinant kinase used in the sion kinase (FAK) and proline-rich tyrosine kinase 2 (PYK2) [35]. In fibroblasts, FAK localises to focal adhe- assay does not require mutation or unnatural ATP ana- sions, whereas PYK2 has a perinuclear distribution. logues as other approaches do [33], making the techni- que applicable to any kinase that can be made and PYK2, which has a kinase domain 60% identical to that purified in an active form. Although we used dimethyl of FAK, can compensate for the loss of FAK provided it labelling for quantitative MS, the technique is fully com- has a focal adhesion targeting sequence. Together these patible with iTRAQ, which allows up to eight samples to results suggest that rather than substrate specificity, it is be compared in a single experiment, meaning that other characteristics, in these cases localisation, that dis- simultaneous in vivo substrate identification and kinase tinguish closely related kinases. Whether the same holds comparisons can be performed. Radioactivity and gel true for the entire kinase family is an intriguing work are not required, but can be used to visualise question. phosphorylations and make qualitative comparisons In addition to p38a’s known role in regulating gene between kinases on one-dimensional gels. The lysate expression at the onset of myoblast differentiation, our requirements are also relatively low; 1.5 to 3 mg were results suggest that it is likely to have a cytosolic role as used in these assays, which may make the technique dif- well, possibly in regulating its own pathway. We have ficult to employ with some primary cells lines, but it is found that p38a is present in the cytosol, active p38 easily applicable to most secondary cell lines and to tis- increases in the cytoplasm with differentiation, p38a can sues as well. We have treated several different lysate phosphorylate several cytosolic proteins, three of the types with FSBA, and complete inhibition of endogen- cytosolic proteins contain predicted D domains, and ous kinase activity occurred in all, meaning that our connections between these proteins and the p38a acti- approach is likely universally applicable. In this study vation pathway have been previously reported. Together our approach for identifying in vivo substrates utilised these results suggest a previously unrecognised role for an inhibitor, but it is more versatile than that, and can p38a in the cytosol. be applied to study phosphorylations triggered by a spe- cific stimulus (using stimulated versus unstimulated Conclusions cells for lysate pools) without requiring a specific inhibi- While the role of the p38a MAPK during myoblast dif- tor be available. Finally, although we have performed ferentiation has been studied extensively with regard to Knight et al. Skeletal Muscle 2012, 2:5 Page 11 of 12 http://www.skeletalmusclejournal.com/content/2/1/5 expertise. RK supervised JDRK. All authors read and approved the final gene regulation, our evidence suggests that this kinase is manuscript. likely involved in other processes as well. The differen- tiation of myoblasts into a mature myotube involves Competing interests The authors declare that they have no competing interests. extensive morphological and functional changes that p38a might regulate, both through the initiation of a Received: 22 August 2011 Accepted: 6 March 2012 myogenic gene program and through direct phosphory- Published: 6 March 2012 lation and functional modification of cytosolic proteins. References No one has previously been able to identify a p38a-spe- 1. Berwick DC, Tavaré JM: Identifying protein kinase substrates: hunting for cific substrate, and our large-scale screen suggests these the organ-grinder’s monkeys. Trends Biochem Sci 2004, 29:227-232. may exist only within the context of cellular localisation. 2. Johnson SA, Hunter T: Kinomics: methods for deciphering the kinome. Nat Methods 2005, 2:17-25. In addition, the substrate screening technique we have 3. Manning BD, Cantley LC: Hitting the target: emerging technologies in the developed should serve as a useful tool for studying the search for kinase substrates. Sci STKE 2002, 2002:pe49. other kinases known to be involved in myogenesis, as 4. Sopko R, Andrews BJ: Linking the kinome and phosphorylome: a comprehensive review of approaches to find kinase targets. Mol Biosyst well as the hundreds of other protein kinases that exist. 2008, 4:920-933. 5. Holt LJ, Tuch BB, Villén J, Johnson AD, Gygi SP, Morgan DO: Global analysis Additional material of Cdk1 substrate phosphorylation sites provides insights into evolution. Science 2009, 325:1682-1686. 6. Lluís F, Perdiguero E, Nebreda AR, Muñoz-Cánoves P: Regulation of skeletal Additional file 1: Figures S1 through S5 and Tables S1 through S4. muscle gene expression by p38 MAP kinases. Trends Cell Biol 2006, 16:36-44. 7. Li Y, Jiang B, Ensign WY, Vogt PK, Han J: Myogenic differentiation requires signalling through both phosphatidylinositol 3-kinase and p38 MAP Acknowledgements kinase. Cell Signal 2000, 12:751-757. JDRK was supported by a Vanier Canada Graduate Scholarship, a Canadian 8. Perdiguero E, Ruiz-Bonilla V, Gresh L, Hui L, Ballestar E, Sousa-Victor P, Institutes of Health Research (CIHR) Canada Graduate Scholarship and a Baeza-Raja B, Jardí M, Bosch-Comas A, Esteller M, Caelles C, Serrano AL, Multiple Sclerosis Society of Canada (MSSC) Studentship. LAM holds the Wagner EF, Muñoz-Cánoves P: Genetic analysis of p38 MAP kinases in Mach Gaensslen Chair in Cardiac Research and was funded by CIHR. ACG is myogenesis: fundamental role of p38α in abrogating myoblast a Canada Research Chair in Functional Proteomics and the Lea Reichmann proliferation. EMBO J 2007, 26:1245-1256. Chair and is funded by CIHR grant MOP-84314. DF acknowledges a Canada 9. Wang H, Xu Q, Xiao F, Jiang Y, Wu Z: Involvement of the p38 mitogen- Research Chair in Proteomics and Systems Biology as well as grants from activated protein kinase α, β, and γ isoforms in myogenic differentiation. the Natural Sciences and Engineering Research Council of Canada and the J- Mol Biol Cell 2008, 19:1519-1528. Louis Lévesque Foundation. RK holds a University Health Research Chair at 10. Enslen H, Raingeaud J, Davis RJ: Selective activation of p38 mitogen- the University of Ottawa and was supported by grants from CIHR and the activated protein (MAP) kinase isoforms by the MAP kinase kinases MSSC. MKK3 and MKK6. J Biol Chem 1998, 273:1741-1748. 11. Boersema PJ, Raijmakers R, Lemeer S, Mohammed S, Heck AJ: Multiplex Author details peptide stable isotope dimethyl labeling for quantitative proteomics. Regenerative Medicine Program, Ottawa Hospital Research Institute, 501 Nat Protoc 2009, 4:484-494. Smyth Road, Ottawa, ON, K1H 8L6, Canada. Department of Cellular and 12. Li QR, Ning ZB, Tang JS, Nie S, Zeng R: Effect of peptide-to-TiO beads Molecular Medicine, University of Ottawa, 451 Smyth Road, Ottawa, ON, K1H ratio on phosphopeptide enrichment selectivity. J Proteome Res 2009, 8M5, Canada. Ottawa Institute of Systems Biology, University of Ottawa, 451 8:5375-5381. Smyth Road, Ottawa, ON, K1H 8M5, Canada. Department of Biochemistry, 13. Wang F, Dong J, Jiang X, Ye M, Zou H: Capillary trap column with strong Microbiology and Immunology, University of Ottawa, 451 Smyth Road, cation-exchange monolith for automated shotgun proteome analysis. Ottawa, ON, K1H 8M5, Canada. CAS Key Lab of Separation Sciences for Anal Chem 2007, 79:6599-6606. Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy 14. Wang F, Chen R, Zhu J, Sun D, Song C, Wu Y, Ye M, Wang L, Zou H: A fully of Sciences, Dalian, 116023, China. Department of Medicine, University of automated system with online sample loading, isotope dimethyl Ottawa, 451 Smyth Road, Ottawa, ON, K1H 8M5, Canada. Samuel Lunenfeld labeling and multidimensional separation for high-throughput Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, ON, quantitative proteome analysis. Anal Chem 2010, 82:3007-3015. M5G 1X5, Canada. Department of Molecular Genetics, University of Toronto, 15. Olsen JV, Blagoev B, Gnad F, Macek B, Kumar C, Mortensen P, Mann M: 1 King’s College Circle, Toronto, ON, M5S 1A8, Canada. Current address: Global, in vivo, and site-specific phosphorylation dynamics in signaling Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University networks. Cell 2006, 127:635-648. Avenue, Toronto, ON, M5G 1X5, Canada. Current address: Department of 16. Crooks GE, Hon G, Chandonia JM, Brenner SE: WebLogo: a sequence logo Cancer Biology, Dana-Farber Cancer Institute, 450 Brookline Avenue, and generator. Genome Res 2004, 14:1188-1190. Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, 17. Hashimoto E, Takio K, Krebs EG: Amino acid sequence at the ATP-binding Boston, MA 02115, USA. site of cGMP-dependent protein kinase. J Biol Chem 1982, 257:727-733. 18. Kamps MP, Taylor SS, Sefton BM: Direct evidence that oncogenic tyrosine Authors’ contributions kinases and cyclic AMP-dependent protein kinase have homologous JDRK and RK conceived of and designed the project. JDRK conceived of the ATP-binding sites. Nature 1984, 310:589-592. substrate-finding technique. RT assisted JDRK with the implementation of 19. Russo MW, Lukas TJ, Cohen S, Staros JV: Identification of residues in the the substrate-finding technique. RECL performed two-dimensional gel work nucleotide binding site of the epidermal growth factor receptor/kinase. and assisted JDRK with the development of the radioactive approach. FW J Biol Chem 1985, 260:5205-5208. assisted with quantitative MS analysis and phosphopeptide validation. AB 20. Zoller MJ, Nelson NC, Taylor SS: Affinity labeling of cAMP-dependent assisted with cell cultures. All other experiments and analysis were protein kinase with p-fluorosulfonylbenzoyl adenosine: covalent performed by JDRK. JDRK wrote the paper, and RK revised and edited it. HZ modification of lysine 71. J Biol Chem 1981, 256:10837-10842. supervised FW. LAM supervised RECL and gave advice on project design. DF 21. Knight JD, Qian B, Baker D, Kothary R: Conservation, variability and the supervised RT and FW, and DF, TP and ACG provided MS facilities and modeling of active protein kinases. PLoS One 2007, 2:e982. Knight et al. Skeletal Muscle 2012, 2:5 Page 12 of 12 http://www.skeletalmusclejournal.com/content/2/1/5 22. Schwartz D, Gygi SP: An iterative statistical approach to the identification of protein phosphorylation motifs from large-scale data sets. Nat Biotechnol 2005, 23:1391-1398. 23. Manning G, Whyte DB, Martinez R, Hunter T, Sudarsanam S: The protein kinase complement of the human genome. Science 2002, 298:1912-1934. 24. Andrés V, Walsh K: Myogenin expression, cell cycle withdrawal, and phenotypic differentiation are temporally separable events that precede cell fusion upon myogenesis. J Cell Biol 1996, 132:657-666. 25. Diebold ML, Koch M, Loeliger E, Cura V, Winston F, Cavarelli J, Romier C: The structure of an Iws1/Spt6 complex reveals an interaction domain conserved in TFIIS, Elongin A and Med26. EMBO J 2010, 29:3979-3991. 26. Yoh SM, Cho H, Pickle L, Evans RM, Jones KA: The Spt6 SH2 domain binds Ser2-P RNAPII to direct Iws1-dependent mRNA splicing and export. Genes Dev 2007, 21:160-174. 27. Asally M, Yasuda Y, Oka M, Otsuka S, Yoshimura SH, Takeyasu K, Yoneda Y: Nup358, a nucleoporin, functions as a key determinant of the nuclear pore complex structure remodeling during skeletal myogenesis. FEBS J 2011, 278:610-621. 28. Obenauer JC, Cantley LC, Yaffe MB: Scansite 2.0: Proteome-wide prediction of cell signaling interactions using short sequence motifs. Nucleic Acids Res 2003, 31:3635-3641. 29. Warde-Farley D, Donaldson SL, Comes O, Zuberi K, Badrawi R, Chao P, Franz M, Grouios C, Kazi F, Lopes CT, Maitland A, Mostafavi S, Montojo J, Shao Q, Wright G, Bader GD, Morris Q: The GeneMANIA prediction server: biological network integration for gene prioritization and predicting gene function. Nucleic Acids Res 2010, , 38 Web server: W214-W220. 30. Knight JD, Kothary R: The myogenic kinome: protein kinases critical to mammalian skeletal myogenesis. Skelet Muscle 2011, 1:29. 31. Huang SY, Tsai ML, Chen GY, Wu CJ, Chen SH: A systematic MS-based approach for identifying in vitro substrates of PKA and PKG in rat uteri. J Proteome Res 2007, 6:2674-2684. 32. Troiani S, Uggeri M, Moll J, Isacchi A, Kalisz HM, Rusconi L, Valsasina B: Searching for biomarkers of Aurora-A kinase activity: identification of in vitro substrates through a modified KESTREL approach. J Proteome Res 2005, 4:1296-1303. 33. Blethrow JD, Glavy JS, Morgan DO, Shokat KM: Covalent capture of kinase- specific phosphopeptides reveals Cdk1-cyclin B substrates. Proc Natl Acad Sci USA 2008, 105:1442-1447. 34. Ruiz-Bonilla V, Perdiguero E, Gresh L, Serrano AL, Zamora M, Sousa-Victor P, Jardí M, Wagner EF, Muñoz-Cánoves P: Efficient adult skeletal muscle regeneration in mice deficient in p38β, p38γ and p38δ MAP kinases. Cell Cycle 2008, 7:2208-2214. 35. Klingbeil CK, Hauck CR, Hsia DA, Jones KC, Reider SR, Schlaepfer DD: Targeting Pyk2 to β1-integrin-containing focal contacts rescues fibronectin-stimulated signaling and haptotactic motility defects of focal adhesion kinase-null cells. J Cell Biol 2001, 152:97-110. doi:10.1186/2044-5040-2-5 Cite this article as: Knight et al.: A novel whole-cell lysate kinase assay identifies substrates of the p38 MAPK in differentiating myoblasts. Skeletal Muscle 2012 2:5. 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A novel whole-cell lysate kinase assay identifies substrates of the p38 MAPK in differentiating myoblasts

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Copyright © 2012 by Knight et al; licensee BioMed Central Ltd.
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Life Sciences; Cell Biology; Developmental Biology; Biochemistry, general; Systems Biology; Biotechnology
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Abstract

Background: The p38a mitogen-activated protein kinase (MAPK) is a critical mediator of myoblast differentiation, and does so in part through the phosphorylation and regulation of several transcription factors and chromatin remodelling proteins. However, whether p38a is involved in processes other than gene regulation during myogenesis is currently unknown, and why other p38 isoforms cannot compensate for its loss is unclear. Methods: To further characterise the involvement of p38a during myoblast differentiation, we developed and applied a simple technique for identifying relevant in vivo kinase substrates and their phosphorylation sites. In addition to identifying substrates for one kinase, the technique can be used in vitro to compare multiple kinases in the same experiment, and we made use of this to study the substrate specificities of the p38a and b isoforms. Results: Applying the technique to p38a resulted in the identification of seven in vivo phosphorylation sites on six proteins, four of which are cytoplasmic, in lysate derived from differentiating myoblasts. An in vitro comparison with p38b revealed that substrate specificity does not discriminate these two isoforms, but rather that their distinguishing characteristic appears to be cellular localisation. Conclusion: Our results suggest p38a has a novel cytoplasmic role during myogenesis and that its unique cellular localisation may be why p38b and other isoforms cannot compensate for its absence. The substrate-finding approach presented here also provides a necessary tool for studying the hundreds of protein kinases that exist and for uncovering the deeper mechanisms of phosphorylation-dependent cell signalling. Keywords: differentiation, FSBA, kinase assay, mitogen-activated protein kinase, myoblast, p38, phosphorylation, quantitative MS Background substrate-finding techniques exist, they are hindered by Protein kinases are well-known regulators of cell signal- problems that prevent them from being easily or readily ling and cellular behaviour that execute their function employed [1-4] and are generally limited to providing in through the covalent attachment of an ATP-derived vitro substrate identifications that may or may not be phosphate to protein substrates. To understand the relevant in vivo. In vivo approaches currently available, function of any protein kinase on a large and cell-wide such as that employed by Holt et al. [5], can associate a scale first requires the development of a substrate kinase with in vivo phosphorylation events, but direct screening technique that allows for the proteins phos- phosphorylation cannot be inferred without additional phorylated by a kinase of interest to be comprehensively experimentation. A simple technique that can identify identified, ideally in a single experiment. Although direct in vivo substrates is an obvious need for the field. The mitogen-activated protein kinase p38a is involved in several cellular processes, but its critical role during * Correspondence: rkothary@ohri.ca differentiation, and particularly the differentiation of Regenerative Medicine Program, Ottawa Hospital Research Institute, 501 Smyth Road, Ottawa, ON, K1H 8L6, Canada myoblasts, has been a major focus. At the initiation of Full list of author information is available at the end of the article © 2012 Knight et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Knight et al. Skeletal Muscle 2012, 2:5 Page 2 of 12 http://www.skeletalmusclejournal.com/content/2/1/5 myoblast differentiation, p38a is known to phosphory- and p38b were acquired from Addgene (Cambridge, late several transcription factors and chromatin remo- MA, USA) [10]. Constructs were transfected into C2C12 delling proteins, thereby inducing the expression of a myoblasts with Lipofectamine 2000 reagent (Invitrogen, myogenic gene program [6]. Although much is known Carlsbad, CA, USA) according to the manufacturer’s about p38a’s role in this process, it is likely very partial, instructions. To inhibit p38 activity, SB 202190 (Pro- and whether p38a plays an important role in other pro- mega, Madison, WI, USA) solubilised in dimethyl sulph- cesses during myoblast differentiation, such as cell oxide (DMSO) or DMSO as control was added to fusion or sarcomere formation, is unknown. At the differentiation media at 48 hours following the induction same time, there are questions regarding the other p38 of differentiation, and medium with inhibitor was chan- isoforms and their role, or lack thereof, in myogenesis. ged daily. p38b is also expressed in myoblasts and is activated in the same manner as p38a, but despite having a kinase Immunofluorescence domain 75% identical to that of p38a (72% sequence Cells were fixed with 4% formaldehyde and stained with identity overall), p38b is unable to compensate for the the following antibodies: Flag M2 (1:1,000 dilution; loss of p38a, even when overexpressed [7-9]. The Sigma-Aldrich, St Louis, MO, USA), myosin heavy chain obvious and suspected explanation is that there are criti- (MyHC) (1:20 dilution; Developmental Studies Hybri- cal myogenic phosphorylations specific to the a isoform, doma Bank, Department of Biology, University of Iowa, but these have yet to be discovered and whether this Iowa City, IA, USA), Alexa Fluor 488 goat anti-mouse assumption is correct is unknown. antibody (1:1,000 dilution; Molecular Probes/Invitrogen) Here we describe a simple approach for substrate find- and Alexa Fluor 555 goat anti-mouse antibody (1:1,000 ing that can be used to identify in vitro and in vivo sub- dilution; Molecular Probes/Invitrogen). The differentia- strates. The technique begins with treatment of cell tion index was calculated as the number of MyHC-posi- lysate to inactivate endogenous kinases, followed by an tive nuclei divided by the total number of nuclei. The in vitro assay using an exogenous kinase of interest, and fusion index was quantified as the number of nuclei per concludes with quantitative mass spectrometry (MS) to MyHC-positive cell. Five fields of view at ×20 magnifica- identify phosphorylation sites specific to the added tion were counted and averaged per replicate, with a kinase. By using lysate derived from vehicle- or inhibi- total of three replicate experiments. tor-treated cells, this in vitro approach can be simulta- neously coupled with biologically relevant information Statistical analysis to identify direct substrates regulated by the kinase of Statistical analyses were performed using StatPlus soft- interest in vivo. Applying this technique to p38a with ware (AnalystSoft Inc; http://www.analystsoft.com/en/ lysate from differentiating myoblasts resulted in the products/statplus/). The data shown are means with SD, identification of several new in vivo substrates that sug- and Student’s t-tests were performed to determine sig- gest novel functions for p38a during myogenesis. We nificance for the differentiation and fusion indices. did not identify a single phosphorylation specific to the p38a isoform compared with p38b,atleast in termsof Western blot analysis in vitro substrate specificity, but we did see a clear dif- For Western blot analysis, cells were lysed in radioim- ference in cellular localisation during myoblast differen- munoprecipitation assay (RIPA) buffer (50 mM tiation. This leads us to propose that although the Tris∙HCl, pH 7.5, 1% Nonidet P-40, 0.1% SDS, 150 mM kinase domains of p38a and b likely have the same NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 50 capacity to phosphorylate substrates, there are major mM NaF, 200 μMNa VO , 1 mM phenylmethanesul- 3 4 differences in actual substrate specificity in an in vivo phonylfluoride (PMSF), 10 μg/ml aprotinin, 10 μg/ml context. leupeptin and 10 μg/ml pepstatin), and 5× Laemmli buf- fer (300 mM Tris, pH 6.8, 0.01% bromophenol blue, Methods 10% SDS, 50% glycerol and 5% b-mercaptoethanol) was Cell culture added to 25 μg of protein per sample to a final concen- C2C12 cells were grown in Dulbecco’s modified Eagle’s tration of 1× for SDS-PAGE. For fractionation, cells medium (DMEM) supplemented with 10% (vol/vol) foe- were lysed in hypotonic buffer (10 mM 4-(2-hydro- tal bovine serum with 100 U/ml penicillin, 100 μg/ml xyethyl)-1-piperazineethanesulphonic acid (HEPES), pH streptomycin and 250 ng/ml amphotericin B. To induce 7.5, 1.5 mM MgCl ,10mMKCl,0.5 mM dithiothreitol differentiation, cells were grown to 85-90% confluence, (DTT),50 mMNaF,200 μMNa VO ,1mM PMSF,10 3 4 and the medium was changed to DMEM with 2% horse μg/ml aprotinin, 10 μg/ml leupeptin and 10 μg/ml pep- serum supplemented with penicillin, streptomycin and statin) and left on ice for 10 minutes. Cells were then amphotericin B as described above. FLAG-tagged p38a passedthrougha25-gaugeneedlethree timesand Knight et al. Skeletal Muscle 2012, 2:5 Page 3 of 12 http://www.skeletalmusclejournal.com/content/2/1/5 centrifuged at 500 g to pellet nuclei and unlysed cells. sample was electrophoresed. For two-dimensional elec- The supernatant was collected as whole cytoplasm. For trophoresis, 17-cm ReadyStrip immobilised pH gradient further fractionation of the cytoplasm, the supernatant (IPG) strips (Bio-Rad Laboratories, Hercules, CA, USA) was centrifuged again at 5,000 g to pellet mitochondria were directly rehydrated with labelled lysate diluted in and membrane fractions. The supernatant was then col- rehydration buffer (7 M urea, 2 M thiourea, 4% 3-[(3- lected and centrifuged at 100,000 g to pellet any remain- cholamidopropyl)dimethylammonio]-1-propanesulpho- ing cell particles, and the resulting supernatant was nate (CHAPS) and 1% DTT) following the manufac- collected as cytosol. RIPA buffer was added to all frac- turer’s directions. Isoelectric focusing was performed on tions to a final concentration of 1× for complete lysis. a PROTEAN IEF Cell (Bio-Rad Laboratories) under the Recombinant phosphorylated p38a (Millipore, Billerica, following conditions: 200 V for 1 hour, 500 V for 1 MA, USA) was dephosphorylated using l-protein phos- hour, 5,000 V ramp for 5 hours and 5,000 V for 80,000 phatase (New England Biolabs, Inc, Ipswich, MA, USA) VH. IPG strips were then equilibrated following the by adding 800 U of phosphatase to 200 ng of p38a manufacturer’sinstructionsand overlaid onto a12% diluted in l-phosphatase buffer, and the sample was SDS-PAGE gel. Following electrophoresis, gels were assayed at 30°C for 1 hour. Antibodies used for blotting dried and imaged. For one-dimensional electrophoresis, were as follows: a-actinin (1:125 dilution; Abcam, Cam- 100 μg of lysate was used per reaction. For two-dimen- bridge, MA, USA), COX IV (1:1,000 dilution; Abcam), sional electrophoresis, 300 μg of lysate was used. GRP78 (1:500 dilution; Cell Signaling Technology, Dan- For in vitro substrate identification, assays were per- vers,MA, USA),lamin A/C (1:500dilution;Abcam), formed as described above with the following modifica- MyHC (1:100 dilution; Developmental Studies Hybri- tions. A quantity of 1.5 mg of lysate was treated with doma Bank), MyoD (1:1,000 dilution; Santa Cruz Bio- FSBA, the sample was desalted and 2× kinase assay buf- technology, Santa Cruz, CA, USA), myogenin (1:100 fer was added (40 mM MOPS, pH 7.2, 50 mM b-glycer- dilution; Developmental Studies Hybridoma Bank), ophosphate,10mM EGTA, 2mMNa VO ,2mM 3 4 neural cell adhesion molecule (1:200 dilution; Abcam), DTT, 50 mM MgCl and 2 mM cold ATP). The sample p38a (1:500 dilution; Cell Signaling Technology), phos- was then split into three 500-μgaliquots, and5 μgof pho-p38 (1:500 dilution; Abcam) and b-tubulin (1:1,000 heat-inactivated p38a was added to the control, 5 μgof dilution; Developmental Studies Hybridoma Bank). An active p38a was added to the second aliquot and 5 μg Alpha Innotech HD2 imaging system (R&D Systems, of active p38b was added to the third aliquot. The sam- Minneapolis, MN, USA) was used to quantify phospho- ples were then assayed for 3 hours at 30°C. p38 and tubulin expression. For in vivo substrate identification, assays were per- formed as above with the following modifications. At 48 FSBA treatment and substrate labelling hours of differentiation, cells were treated with 5 μMof Cells were lysed in Nonidet P-40 buffer (50 mM SB 202190 or an equivalent amount of DMSO as vehi- Tris∙HCl, pH 7.8, 150 mM NaCl, 1% (vol/vol) Nonidet cle. Twenty-four hours later the cells were lysed in Non- P-40, 1 mM PMSF, 10 μg/ml aprotinin, 10 μg/ml leu- idet P-40 buffer. Lysate (1 mg) from DMSO-treated cells peptin and 10 μg/ml pepstatin). Lysate was treated at a or 2× 1 mg of lysate from SB 202190-treated cells was concentration of 2 mg/ml with 20 mM 5’-4-fluorosul- inhibited with FSBA, the samples were desalted and 2× phonylbenzoyladenosine (FSBA) solubilised in DMSO kinase assay buffer was added to each. A quantity of 5 and placed at 30°C for 1 hour. The sample was then μgofactivep38a was added to one of the lysate ali- diluted down to 1:5 with Nonidet P-40 buffer minus quots from SB-treated cells. The samples were then protease inhibitors and desalted using Millipore Amicon assayed for 3 hours at 30°C. ultrafiltration columns with a 10 kDa molecular weight cutoff. Following concentration, the sample was diluted Dimethyl labelling to 4 mg/ml with Nonidet P-40 buffer and diluted 1:2 After assaying the samples, they were precipitated by with 2× kinase assay buffer (40 mM 3-morpholinopro- methanol chloroform, then redissolved in 200 μlof 8 M pane-1-sulphonic acid (MOPS), pH 7.2, 50 mM b-gly- urea and 50 mM Tris∙HCl, pH 8.1, with sonication. The cerophosphate, 10 mM ethylene glycol tetraacetic acid samples were then reduced with 20 mM DTT for 1 (EGTA), 2 mM Na VO , 2 mM DTT, 50 mM MgCl , hour at 60°C and alkylated by 100 mM iodoacetamide 3 4 2 400 μMcoldATP and5 μCi [g- P]ATP). Recombinant for 30 minutes at room temperature in the dark. Subse- p38a or p38b (Millipore) was added to a final concen- quently, the samples were diluted to 2 M urea with 50 tration of 0.5% (wt/wt) total protein. Control and mM Tris∙HCl, pH 8.1, and digested with trypsin at a kinase-added samples were assayed at 30°C for 1.5 protein-to-trypsin ratio of 50:1 (wt/wt) for 16 hours at hours. For one-dimensional SDS-PAGE, 5× Laemmli 37°C. Next, the digested samples were acidified to pH 2 buffer was added following the assay to 1×, and the using 10% (vol/vol) formic acid. Dimethyl labelling of Knight et al. Skeletal Muscle 2012, 2:5 Page 4 of 12 http://www.skeletalmusclejournal.com/content/2/1/5 the samples was performed as reported previously [11] and MS/MS spectra were acquired in a data-dependent and is described briefly as follows. The acidified peptides mode, and one full MS scan was followed by ten MS/ were loaded onto C18 solid phase extraction (SPE) col- MS scans. The resolution was set at 60,000 at m/z 400 umns (50 mg of packing material). After brief washing after accumulation to a target value of 500,000. with 50 mM sodium phosphate buffer, pH 7.5, 3 ml of light, intermediate and heavy labelling reagents were Protein identification and quantification loaded onto C18 SPE columns trapped with control, All MS/MS spectra in one acquired raw file were con- p38a-and p38b-labelled samples, respectively. After verted to a single *.mgf file using DTASuperCharge ver- being washed with 0.1% (vol/vol) formic acid, the sion 2.0a7 (Matrix Science, Boston, MA, USA). The *. labelled samples were eluted with 80% acetonitrile mgf file was queried against the International Protein (ACN) (vol/vol) and 0.1% (vol/vol) formic acid, then Index mouse database version 3.52 (EMBL-EBI; http:// dried by vacuum centrifugation. www.ebi.ac.uk/IPI/IPIhelp.html) using Mascot version 2.1 (Matrix Science). To evaluate the false discovery rate Phosphopeptide enrichment (FDR), reversed sequences were appended to the data- Phosphopeptide enrichment by TiO was carried out as base. Cysteine residues were searched as a static modifi- reported previously [12] with modifications. The dried cation of +57.0215 Da; methionine residues were samples were redissolved with 65% ACN/2% trifluoroa- searched with a variable modification of +15.9949 Da; cetic acid (TFA)/saturated glutamic acid and combined. and serine, threonine and tyrosine residues were TiO beads suspended in 65% ACN/2% TFA/saturated searched with a variable modification of +79.9663 Da. glutamic acid were added into the above samples with a Light, intermediate and heavy dimethylation of peptide peptide to TiO bead ratio of 1:4 (wt/wt). After being amino termini and lysine residues were set as variable nutated for 40 minutes, the TiO beads were recovered modifications of +28.0313 Da, +32.0564 Da and by centrifugation and washed thoroughly with 65% +36.0757 Da, respectively. Peptides were queried using ACN/2% TFA. Finally, the enriched phosphopeptides full tryptic cleavage constraints with up to two missed were eluted with 10% (vol/vol) NH ·H Oand dried by cleavage sites. The mass tolerances were 7 ppm for par- 3 2 vacuum centrifugation. ent masses and 0.5 Da for fragment masses. Phospho- peptides with a Mascot score ≥ 30 (rank 1, P ≤ 0.05, Online liquid chromatography tandem mass spectrometry bold red required) were selected and quantified (FDR < analysis 0.01). Phosphorylation site localisation and phosphopep- Online liquid chromatography tandem mass spectrome- tide quantification were performed using a dimethyl- adapted version of MSQuant version 2.0a81. For each try (LC-MS/MS) analysis was performed as reported previously [13,14] with modifications. The dried sample peptide, the putative site of phosphorylation yielding the was redissolved with 0.1% formic acid and loaded onto a highest posttranslational modification (PTM) score was biphasic trap column (200 μmID×10 cm;5-cm accepted (PTM score > 13 required, as described pre- reversed phase column packed with ReproSil-Pur C18 viously [15]). Peptide ratios were obtained by calculating resin (5 μm at 200 Å; Dr.Maisch GmbH, Ammerbuch- the extracted ion chromatograms of the light, medium Entringen, Germany) and a 5-cm monolith strong cation and heavy forms of the peptide using the monoisotopic exchange (SCX) column). The trapped phosphopeptides peaks only, and protein ratios were calculated from the were eluted from the trap column onto a C18 tip col- average of all quantified peptides. All MSQuant outputs umn (75 μmID × 20 cm,3 μmat200 Å; Dr.Maisch of the same online multidimensional separation were GmbH) by a series of salt washes at increasing concen- then imported into StatQuant version 1.2.2, and the trations (0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, quantified phosphopeptides were sorted together and 70, 80, 90, 100, 150, 200 and 1,000 mM). Each fraction exported. was then separated by reversed phase-based gradient elution and detected using an LTQ Orbitrap XL Fourier Substrate validation transform mass spectrometer (Thermo Scientific, Wal- 1 μg recombinant HSP27 (Enzo Life Sciences, Farming- tham, MA, USA). The reversed phase gradient was set dale,NY, USA),recombinantRps27(Abnova,Taipei as follows: 0% to 5% ACN for 2 minutes, 5% to 30% City, Taiwan) or recombinant Smad9 (Abnova) were ACN for 90 minutes and 30% to 80% ACN for 5 min- diluted in 30 μl of 1× kinase assay buffer (20 mM utes. After flushing with 80% ACN for 10 minutes, the MOPS,pH7.2,25mM b-glycerophosphate, 5 mM column was equilibrated with 0.1% formic acid aqueous EGTA, 1 mM Na VO , 1 mM DTT, 25 mM MgCl , 200 3 4 2 solution for 13 minutes. The LTQ Orbitrap XL Fourier μMcoldATP and2.5 μCi [g- P]ATP). A quantity of transform mass spectrometer was operated in positive 500 ng of p38a or p38b was then added, and the sam- ionization mode. A voltage of 1.8 kV was applied. MS ples were assayed at 30°C for 1 hour. Laemmli buffer at Knight et al. Skeletal Muscle 2012, 2:5 Page 5 of 12 http://www.skeletalmusclejournal.com/content/2/1/5 5× concentration was added to 1× to terminate the assays, then the samples were electrophoresed and the gels were dried and imaged. Peptide assays were per- formed similarly with 5 μgofthe followingpeptides synthesised by Biomatik (Cambridge, ON, Canada): HSP27 S180-APLPKAVTQSAEITIPVTF, HSP27 A180- APLPKAVTQAAEITIPVTF, Rps27 S27-KHKKKRLVQ SPNSYFMDVK, Rps27 A27-KHKKKRLVQAPNSYF MDVK, Smad9 T136-NPYHYQRVETPVLPPVLVP and Smad9 A136-NPYHYQRVEAPVLPPVLVP. The fre- quency logo was generated using WebLogo [16]. Results FSBA inhibits endogenous protein kinases A substrate-finding approach that works with cell lysate must overcome the obstacle of endogenous pro- tein kinase activity. Lysate contains tens if not hun- dreds of active kinases, making it difficult to attribute individual phosphorylations that occur during a lysate- based assay to a particular kinase. 5’-4-fluorosulphonyl- benzoyladenosine (FSBA) offers a simple solution. FSBA is an ATP analogue that inhibits protein kinases by occupying the ATP binding site and covalently attaching to an invariant lysine [17-20], the fully func- tionally conserved and so-called catalytic lysine [21]. As FSBA irreversibly occupies the ATP binding site, a bound kinase will permanently lose activity. Treatment of whole-cell C2C12 myoblast lysate with this com- pound can completely eliminate the endogenous kinase signal present (Figure 1A). Kinase-specific substrate labelling Cell lysate treated with FSBA can be desalted to remove any unbound inhibitor and a pool of protein with no inherent kinase activity is generated. A kinase of interest can then be added with a kinase assay buf- fer, and any labelling that subsequently occurs is due to the added kinase as opposed to an endogenous one. To specifically label substrates of p38a,akinase assay buffer containing [g- P]ATP was added to FSBA-trea- ted C2C12 lysate, along with recombinant p38a,and the sample was assayed at 30°C. Substrates labelled by Figure 1 FSBA is a pan-kinase inhibitor that allows for kinase- p38a appeared as bands following one-dimensional gel specific substrate labelling of cell lysate. (A) C2C12 cell lysate was treated with either nothing (lane 1), dimethyl sulphoxide electrophoresis or as spots in two-dimensional gel elec- (DMSO) or 5’-4-fluorosulphonylbenzoyladenosine (FSBA) solubilised trophoresis with no contaminating signal from endo- in DMSO. Subsequently, samples were desalted, kinase assay buffer genous kinases (Figure 1B and Additional file 1 Figure 32 containing P-g-ATP was added and the samples were assayed for S1). Although this type of approach is excellent for 1.5 hours. Treatment with FSBA abolished the labelling of visualising phosphorylation, it is very difficult to iden- endogenous protein kinase substrates. (B) After pretreatment of C2C12 cell lysate with FSBA, purified p38a was added with a kinase tify phosphorylated proteins through spot-picking and assay buffer to specifically label its substrates and visualised using MS. We therefore sought an alternative gel- and radio- one-dimensional SDS-PAGE or two-dimensional gel electrophoresis active-free approach for identifying phosphorylated (Additional file 1 Figure S1). proteins. Knight et al. Skeletal Muscle 2012, 2:5 Page 6 of 12 http://www.skeletalmusclejournal.com/content/2/1/5 dimethyl labels. As a final step before MS, the samples are combined and an enrichment for phosphopeptides is performed using either TiO for serine/threonine phos- phorylation or a phosphotyrosine antibody for tyrosine phosphorylation. Phosphopeptides are then identified by performing LC-MS/MS, and their relative abundance between samples quantified by the differential dimethyl labelling of peptides. Phosphopeptides that are more abundant in the kinase-added sample are from proteins labelled by the added kinase during the in vitro assay, and by this means substrates can be identified. This approach results not only in substrate identification but also in the identification of the site of phosphorylation. Because up to three samples can be compared using dimethyl labelling, the phosphorylation profile of two kinases (plus a control) can be compared in a single experiment. We began our initial in vitro substrate-finding proce- dure by treating 1.5 mg of C2C12 lysate with FSBA. For the assay, the sample was split into three equal parts, with active p38a and b added to the second and third samples, respectively. The rationale behind comparing p38a with p38b was to identify specific p38a phosphor- ylations that might explain why p38b cannot compen- sate for the loss of p38a in differentiating myoblasts. In total, 387 unique serine/threonine phosphopeptides were identified (Additional file 1 Table S1). A histogram of their relative abundance ratios (p38a/control) is shown in Additional file 1 Figure S2. A threefold increase in the abundance ratio was selected as a cutoff for high-confidence in vitro substrates as this ratio is beyond the range of inherent variability, and 158 phos- phorylation sites from 94 different proteins showed at least a threefold increase in the p38a-assayed sample relative to the control. The list of p38a phosphorylation Figure 2 Methodology used to identify in vitro kinase substrates using FSBA and quantitative mass spectrometry. The sites is presented in Additional file 1 Table S2. We iden- differential tagging of peptides with dimethyl labels allows for the tified five previously known substrates (caldesmon, his- relative quantification and identification of phosphopeptides tone H2B, Psmd1, SAKS1 and Smad3) and eighty-nine simultaneously in a high-mass accuracy mass spectrometer. An that were previously unknown. Three of these previously example/theoretical substrate identification for the added kinase (green) is shown and is based on the higher intensity of the unknown targets were validated to determine if the phosphopeptide in MS. technique was discovering true in vitro p38a substrates. Recombinant forms of these three proteins could be phosphorylated in vitro by p38a (Figure 3A), and a vali- dation of peptides confirmed the site of phosphorylation Quantitative MS coupled with a phosphopeptide (Figure 3B). After aligning substrates on their phosphor- enrichment to identify substrates ylation sites, we found that a consensus phosphorylation The approach we devised to identify substrates is out- motif was present in many substrates, although it was lined in Figure 2. Cell lysate is treated with FSBA and not an absolute requirement (Figure 3C). The motif desalted as described in the previous section. This is fol- contains a proline immediately downstream from the lowed by the addition of a nonradioactive kinase assay target serine or threonine and an aliphatic residue two buffer, and the sample is split in two, to one of which is residues upstream. This motif is in agreement with that added active kinase (kinase-added). After assaying at 30° previously described for p38 [22], providing further sup- C, the two samples are digested, and peptides from each port for our substrate-finding approach. sample are differentially tagged using isotopomeric Knight et al. Skeletal Muscle 2012, 2:5 Page 7 of 12 http://www.skeletalmusclejournal.com/content/2/1/5 Figure 3 Validation of newly discovered p38a substrates. (A) Validation of three newly identified targets as in vitro p38a substrates was performed using full-length substrate incubated with a kinase assay buffer and p38a. (B) Wild-type peptides containing the phosphorylation site or peptides harbouring an S/T-to-A mutation of the phosphorylation site were incubated with p38a and a kinase assay buffer to confirm the location of the phosphorylation site. (C) The 158 phosphorylation sites identified were aligned, and a frequency logo was generated. p38a has a consensus phosphorylation motif of aXS/TP, where “a” is an aliphatic residue and “X” is any amino acid. Substrate specificity does not distinguish p38a and p38b We first assayed p38a and p38b on cell lysate using the radioactive approach and were surprised to see no obvious differences in the substrate banding patterns Figure 4 Localisation, not substrate specificity, distinguishes produced by the two isoforms (Figure 4A). Consistent p38a from p38b. (A) FSBA-treated C2C12 cell lysate was incubated with this observation, of the 158 p38a phosphorylations with a kinase assay buffer and p38a, p38b or no kinase as a control. identified using the quantitative approach, none Both isoforms produced similar banding patterns. The prominent appeared to be specific to this isoform. The 158 quanti- bands marked with the asterisks in each of the p38a and p38b lanes represent autophosphorylation of the added recombinant tative values for the p38a phosphorylations are plotted kinase. (B) The quantitative values for all 158 identified p38a from highest to lowest in Figure 4B (blue line for p38a). phosphorylations are plotted in blue on a log scale, with the The red line represents the corresponding p38b values. corresponding values for p38b shown in red. The dashed line Although p38a and p38b showed apparent differences represents the fold cutoff for accepted substrates. Four p38b in preference, they had very similar overall profiles. phosphorylations fell appreciably below the fold cutoff (point 2 corresponds to serine 27 of the 40S ribosomal protein S27-Rps27). There were only four phosphorylations that fell above (C) Incubation of p38a or p38b with purified Rps27 and a kinase the threefold cutoff for p38a,whereas for p38b they assay buffer. (D) Incubation of p38a or p38b with a peptide from were well below. However, the values were still positive Rps27 containing serine 27 (or mutation of serine 27 to alanine) and for p38b, suggesting that these could be real phosphory- a kinase assay buffer. (C) and (D) demonstrate that serine 27 of lations but there is simply less confidence in them. To Rps27 is not a specific p38a phosphorylation site. (E) FLAG-tagged p38a or p38b were transfected into C2C12 cells, and at 48 hours of determine if these might represent specific phosphoryla- differentiation immunofluorescence staining for FLAG was tions, we performed in vitro assays with either p38a or performed. p38a has a ubiquitous distribution, whereas p38b is p38b using purified substrate for one candidate. The found solely at the cell periphery. Cells were imaged using a Zeiss purified substrate was the 40S ribosomal protein S27 LSM 510 META confocal microscope (Carl Zeiss MicroImaging (Rps27), and both p38 isoforms were able to phosphory- GmbH, Jena, Germany). Scale bar = 20 μm. late this protein at the same site (Figure 4C and 4D). Knight et al. Skeletal Muscle 2012, 2:5 Page 8 of 12 http://www.skeletalmusclejournal.com/content/2/1/5 Therefore, none of the 158 phosphorylations found for p38a appear to be specific to this isoform. If substrate specificity does not distinguish the two p38 isoforms, and yet p38b is unable to compensate for the loss of p38a, there must be an alternative character- istic that discriminates them. An obvious possibility is cellular localisation. If p38a and p38b localise differently within the cell, then p38b wouldsimplybe unableto fulfil p38a’s role, even though it might have the catalytic potential to do so. To study this, we overexpressed FLAG-tagged p38a and p38b in C2C12 cells and assessed their localisation during differentiation (Figure 4E). Whereas p38a has a ubiquitous localisation pattern, p38b is found only at the periphery of the cell. p38a therefore has access to a substrate pool that p38b does not, highlighting a major reason why p38b cannot com- pensate for the loss of p38a. Identification of novel in vivo p38a substrates Although the approach we have outlined can identify in vitro kinase substrates, such in vitro approaches will also turn up irrelevant substrates that are phosphorylated only because of the absence of the appropriate cellular context. For example, we identified just over 400 phos- phorylation sites in the screen described above, 158 of which are in vitro p38 targets. If these numbers are representative, it suggests that p38 could be responsible for approximately 40% of all cellular phosphorylations in differentiating myoblasts, a number that seems unlikely, Figure 5 Methodology used to identify in vivo kinase given that there are about 500 mouse and human substrates using FSBA and quantitative MS. In vivo substrates are kinases [23]. identified based on the higher phosphopeptide intensity in MS for To overcome the drawbacks associated with in vitro both the vehicle-treated (blue) and kinase-added (green) samples. A substrate identification, we modified the approach to higher phosphopeptide abundance in MS for the vehicle-treated (blue) sample relative to the inhibitor-treated (red) sample indicates allow for the identification of relevant, direct in vivo a phosphorylation site regulated by the kinase of interest in vivo.A substrates (outlined in Figure 5). For our purposes, dif- higher phosphopeptide abundance for the kinase-added (green) ferentiating C2C12 cells were treated either with the sample relative to the inhibitor-treated (red) sample indicates a p38 inhibitor SB 202190 or with DMSO as vehicle prior phosphorylation site directly targeted by the kinase in vitro. to lysis. SB inhibitor or vehicle treatment began at 48 hours of differentiation, and the cells were lysed 24 hours later. The 48-hour time point was chosen because aliquots were taken from the lysate of inhibitor-treated of our interest in identifying novel functions for p38a cells. All three aliquots were treated with FSBA and during the middle stages of myoblast differentiation. desalted to remove unbound FSBA and SB inhibitor, Myogenic gene activation occurs within the first 48 and a kinase assay buffer was added to each. Active hours of differentiation [24] and is followed by cell p38a was added to one of the inhibitor-treated aliquots, fusion, sarcomere formation and other processes. If dif- and the samples were assayed at 30°C. Following the ferentiating C2C12 myoblasts are treated with a p38 assay, the samples were digested, dimethyl-labelled and inhibitor at 48 hours, there is a reduction in cell fusion phosphopeptides enriched as described above. What we and overall differentiation (Additional file 1 Figure S3), sought to identify were phosphorylation sites that indicating that there is a requirement for long-term p38 decreased on inhibitor treatment and could be elevated activity for efficient myogenesis beyond the initial stage back up by direct in vitro p38a labelling of SB inhibitor- of myogenic gene activation, possibly both for maintain- treated lysate. These would be direct phosphorylation ing such gene activation and for fusion-related pro- sites regulated by p38a in vivo. cesses. Following cell lysis, a 1-mg aliquot was taken By using this approach, we identified a total of 717 from the lysate of vehicle-treated cells and two 1-mg phosphorylation sites (Additional file 1 Table S3), 73 of Knight et al. Skeletal Muscle 2012, 2:5 Page 9 of 12 http://www.skeletalmusclejournal.com/content/2/1/5 Table 1 Newly discovered in vivo p38a substrates Genes Protein names Sites Ahnak Neuroblast differentiation-associated protein AHNAK S4906 Iws1 Protein IWS1 homolog S183, S185 Grp78 78 kDa glucose-regulated protein T649 Pgrmc Membrane-associated progesterone receptor S181 component 1 Prdx6 Peroxiredoxin 6 T93 Ranbp2 E3 SUMO protein ligase RanBP2 S2505 SUMO, small ubiquitin-like modifier. which showed a threefold decrease following SB inhibi- tor treatment, and seven of these sites were direct p38a phosphorylation sites (that is, showed at least a threefold increase in the p38a-labelled sample relative to the lysate from inhibitor-treated cells). In contrast to the pure in vitro approach, which suggested that approxi- mately 40% of all cellular phosphorylations might be p38 target sites, the numbers derived from the in vivo study suggest that the actual level is closer to about 1%. The seven in vivo sites (on six substrates) that we iden- tified are listed in Table 1. The substrate Iws1 is a largely nuclear protein involved in several processes, such as chromatin remo- Figure 6 Cytoplasmic characterization of p38a during C2C12 delling and mRNA export [25,26], functions with which cell differentiation. (A) C2C12 cells were fractionated over a p38 has previously been associated. Ranbp2 localises differentiation time course. C, control whole-cell lysate; P, predominantly to the nuclear pore complex and is criti- proliferating lysate. Western blotting for phospho-p38 shows a clear cal for myotube formation [27]. The other four sub- increase in the cytoplasm as differentiation proceeds. Nuclei were removed in the initial step of fractionation, and lamin A/C was used strates are cytoplasmic proteins without known or to demonstrate that there were no contaminating nuclei in suggested nuclear functions (Ahnak, Grp78, Pgrmc and cytoplasmic extracts. Quantification of phospho-p38 expression is Prdx6). On the basis of fractionations and Western blot shown in Additional file 1 Figure S4. (B) After 48 hours of analysis, we found that p38a was indeed present in the differentiation, C2C12 cells were lysed and the cytoplasmic fraction cytoplasm during differentiation (Figure 6A), which is in was collected and further fractionated into cytosolic (including the cytoskeleton) and noncytosolic fractions. Nuclear marker: lamin A/C; agreement with the immunofluorescence staining of cytoskeletal marker: a-actinin; mitochondrial marker: COX IV; FLAG-tagged p38a. The levels of phosphorylated p38, membrane marker: neural cell adhesion molecule (NCAM); the active form, increased in the cytoplasm with differ- endoplasmic reticulum marker: GRP78. (C) Interconnections entiation (Figure 6A and Additional file 1 Figure S4), between the p38a activation pathway and newly discovered in vivo substrates. Proteins previously associated with p38a in its activation suggesting that p38a might play an important role pathway are indicated as blue nodes. New substrates are indicated there. Validation of the phospho-p38 antibody is shown as red nodes. Edge colouring indicates the type of association in Additional file 1 Figure S5. Further fractionation of between nodes. the cytoplasm revealed that p38a and phospho-p38 were present only in the cytosolic fraction (Figure 6B), meaning that all four of the in vivo cytoplasmic phos- 6C), providing additional support that these targets are phorylation sites we identified would be accessible to relevant and that their phosphorylation could be critical p38a. Using Scansite [28], we found that five of the six for pathway function. substrates contained one or more predicted D domains, MAPK docking domains found on many in vivo sub- Discussion strates (D domain sites and scores are listed in Addi- We have described here a simple technique for kinase tional file 1 Table S4). We next used Genemania [29] to substrate finding that uses whole-cell lysate, can identify identify previously known associations between these sites of phosphorylation in addition to the protein phos- substrates and the p38 activation pathway we recently phorylated, has the ability to identify both in vivo and in reviewed and outlined [30]. Four of the substrates have vitro substrates, and can be used to compare the in vitro previously known associations with this pathway (Figure substrate specificity of two kinases in the same Knight et al. Skeletal Muscle 2012, 2:5 Page 10 of 12 http://www.skeletalmusclejournal.com/content/2/1/5 experiment. Performing the assay with p38a and p38b the in vivo approach on unfractionated lysate, when revealed that, although differences in preference are combined with fractionation prior to LC-MS/MS, the apparent, there are no obvious phosphorylation sites number of substrates identified could be much greater specific to p38a. However, a major characteristic that than what we have demonstrated in principle here. distinguishes p38a from p38b during myoblast differen- As mentioned, a major advantage of the technique tiation is localisation, with p38a located ubiquitously presented here is that it can be used to compare the substrate profiles of two or more kinases, and we used throughout the cell while p38b is found only at the cell this to study the substrate specificity of the p38a and b periphery. We also have shown that p38a can phos- isoforms. However, we were unable to identify any phos- phorylate several cytosolic phosphorylation sites in vivo and that this may be a critical but previously unknown phorylations that were specific to p38a.There are function for it. These results demonstrate the utility of apparent differences in preference for substrates the substrate screening technique we have developed between p38a and p38b, which could be functionally and how its use not only can find proteins phosphory- relevant. Assuming that p38b had the same ubiquitous lated by a kinase of interest but also can answer particu- localisation pattern as p38a, a low preference for certain lar questions and propose novel avenues of research. critical myogenic phosphorylations could result in a dif- The technique we have developed has several advan- ference in cell behaviour if p38b were theonlyisoform tages over existing substrate finding approaches. It can present. However, it seems likely that differentiation provide direct substrate identifications and information would still occur, but possibly at a reduced rate or in a on in vivo relevance in a single experiment. Whole-cell compromised way. This is not the case, as p38b-con- lysate can be used as a source of candidate substrates, taining myoblasts that lack p38a fail to differentiate at allowing screens to be performed on samples of specific all [7-9,34]. Rather, we believe what truly distinguishes relevance to the area of interest instead of on protein or the two isoforms is their localisation. p38a has a known peptide arrays. Our approach for eliminating endogen- critical role in the nucleus, and our results suggest that ous kinase activity in lysate is nondenaturing, decreasing the same may be true for the cytoplasm; therefore, p38b the likelihood of false-positive and false-negative sub- would be unable to compensate because it is found strate phosphorylations that would result from the heat- solely at the periphery of the cell. A similar distinction inactivating approach that others have employed [31,32]. exists for two other closely related kinases, focal adhe- At the same time, the recombinant kinase used in the sion kinase (FAK) and proline-rich tyrosine kinase 2 (PYK2) [35]. In fibroblasts, FAK localises to focal adhe- assay does not require mutation or unnatural ATP ana- sions, whereas PYK2 has a perinuclear distribution. logues as other approaches do [33], making the techni- que applicable to any kinase that can be made and PYK2, which has a kinase domain 60% identical to that purified in an active form. Although we used dimethyl of FAK, can compensate for the loss of FAK provided it labelling for quantitative MS, the technique is fully com- has a focal adhesion targeting sequence. Together these patible with iTRAQ, which allows up to eight samples to results suggest that rather than substrate specificity, it is be compared in a single experiment, meaning that other characteristics, in these cases localisation, that dis- simultaneous in vivo substrate identification and kinase tinguish closely related kinases. Whether the same holds comparisons can be performed. Radioactivity and gel true for the entire kinase family is an intriguing work are not required, but can be used to visualise question. phosphorylations and make qualitative comparisons In addition to p38a’s known role in regulating gene between kinases on one-dimensional gels. The lysate expression at the onset of myoblast differentiation, our requirements are also relatively low; 1.5 to 3 mg were results suggest that it is likely to have a cytosolic role as used in these assays, which may make the technique dif- well, possibly in regulating its own pathway. We have ficult to employ with some primary cells lines, but it is found that p38a is present in the cytosol, active p38 easily applicable to most secondary cell lines and to tis- increases in the cytoplasm with differentiation, p38a can sues as well. We have treated several different lysate phosphorylate several cytosolic proteins, three of the types with FSBA, and complete inhibition of endogen- cytosolic proteins contain predicted D domains, and ous kinase activity occurred in all, meaning that our connections between these proteins and the p38a acti- approach is likely universally applicable. In this study vation pathway have been previously reported. Together our approach for identifying in vivo substrates utilised these results suggest a previously unrecognised role for an inhibitor, but it is more versatile than that, and can p38a in the cytosol. be applied to study phosphorylations triggered by a spe- cific stimulus (using stimulated versus unstimulated Conclusions cells for lysate pools) without requiring a specific inhibi- While the role of the p38a MAPK during myoblast dif- tor be available. Finally, although we have performed ferentiation has been studied extensively with regard to Knight et al. Skeletal Muscle 2012, 2:5 Page 11 of 12 http://www.skeletalmusclejournal.com/content/2/1/5 expertise. RK supervised JDRK. All authors read and approved the final gene regulation, our evidence suggests that this kinase is manuscript. likely involved in other processes as well. The differen- tiation of myoblasts into a mature myotube involves Competing interests The authors declare that they have no competing interests. extensive morphological and functional changes that p38a might regulate, both through the initiation of a Received: 22 August 2011 Accepted: 6 March 2012 myogenic gene program and through direct phosphory- Published: 6 March 2012 lation and functional modification of cytosolic proteins. References No one has previously been able to identify a p38a-spe- 1. Berwick DC, Tavaré JM: Identifying protein kinase substrates: hunting for cific substrate, and our large-scale screen suggests these the organ-grinder’s monkeys. Trends Biochem Sci 2004, 29:227-232. may exist only within the context of cellular localisation. 2. Johnson SA, Hunter T: Kinomics: methods for deciphering the kinome. Nat Methods 2005, 2:17-25. In addition, the substrate screening technique we have 3. Manning BD, Cantley LC: Hitting the target: emerging technologies in the developed should serve as a useful tool for studying the search for kinase substrates. Sci STKE 2002, 2002:pe49. other kinases known to be involved in myogenesis, as 4. Sopko R, Andrews BJ: Linking the kinome and phosphorylome: a comprehensive review of approaches to find kinase targets. Mol Biosyst well as the hundreds of other protein kinases that exist. 2008, 4:920-933. 5. Holt LJ, Tuch BB, Villén J, Johnson AD, Gygi SP, Morgan DO: Global analysis Additional material of Cdk1 substrate phosphorylation sites provides insights into evolution. Science 2009, 325:1682-1686. 6. Lluís F, Perdiguero E, Nebreda AR, Muñoz-Cánoves P: Regulation of skeletal Additional file 1: Figures S1 through S5 and Tables S1 through S4. muscle gene expression by p38 MAP kinases. Trends Cell Biol 2006, 16:36-44. 7. Li Y, Jiang B, Ensign WY, Vogt PK, Han J: Myogenic differentiation requires signalling through both phosphatidylinositol 3-kinase and p38 MAP Acknowledgements kinase. Cell Signal 2000, 12:751-757. JDRK was supported by a Vanier Canada Graduate Scholarship, a Canadian 8. Perdiguero E, Ruiz-Bonilla V, Gresh L, Hui L, Ballestar E, Sousa-Victor P, Institutes of Health Research (CIHR) Canada Graduate Scholarship and a Baeza-Raja B, Jardí M, Bosch-Comas A, Esteller M, Caelles C, Serrano AL, Multiple Sclerosis Society of Canada (MSSC) Studentship. LAM holds the Wagner EF, Muñoz-Cánoves P: Genetic analysis of p38 MAP kinases in Mach Gaensslen Chair in Cardiac Research and was funded by CIHR. ACG is myogenesis: fundamental role of p38α in abrogating myoblast a Canada Research Chair in Functional Proteomics and the Lea Reichmann proliferation. EMBO J 2007, 26:1245-1256. Chair and is funded by CIHR grant MOP-84314. DF acknowledges a Canada 9. Wang H, Xu Q, Xiao F, Jiang Y, Wu Z: Involvement of the p38 mitogen- Research Chair in Proteomics and Systems Biology as well as grants from activated protein kinase α, β, and γ isoforms in myogenic differentiation. the Natural Sciences and Engineering Research Council of Canada and the J- Mol Biol Cell 2008, 19:1519-1528. Louis Lévesque Foundation. RK holds a University Health Research Chair at 10. Enslen H, Raingeaud J, Davis RJ: Selective activation of p38 mitogen- the University of Ottawa and was supported by grants from CIHR and the activated protein (MAP) kinase isoforms by the MAP kinase kinases MSSC. MKK3 and MKK6. J Biol Chem 1998, 273:1741-1748. 11. Boersema PJ, Raijmakers R, Lemeer S, Mohammed S, Heck AJ: Multiplex Author details peptide stable isotope dimethyl labeling for quantitative proteomics. Regenerative Medicine Program, Ottawa Hospital Research Institute, 501 Nat Protoc 2009, 4:484-494. Smyth Road, Ottawa, ON, K1H 8L6, Canada. Department of Cellular and 12. Li QR, Ning ZB, Tang JS, Nie S, Zeng R: Effect of peptide-to-TiO beads Molecular Medicine, University of Ottawa, 451 Smyth Road, Ottawa, ON, K1H ratio on phosphopeptide enrichment selectivity. J Proteome Res 2009, 8M5, Canada. Ottawa Institute of Systems Biology, University of Ottawa, 451 8:5375-5381. Smyth Road, Ottawa, ON, K1H 8M5, Canada. Department of Biochemistry, 13. Wang F, Dong J, Jiang X, Ye M, Zou H: Capillary trap column with strong Microbiology and Immunology, University of Ottawa, 451 Smyth Road, cation-exchange monolith for automated shotgun proteome analysis. Ottawa, ON, K1H 8M5, Canada. CAS Key Lab of Separation Sciences for Anal Chem 2007, 79:6599-6606. Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy 14. Wang F, Chen R, Zhu J, Sun D, Song C, Wu Y, Ye M, Wang L, Zou H: A fully of Sciences, Dalian, 116023, China. Department of Medicine, University of automated system with online sample loading, isotope dimethyl Ottawa, 451 Smyth Road, Ottawa, ON, K1H 8M5, Canada. Samuel Lunenfeld labeling and multidimensional separation for high-throughput Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, ON, quantitative proteome analysis. Anal Chem 2010, 82:3007-3015. M5G 1X5, Canada. Department of Molecular Genetics, University of Toronto, 15. Olsen JV, Blagoev B, Gnad F, Macek B, Kumar C, Mortensen P, Mann M: 1 King’s College Circle, Toronto, ON, M5S 1A8, Canada. Current address: Global, in vivo, and site-specific phosphorylation dynamics in signaling Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University networks. Cell 2006, 127:635-648. Avenue, Toronto, ON, M5G 1X5, Canada. Current address: Department of 16. Crooks GE, Hon G, Chandonia JM, Brenner SE: WebLogo: a sequence logo Cancer Biology, Dana-Farber Cancer Institute, 450 Brookline Avenue, and generator. Genome Res 2004, 14:1188-1190. Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, 17. Hashimoto E, Takio K, Krebs EG: Amino acid sequence at the ATP-binding Boston, MA 02115, USA. site of cGMP-dependent protein kinase. J Biol Chem 1982, 257:727-733. 18. Kamps MP, Taylor SS, Sefton BM: Direct evidence that oncogenic tyrosine Authors’ contributions kinases and cyclic AMP-dependent protein kinase have homologous JDRK and RK conceived of and designed the project. JDRK conceived of the ATP-binding sites. Nature 1984, 310:589-592. substrate-finding technique. RT assisted JDRK with the implementation of 19. Russo MW, Lukas TJ, Cohen S, Staros JV: Identification of residues in the the substrate-finding technique. RECL performed two-dimensional gel work nucleotide binding site of the epidermal growth factor receptor/kinase. and assisted JDRK with the development of the radioactive approach. FW J Biol Chem 1985, 260:5205-5208. assisted with quantitative MS analysis and phosphopeptide validation. AB 20. Zoller MJ, Nelson NC, Taylor SS: Affinity labeling of cAMP-dependent assisted with cell cultures. All other experiments and analysis were protein kinase with p-fluorosulfonylbenzoyl adenosine: covalent performed by JDRK. JDRK wrote the paper, and RK revised and edited it. HZ modification of lysine 71. J Biol Chem 1981, 256:10837-10842. supervised FW. LAM supervised RECL and gave advice on project design. DF 21. Knight JD, Qian B, Baker D, Kothary R: Conservation, variability and the supervised RT and FW, and DF, TP and ACG provided MS facilities and modeling of active protein kinases. PLoS One 2007, 2:e982. Knight et al. Skeletal Muscle 2012, 2:5 Page 12 of 12 http://www.skeletalmusclejournal.com/content/2/1/5 22. Schwartz D, Gygi SP: An iterative statistical approach to the identification of protein phosphorylation motifs from large-scale data sets. Nat Biotechnol 2005, 23:1391-1398. 23. Manning G, Whyte DB, Martinez R, Hunter T, Sudarsanam S: The protein kinase complement of the human genome. Science 2002, 298:1912-1934. 24. Andrés V, Walsh K: Myogenin expression, cell cycle withdrawal, and phenotypic differentiation are temporally separable events that precede cell fusion upon myogenesis. J Cell Biol 1996, 132:657-666. 25. Diebold ML, Koch M, Loeliger E, Cura V, Winston F, Cavarelli J, Romier C: The structure of an Iws1/Spt6 complex reveals an interaction domain conserved in TFIIS, Elongin A and Med26. EMBO J 2010, 29:3979-3991. 26. Yoh SM, Cho H, Pickle L, Evans RM, Jones KA: The Spt6 SH2 domain binds Ser2-P RNAPII to direct Iws1-dependent mRNA splicing and export. Genes Dev 2007, 21:160-174. 27. Asally M, Yasuda Y, Oka M, Otsuka S, Yoshimura SH, Takeyasu K, Yoneda Y: Nup358, a nucleoporin, functions as a key determinant of the nuclear pore complex structure remodeling during skeletal myogenesis. FEBS J 2011, 278:610-621. 28. Obenauer JC, Cantley LC, Yaffe MB: Scansite 2.0: Proteome-wide prediction of cell signaling interactions using short sequence motifs. Nucleic Acids Res 2003, 31:3635-3641. 29. Warde-Farley D, Donaldson SL, Comes O, Zuberi K, Badrawi R, Chao P, Franz M, Grouios C, Kazi F, Lopes CT, Maitland A, Mostafavi S, Montojo J, Shao Q, Wright G, Bader GD, Morris Q: The GeneMANIA prediction server: biological network integration for gene prioritization and predicting gene function. Nucleic Acids Res 2010, , 38 Web server: W214-W220. 30. Knight JD, Kothary R: The myogenic kinome: protein kinases critical to mammalian skeletal myogenesis. Skelet Muscle 2011, 1:29. 31. Huang SY, Tsai ML, Chen GY, Wu CJ, Chen SH: A systematic MS-based approach for identifying in vitro substrates of PKA and PKG in rat uteri. J Proteome Res 2007, 6:2674-2684. 32. Troiani S, Uggeri M, Moll J, Isacchi A, Kalisz HM, Rusconi L, Valsasina B: Searching for biomarkers of Aurora-A kinase activity: identification of in vitro substrates through a modified KESTREL approach. J Proteome Res 2005, 4:1296-1303. 33. Blethrow JD, Glavy JS, Morgan DO, Shokat KM: Covalent capture of kinase- specific phosphopeptides reveals Cdk1-cyclin B substrates. Proc Natl Acad Sci USA 2008, 105:1442-1447. 34. Ruiz-Bonilla V, Perdiguero E, Gresh L, Serrano AL, Zamora M, Sousa-Victor P, Jardí M, Wagner EF, Muñoz-Cánoves P: Efficient adult skeletal muscle regeneration in mice deficient in p38β, p38γ and p38δ MAP kinases. Cell Cycle 2008, 7:2208-2214. 35. Klingbeil CK, Hauck CR, Hsia DA, Jones KC, Reider SR, Schlaepfer DD: Targeting Pyk2 to β1-integrin-containing focal contacts rescues fibronectin-stimulated signaling and haptotactic motility defects of focal adhesion kinase-null cells. J Cell Biol 2001, 152:97-110. doi:10.1186/2044-5040-2-5 Cite this article as: Knight et al.: A novel whole-cell lysate kinase assay identifies substrates of the p38 MAPK in differentiating myoblasts. Skeletal Muscle 2012 2:5. 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Skeletal MuscleSpringer Journals

Published: Mar 6, 2012

References