Reconstructing the Regulatory Kinase Pathways of Myogenesis from Phosphopeptide Data

Multiple kinase activities are required for skeletal muscle differentiation. However, the mechanisms by which these kinase pathways converge to coordinate the myogenic process are unknown. Using multiple phosphoprotein and phosphopeptide enrichment techniques we obtained phosphopeptides from growing and differentiating C2C12 muscle cells and determined specific peptide sequences using LC-MS/MS. To place these phosphopeptides into a rational context, a bioinformatics approach was used. Phosphorylation sites were matched to known site-specific and to site non-specific kinase-substrate interactions, and then other substrates and upstream regulators of the implicated kinases were incorporated into a model network of protein-protein interactions. The model network implicated several kinases of known relevance to myogenesis including AKT, GSK3, CDK5, p38, DYRK, and MAPKAPK2 kinases. This combination of proteomics and bioinformatics technologies should offer great utility as the volume of protein-protein and kinase-substrate information continues to increase. Multiple kinase activities are required for skeletal muscle differentiation. However, the mechanisms by which these kinase pathways converge to coordinate the myogenic process are unknown. Using multiple phosphoprotein and phosphopeptide enrichment techniques we obtained phosphopeptides from growing and differentiating C2C12 muscle cells and determined specific peptide sequences using LC-MS/MS. To place these phosphopeptides into a rational context, a bioinformatics approach was used. Phosphorylation sites were matched to known site-specific and to site non-specific kinase-substrate interactions, and then other substrates and upstream regulators of the implicated kinases were incorporated into a model network of protein-protein interactions. The model network implicated several kinases of known relevance to myogenesis including AKT, GSK3, CDK5, p38, DYRK, and MAPKAPK2 kinases. This combination of proteomics and bioinformatics technologies should offer great utility as the volume of protein-protein and kinase-substrate information continues to increase. The process of skeletal muscle formation, or myogenesis, has been intensely studied because of the medical significance of muscle self-repair and as a model for cellular differentiation in general. During embryonic development, secreted factors including Shh and Wnts drive the commitment and formation of myogenic progenitor cells (1Huh M.S. Smid J.K. Rudnicki M.A. Muscle function and dysfunction in health and disease.Birth Defects Res. 2005; 75: 180-192Crossref PubMed Scopus (12) Google Scholar). Myoblasts, proliferating myogenic progenitors that express MyoD and Myf5, differentiate into non-proliferating myocytes that express myogenin and MRF4 (2Chargé S.B. Rudnicki M.A. Cellular and molecular regulation of muscle regeneration.Physiol. Rev. 2004; 84: 209-238Crossref PubMed Scopus (1987) Google Scholar). Subsequently these myocytes fuse into multinucleated syncytia that mature into contractile myofibers. This process of myoblast proliferation, cell cycle arrest, and myocyte fusion is recapitulated in injury repair and in cell culture models of myoblast differentiation such as the murine C2C12 muscle cell line. Transcriptional control of myogenesis has been extensively studied, firmly establishing the essential role of the myogenic regulatory factors (MRFs) 1The abbreviations used are: MRF, myogenic regulatory factor; 2D-GE, two-dimensional gel electrophoresis; CDK, cyclin-dependent kinase; CRHSP, calcium-regulated heat stable protein; CRMP, collapsin response mediator protein; dH2O, deionized water; DYRK, dual specificity tyrosine (Y) phosphorylation-regulated kinase; ERK, extracellular signal-regulated kinase; FOXO, forkhead box (transcription factor); GSK, glycogen synthase kinase; IGF, insulin-like growth factor; JNK, c-Jun NH2-terminal kinase; MAPKAPK, mitogen-activated protein kinase-activated protein kinase; PEA, phosphoprotein enriched in astrocytes; PKC, protein kinase C; STAT, signal transducer and activator of transcription; HNRNPK, heterogeneous nuclear ribonucleoprotein K; CK, casein kinase. Myf5, MyoD, myogenin, and MRF4. Accordingly recent interest has focused on defining the kinase signal transduction mechanisms that also modulate this process. The best characterized kinase signaling factors in myogenesis are the p38 mitogen-activated protein kinases. Inhibitors of p38 signaling block muscle-specific gene expression and myotube formation (3Cuenda A. Cohen P. Stress-activated protein kinase-2/p38 and a rapamycin-sensitive pathway are required for C2C12 myogenesis.J. Biol. Chem. 1999; 274: 4341-4346Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar, 4Zetser A. Gredinger E. Bengal E. p38 mitogen-activated protein kinase pathway promotes skeletal muscle differentiation. Participation of the Mef2c transcription factor.J. Biol. Chem. 1999; 274: 5193-5200Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar). There is evidence that p38 acts through multiple mechanisms including phosphorylation of the MRF cofactor myocyte enhancer factor 2 (4Zetser A. Gredinger E. Bengal E. p38 mitogen-activated protein kinase pathway promotes skeletal muscle differentiation. Participation of the Mef2c transcription factor.J. Biol. Chem. 1999; 274: 5193-5200Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar, 5de Angelis L. Zhao J. Andreucci J.J. Olson E.N. Cossu G. McDermott J.C. Regulation of vertebrate myotome development by the p38 MAP kinase-MEF2 signaling pathway.Dev. Biol. 2005; 283: 171-179Crossref PubMed Scopus (82) Google Scholar, 6Han J. Jiang Y. Li Z. Kravchenko V.V. Ulevitch R.J. Activation of the transcription factor MEF2C by the MAP kinase p38 in inflammation.Nature. 1997; 386: 296-299Crossref PubMed Scopus (685) Google Scholar), phosphorylation of the MyoD cofactor E47, and enhanced recruitment of the SWI/SNF chromatin-remodeling complex to MRF-targeted promoters (7Lluis F. Perdiguero E. Nebreda A.R. Munoz-Canoves P. Regulation of skeletal muscle gene expression by p38 MAP kinases.Trends Cell Biol. 2006; 16: 36-44Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar). Another key signaling kinase in muscle is AKT1. Stimulation of AKT1 by IGF-1 or insulin induces muscle hypertrophy, whereas inhibition of AKT1 by glucocorticoids promotes muscle atrophy. Hypertrophy is associated with AKT-mediated mTOR (mammalian target of rapamycin) activation and GSK3B repression (8Rommel C. Bodine S.C. Clarke B.A. Rossman R. Nunez L. Stitt T.N. Yancopoulos G.D. Glass D.J. Mediation of IGF-1-induced skeletal myotube hypertrophy by PI (3) K/Akt/mTOR and PI (3) K/Akt/GSK3 pathways.Nat. Cell Biol. 2001; 3: 1009-1013Crossref PubMed Scopus (1213) Google Scholar). Conversely AKT1 inhibition leads to dephosphorylation and activation of FOXO transcription factors resulting in atrophy (9Hoffman E.P. Nader G.A. Balancing muscle hypertrophy and atrophy.Nat. Med. 2004; 10: 584-585Crossref PubMed Scopus (91) Google Scholar). Although some kinase activities are required for normal muscle development, others must be suppressed. JNK1 is normally inactive during myogenesis, and its activation leads to muscle pathology (10Kolodziejczyk S.M. Walsh G.S. Balazsi K. Seale P. Sandoz J. Hierlihy A.M. Rudnicki M.A. Chamberlain J.S. Miller F.D. Megeney L.A. Activation of JNK1 contributes to dystrophic muscle pathogenesis.Curr. Biol. 2001; 11: 1278-1282Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). In addition to these kinases numerous other kinase proteins have been implicated as regulators of the differentiation process. For example CDK5 expression and activity increases during early myogenesis, and expression of dominant-negative forms of CDK5 inhibits muscle formation (11Lazaro J.B. Kitzmann M. Poul M.A. Vandromme M. Lamb N.J. Fernandez A. Cyclin dependent kinase 5, cdk5, is a positive regulator of myogenesis in mouse C2 cells.J. Cell Sci. 1997; 110: 1251-1260Crossref PubMed Google Scholar, 12Philpott A. Porro E.B. Kirschner M.W. Tsai L.H. The role of cyclin-dependent kinase 5 and a novel regulatory subunit in regulating muscle differentiation and patterning.Genes Dev. 1997; 11: 1409-1421Crossref PubMed Scopus (103) Google Scholar). However, an important unresolved question is how these different pathways are integrated into an overall network of kinase-substrate interactions to control myogenic differentiation. Previously we profiled changes to protein phosphorylation during myogenesis on a proteome-wide scale by using phosphoprotein enrichment and comparative two-dimensional gel electrophoresis (13Puente L.G. Carriere J.F. Kelly J.F. Megeney L.A. Comparative analysis of phosphoprotein-enriched myocyte proteomes reveals widespread alterations during differentiation.FEBS Lett. 2004; 574: 138-144Crossref PubMed Scopus (16) Google Scholar). Here we extend those findings by mapping sequence-specific sites of protein phosphorylation in growing and differentiating C2C12 cells. To place these results in a systems context, we took advantage of recent advances in the availability of protein-protein interaction databases. Databases of protein-protein interactions have been constructed from yeast, fruit fly, worm, and human cells using yeast two-hybrid, tag/pulldown, and literature search approaches (14Bork P. Jensen L.J. von Mering C. Ramani A.K. Lee I. Marcotte E.M. Protein interaction networks from yeast to human.Curr. Opin. Struct. Biol. 2004; 14: 292-299Crossref PubMed Scopus (283) Google Scholar). Subsets of the total protein-protein interaction set such as kinase-substrate interaction maps have also been constructed (15Ptacek J. Devgan G. Michaud G. Zhu H. Zhu X. Fasolo J. Guo H. Jona G. Breitkreutz A. Sopko R. McCartney R.R. Schmidt M.C. Rachidi N. Lee S.J. Mah A.S. Meng L. Stark M.J. Stern D.F. De Virgilio C. Tyers M. Andrews B. Gerstein M. Schweitzer B. Predki P.F. Snyder M. Global analysis of protein phosphorylation in yeast.Nature. 2005; 438: 679-684Crossref PubMed Scopus (820) Google Scholar). To date, however, relatively few studies have leveraged this type of information to assist in the interpretation of results. Here we used kinase-substrate interaction databases to bioinformatically reconstruct a kinase signaling network based upon our experimentally identified phosphorylation events. This approach yielded a model kinase-substrate network that predicted several known features of myogenic signaling and revealed the potential relevance of newly identified phosphorylation events in relation to known muscle-related biochemical pathways. C2C12 myoblasts were cultured in growth medium (10% fetal bovine serum, Dulbecco’s modified Eagle’s medium), and whole cell lysates were collected either 0 or 24 h after a change to differentiation medium (2% horse serum, Dulbecco’s modified Eagle’s medium) as described previously (13Puente L.G. Carriere J.F. Kelly J.F. Megeney L.A. Comparative analysis of phosphoprotein-enriched myocyte proteomes reveals widespread alterations during differentiation.FEBS Lett. 2004; 574: 138-144Crossref PubMed Scopus (16) Google Scholar). Phosphoprotein enrichment was performed by affinity column purification of total myoblast proteins using PhosphoProtein Purification kits (Qiagen) as described previously (13Puente L.G. Carriere J.F. Kelly J.F. Megeney L.A. Comparative analysis of phosphoprotein-enriched myocyte proteomes reveals widespread alterations during differentiation.FEBS Lett. 2004; 574: 138-144Crossref PubMed Scopus (16) Google Scholar). Phosphoprotein-enriched samples (typically 100–150 μg) were washed in water and concentrated using Amicon 10,000 molecular weight cutoff ultrafiltration columns (Millipore). Aliquots of retentate were separated by SDS-PAGE (12.5% acrylamide “mini” format). Gels were stained with Bio-Safe colloidal G-250 Coomassie Blue (Bio-Rad). 23 1-mm gel sections were excised from each lane, and in-gel digest and extraction of proteins was performed. Gel pieces cut into 1-mm cubes were destained in 100 mm ammonium bicarbonate (NH4HCO3) in 30% ACN. Destained gel pieces were washed in dH2O and shrunk in ACN. Gel pieces were incubated for 1 h at 56 °C in 10 mm DTT, 50 mm NH4HCO3; washed in 50 mm NH4HCO3; and incubated for 1 h at room temperature in 55 mm iodoacetamide, 50 mm NH4HCO3. Gel pieces were shrunk with ACN and reswollen in 50 mm NH4HCO3 with Promega modified trypsin (10 ng/μl). Sufficient 50 mm NH4HCO3 was added to cover the gel pieces, and sealed tubes were incubated overnight at 37 °C. Approximately 15 of phosphoprotein-enriched protein was in 50 mm NH4HCO3 Promega modified trypsin (10 ng/μl). overnight at 37 °C digest were concentrated to and through with of as described previously A. Jensen of from by affinity with 2001; PubMed Scopus Google Scholar). were washed by of and by of was by and by of the of or was in was washed in by 50 mm washed in dH2O, and then washed in was incubated in 100 mm for 15 with was in 30% and then in of was added to the peptide in mm in and for 15 into a was by of a of 100 mm in 30% and and in by mm ammonium In the 5 was into the and for 5 to the In the 15 of the was through the The of was by and the phosphopeptides were in of in columns were used to the with the Using of a of as the were to a for 15 at room and then for 1 at The was with 50 of 100 mm in with 30% in and with dH2O and then by of of mm ammonium were and by and phosphopeptides were in of of each digest (typically was by using a to a were separated on a column using the in and ACN in 5 The was set to on and The was the was the an was previously that to and a of each digest was with the set to the during the was using the for In addition to and was to search for the potential of on or tyrosine by in the for the of a on these or for resulting from a of or and phosphopeptide identified by were for the of a and the peptide In the of the modified Previously phosphoprotein-enriched samples were separated by 2D-GE, and proteins were identified by (13Puente L.G. Carriere J.F. Kelly J.F. Megeney L.A. Comparative analysis of phosphoprotein-enriched myocyte proteomes reveals widespread alterations during differentiation.FEBS Lett. 2004; 574: 138-144Crossref PubMed Scopus (16) Google Scholar). of these proteins evidence of phosphorylation the and differentiating (13Puente L.G. Carriere J.F. Kelly J.F. Megeney L.A. Comparative analysis of phosphoprotein-enriched myocyte proteomes reveals widespread alterations during differentiation.FEBS Lett. 2004; 574: 138-144Crossref PubMed Scopus (16) Google Scholar). was extensively the using to and the protein and phosphorylation phosphopeptide were For each experimentally determined phosphorylation known to the site in or in was identified using the bioinformatics and In a of the kinase was predicted using J.C. of cell signaling interactions using Res. PubMed Scopus Google Scholar). was for other known substrates of each kinase. The process of mapping substrates to kinases was then for proteins in the including the kinases results were and by of the The identified interactions were then using the bioinformatics Megeney L.A. The yeast scale with 2005; PubMed Scopus Google Scholar). phosphopeptides were identified from C2C12 cell growth or differentiation 1 and 2 for phosphopeptides were identified after after gel electrophoresis and after and LC-MS/MS. phosphopeptides were by multiple that each a into the of The of the a from or the of such performed. phosphopeptides in were matched to known or predicted and in were identified that the were in either growing or differentiating cells the 55 identified at phosphorylation sites that have been by the of phosphoprotein-enriched samples that this phosphopeptide in the of of the peptide in on the protein phosphorylation of this site has previously been this phosphopeptide was in C2C12 cell this phosphopeptide was in differentiating C2C12 cell this phosphopeptide was after phosphoprotein enrichment and two-dimensional gel electrophoresis; this phosphopeptide was after phosphoprotein enrichment and this phosphopeptide was after phosphoprotein enrichment and kinases are those that have been to the specific site in other studies or that are predicted to the target predicted interactions are with an phosphopeptide was previously by is for the of phosphoprotein-enriched samples that this phosphopeptide in the of of the peptide in on the protein phosphorylation of this site has previously been this phosphopeptide was in C2C12 cell this phosphopeptide was in differentiating C2C12 cell this phosphopeptide was after phosphoprotein enrichment and two-dimensional gel electrophoresis; this phosphopeptide was after phosphoprotein enrichment and this phosphopeptide was after phosphoprotein enrichment and kinases are those that have been to the specific site in other studies or that are predicted to the target predicted interactions are with an This phosphopeptide was previously by (13Puente L.G. Carriere J.F. Kelly J.F. Megeney L.A. Comparative analysis of phosphoprotein-enriched myocyte proteomes reveals widespread alterations during differentiation.FEBS Lett. 2004; 574: 138-144Crossref PubMed Scopus (16) Google is for in a differentiating in a in systems is to into To this we to a novel bioinformatics that because phosphopeptides are the of protein kinase activity we a kinase-substrate interaction network based upon our To this experimentally for which the kinase was known or predicted were used as the to an network of kinase-substrate interactions also other known substrates of those as as interactions upstream of each were determined and incorporated into the model For the and kinases and were from the was in phosphorylation was to a specific The of myogenic signaling is that AKT, p38, and CDK5 activity cell cycle and myotube formation, whereas and activity are associated with cell and of the kinases in in our and this AKT, CDK5, GSK3, and MAPKAPK2 kinases some kinase-substrate interactions known to be in myogenic signaling such as enhancer factor 2 and were by the the of phosphopeptides context, the kinase-substrate network a of features of such as the of kinases as interactions were in the model of kinases is required for muscle differentiation X. B. M. E. is a kinase in skeletal muscle Biol. Chem. Full Text Full Text PDF PubMed Scopus Google at in phosphorylation and regulation of X. E. the nuclear of during skeletal muscle Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). Here we that the substrates and are also in C2C12 and revealed interactions glycogen and mechanisms by which to of the phosphorylation sites to activity are to be sites for In to the site for a of at and A.R. A. L.A. C. phosphorylation of the collapsin response mediator proteins in Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). In C2C12 cells site-specific and phosphorylation (13Puente L.G. Carriere J.F. Kelly J.F. Megeney L.A. Comparative analysis of phosphoprotein-enriched myocyte proteomes reveals widespread alterations during differentiation.FEBS Lett. 2004; 574: 138-144Crossref PubMed Scopus (16) Google of was in differentiating whereas is in Although has been studied in the of these and the that the a site J.K. P. and analysis of the human phosphoprotein required for expression in and Biol. Chem. Full Text Full Text PDF PubMed Scopus Google that proteins an important role in muscle differentiation. factor is also a of and phosphorylation at is to phosphorylation of by to repression of Cohen P. R. M. X. The kinase factor at and the protein at potential role for as a glycogen synthase kinase J. 2001; PubMed Scopus Google Scholar). muscle hypertrophy is associated with inhibition and this that of be by are with the model that activity is and is during In with this several proteins that were and evidence of phosphorylation in specific phosphorylation sites were determined (13Puente L.G. Carriere J.F. Kelly J.F. Megeney L.A. Comparative analysis of phosphoprotein-enriched myocyte proteomes reveals widespread alterations during differentiation.FEBS Lett. 2004; 574: 138-144Crossref PubMed Scopus (16) Google Scholar). is phosphoprotein of at and in and to in is in human embryonic cells N. Cohen P. of calcium-regulated protein of 24 as a for and using J. 2005; PubMed Scopus Google Scholar). phosphorylation at and in differentiating C2C12 and phosphorylation at Phosphorylation of at to was also an important role in IGF-1 which muscle hypertrophy, phosphorylation of the site in human embryonic cells. is a M. M.J. J. Purification and of a novel for in cells.J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). activity is important in normal muscle development, and activation has been to the of S.J. S.M. J.C. Megeney L.A. dystrophic pathology by activation of the J. 2004; PubMed Scopus Google Scholar). of phosphorylation in C2C12 (13Puente L.G. Carriere J.F. Kelly J.F. Megeney L.A. Comparative analysis of phosphoprotein-enriched myocyte proteomes reveals widespread alterations during differentiation.FEBS Lett. 2004; 574: 138-144Crossref PubMed Scopus (16) Google was and with evidence of a phosphorylation site was in growing cells in differentiating cells. This is with the recent that the of promotes cell cycle of J. A. E. Phosphorylation of phosphoprotein enriched in extracellular signal-regulated transcription and cell Biol. 2005; 16: PubMed Scopus Google Scholar), and activity is a for myoblast differentiation inhibits myogenesis through a 1999; PubMed Scopus Google Scholar). potential role for was in the model and the potential substrates and were in phosphoprotein enrichment (13Puente L.G. Carriere J.F. Kelly J.F. Megeney L.A. Comparative analysis of phosphoprotein-enriched myocyte proteomes reveals widespread alterations during differentiation.FEBS Lett. 2004; 574: 138-144Crossref PubMed Scopus (16) Google Scholar). Although site-specific evidence of phosphorylation is these are in of a recent that promotes whereas inhibits of myoblasts G. R. I. I. P. L. growth signaling in cultured human skeletal muscle cells and in C2C12 role of protein kinase 2006; PubMed Scopus Google Scholar). as as and also which is to myoblast and MyoD function and muscle differentiation C. H. I. I. S.C. J. factor early differentiation of skeletal muscle cells by 2005; PubMed Scopus Google Scholar, K. E. In activation of signaling in cells and in skeletal PubMed Scopus Google Scholar, Y. I. E. Y. A. K. A. Y. inhibition MyoD and in the regulation of growth and differentiation of Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the substrates multiple phosphorylation studies be required to the role of the cover a of the is important to the of a model kinase network these The used on that substrates have a of that kinases have a of and that the overall of the kinase-substrate network is a has potential kinases then of specific kinase activities is a kinase has few substrates then the that of those substrates be experimentally is The the model is to features of the in network or reveals In a network a as mapping a few in a the overall of the in a on the network are by a of interactions, and the overall as a of the of at the on kinase-substrate interactions in yeast these of and kinases by (15Ptacek J. Devgan G. Michaud G. Zhu H. Zhu X. Fasolo J. Guo H. Jona G. Breitkreutz A. Sopko R. McCartney R.R. Schmidt M.C. Rachidi N. Lee S.J. Mah A.S. Meng L. Stark M.J. Stern D.F. De Virgilio C. Tyers M. Andrews B. Gerstein M. Schweitzer B. Predki P.F. Snyder M. Global analysis of protein phosphorylation in yeast.Nature. 2005; 438: 679-684Crossref PubMed Scopus (820) Google that of substrates were associated with or kinases that kinases multiple of the kinase-substrate of the protein-protein interaction for yeast that this network is Megeney L.A. The yeast scale with 2005; PubMed Scopus Google Scholar). Another was protein myogenesis in protein phosphorylation in a of proteins a of then results have been However, we previously that the of the that is for on in during early myogenesis (13Puente L.G. Carriere J.F. Kelly J.F. Megeney L.A. Comparative analysis of phosphoprotein-enriched myocyte proteomes reveals widespread alterations during differentiation.FEBS Lett. 2004; 574: 138-144Crossref PubMed Scopus (16) Google Scholar). The of obtained from two-dimensional was in a Comparative analysis of from growing differentiating myocytes to the analysis on proteins that evidence of during of these factors to the to a model network that features of However, a of because is of of phosphorylation be as evidence of the results are the of relatively kinase-substrate databases are from the of protein-protein interaction is that should or the results kinase networks to be Megeney L.A. The yeast scale with 2005; PubMed Scopus Google Scholar), the of substrates the of changes in the overall of the However, this to be experimentally Another to be is the role of in the this of a is the in the literature with the of for activity or dephosphorylation events. phosphopeptide and bioinformatics databases of kinase-substrate interactions were used to a model signaling network for C2C12 a relatively of a complex network was constructed that the of several known features of myogenic In for newly described in signaling were protein-protein interaction including kinase-substrate are at an and the utility of this approach is to as

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