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W2023488851 abstract "Brain development and spinal cord regeneration require neurite sprouting and growth cone navigation in response to extension and collapsing factors present in the extracellular environment. These external guidance cues control neurite growth cone extension and retraction processes through intracellular protein phosphorylation of numerous cytoskeletal, adhesion, and polarity complex signaling proteins. However, the complex kinase/substrate signaling networks that mediate neuritogenesis have not been investigated. Here, we compare the neurite phosphoproteome under growth and retraction conditions using neurite purification methodology combined with mass spectrometry. More than 4000 non-redundant phosphorylation sites from 1883 proteins have been annotated and mapped to signaling pathways that control kinase/phosphatase networks, cytoskeleton remodeling, and axon/dendrite specification. Comprehensive informatics and functional studies revealed a compartmentalized ERK activation/deactivation cytoskeletal switch that governs neurite growth and retraction, respectively. Our findings provide the first system-wide analysis of the phosphoprotein signaling networks that enable neurite growth and retraction and reveal an important molecular switch that governs neuritogenesis. Brain development and spinal cord regeneration require neurite sprouting and growth cone navigation in response to extension and collapsing factors present in the extracellular environment. These external guidance cues control neurite growth cone extension and retraction processes through intracellular protein phosphorylation of numerous cytoskeletal, adhesion, and polarity complex signaling proteins. However, the complex kinase/substrate signaling networks that mediate neuritogenesis have not been investigated. Here, we compare the neurite phosphoproteome under growth and retraction conditions using neurite purification methodology combined with mass spectrometry. More than 4000 non-redundant phosphorylation sites from 1883 proteins have been annotated and mapped to signaling pathways that control kinase/phosphatase networks, cytoskeleton remodeling, and axon/dendrite specification. Comprehensive informatics and functional studies revealed a compartmentalized ERK activation/deactivation cytoskeletal switch that governs neurite growth and retraction, respectively. Our findings provide the first system-wide analysis of the phosphoprotein signaling networks that enable neurite growth and retraction and reveal an important molecular switch that governs neuritogenesis. IntroductionNeuritogenesis is a dynamic process involving the extension of long, thin protrusions called neurites that will subsequently differentiate into long axons or an elaborate dendritic arbor (1Giannone G. Mège R.M. Thoumine O. Trends Cell Biol. 2009; 19: 475-486Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 2Drees F. Gertler F.B. Curr. Opin. Neurobiol. 2008; 18: 53-59Crossref PubMed Scopus (81) Google Scholar, 3Koh C.G. Neurosignals. 2006; 15: 228-237Crossref PubMed Scopus (76) Google Scholar, 4Allen J. Chilton J.K. Dev. Biol. 2009; 327: 4-11Crossref PubMed Scopus (21) Google Scholar). This highly polarized process occurs through the segmentation from the soma periphery of a microtubule-rich shaft capped with a growth cone, which itself is characterized by an actin-rich lamellipodium with numerous filopodial extensions and integrin-mediated adhesive contacts. Understanding this process is crucial, as it is necessary for proper wiring of the brain and nerve regeneration and has been linked to numerous neurodegenerative diseases.Although cultured neurons randomly form neurites in vitro, in vivo this process is orchestrated by gradients of chemoattractants, extracellular matrix proteins, and collapsing factors that precisely guide neurite initiation and advancement (5Arimura N. Kaibuchi K. Neuron. 2005; 48: 881-884Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 6Fukata M. Nakagawa M. Kaibuchi K. Curr. Opin. Cell Biol. 2003; 15: 590-597Crossref PubMed Scopus (382) Google Scholar). This occurs in a polarized and highly controlled manner and relies on spatially regulated mechanisms for gradient sensing, membrane trafficking, integrin-mediated adhesion, and organization of the actin-microtubule cytoskeleton (5Arimura N. Kaibuchi K. Neuron. 2005; 48: 881-884Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 6Fukata M. Nakagawa M. Kaibuchi K. Curr. Opin. Cell Biol. 2003; 15: 590-597Crossref PubMed Scopus (382) Google Scholar, 7Nikolic M. Int. J. Biochem. Cell Biol. 2002; 34: 731-745Crossref PubMed Scopus (117) Google Scholar, 8Dent E.W. Gertler F.B. Neuron. 2003; 40: 209-227Abstract Full Text Full Text PDF PubMed Scopus (668) Google Scholar). These events are largely orchestrated by numerous surface guidance/repulsion and adhesion receptors that signal to the cell interior through multiple kinase activation and substrate phosphorylation events (7Nikolic M. Int. J. Biochem. Cell Biol. 2002; 34: 731-745Crossref PubMed Scopus (117) Google Scholar, 9Jeon S. Park J.K. Bae C.D. Park J. Neurochem. Int. 2010; 56: 810-818Crossref PubMed Scopus (25) Google Scholar, 10Dehmelt L. Halpain S. Curr. Biol. 2007; 17: R611-R614Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 11Yamauchi J. Miyamoto Y. Sanbe A. Tanoue A. Exp. Cell Res. 2006; 312: 2954-2961Crossref PubMed Scopus (34) Google Scholar, 12Nusser N. Gosmanova E. Makarova N. Fujiwara Y. Yang L. Guo F. Luo Y. Zheng Y. Tigyi G. Cell. Signal. 2006; 18: 704-714Crossref PubMed Scopus (40) Google Scholar, 13Jørgensen C. Sherman A. Chen G.I. Pasculescu A. Poliakov A. Hsiung M. Larsen B. Wilkinson D.G. Linding R. Pawson T. Science. 2009; 326: 1502-1509Crossref PubMed Scopus (176) Google Scholar, 14Round J. Stein E. Curr. Opin. Neurobiol. 2007; 17: 15-21Crossref PubMed Scopus (105) Google Scholar, 15Woodring P.J. Litwack E.D. O'Leary D.D. Lucero G.R. Wang J.Y. Hunter T. J. Cell Biol. 2002; 156: 879-892Crossref PubMed Scopus (125) Google Scholar, 16Spencer T.K. Mellado W. Filbin M.T. Mol. Cell. Neurosci. 2008; 38: 110-116Crossref PubMed Scopus (46) Google Scholar, 17Hirose M. Ishizaki T. Watanabe N. Uehata M. Kranenburg O. Moolenaar W.H. Matsumura F. Maekawa M. Bito H. Narumiya S. J. Cell Biol. 1998; 141: 1625-1636Crossref PubMed Scopus (401) Google Scholar). Protein phosphorylation is a critical posttranslational modification that regulates protein-protein interactions, enzymatic activity, and subcellular localization. The reversible addition of PO43− to serine, threonine, and tyrosine amino acid residues is mediated by more than 500 kinases and more than 150 phosphatases (18Manning G. Whyte D.B. Martinez R. Hunter T. Sudarsanam S. Science. 2002; 298: 1912-1934Crossref PubMed Scopus (5567) Google Scholar, 19Alonso A. Sasin J. Bottini N. Friedberg I. Friedberg I. Osterman A. Godzik A. Hunter T. Dixon J. Mustelin T. Cell. 2004; 117: 699-711Abstract Full Text Full Text PDF PubMed Scopus (1395) Google Scholar, 20Manning G. Plowman G.D. Hunter T. Sudarsanam S. Trends Biochem. Sci. 2002; 27: 514-520Abstract Full Text Full Text PDF PubMed Scopus (654) Google Scholar). In many cases, kinases phosphorylate specific amino acids in the context of a consensus recognition sequence. For example, ERK is a proline-directed kinase that phosphorylates Ser/Thr residues with the consensus sequence PX(S/T)P, where X can be a neutral or basic amino acid (21Kim E.K. Choi E.J. Biochim. Biophys. Acta. 2010; 1802: 396-405Crossref PubMed Scopus (1273) Google Scholar, 22Yee K.L. Weaver V.M. Hammer D.A. IET Syst. Biol. 2008; 2: 8-15Crossref PubMed Scopus (80) Google Scholar, 23Ramos J.W. Int. J. Biochem. Cell Biol. 2008; 40: 2707-2719Crossref PubMed Scopus (359) Google Scholar, 24Pullikuth A.K. Catling A.D. Cell. Signal. 2007; 19: 1621-1632Crossref PubMed Scopus (115) Google Scholar). The identification of phosphorylation sites in the context of a specific kinase recognition sequence of given substrates has proven quite useful in the predication of kinase activation events in the cell (25Linding R. Jensen L.J. Ostheimer G.J. van Vugt M.A. Jørgensen C. Miron I.M. Diella F. Colwill K. Taylor L. Elder K. Metalnikov P. Nguyen V. Pasculescu A. Jin J. Park J.G. Samson L.D. Woodgett J.R. Russell R.B. Bork P. Yaffe M.B. Pawson T. Cell. 2007; 129: 1415-1426Abstract Full Text Full Text PDF PubMed Scopus (553) Google Scholar, 26Jørgensen C. Linding R. Curr. Opin. Genet. Dev. 2010; 20: 15-22Crossref PubMed Scopus (0) Google Scholar, 27Tan C.S. Linding R. Proteomics. 2009; 9: 5233-5242Crossref PubMed Scopus (0) Google Scholar). Also, kinases themselves can be phosphorylated at specific sites that are known to promote enzymatic activation, which can be used to predict kinase activity in the cell. This is further enriched by computational models that predict higher order contextual features of kinases such as protein scaffolding, subcellular localization, and expression patterns (13Jørgensen C. Sherman A. Chen G.I. Pasculescu A. Poliakov A. Hsiung M. Larsen B. Wilkinson D.G. Linding R. Pawson T. Science. 2009; 326: 1502-1509Crossref PubMed Scopus (176) Google Scholar, 25Linding R. Jensen L.J. Ostheimer G.J. van Vugt M.A. Jørgensen C. Miron I.M. Diella F. Colwill K. Taylor L. Elder K. Metalnikov P. Nguyen V. Pasculescu A. Jin J. Park J.G. Samson L.D. Woodgett J.R. Russell R.B. Bork P. Yaffe M.B. Pawson T. Cell. 2007; 129: 1415-1426Abstract Full Text Full Text PDF PubMed Scopus (553) Google Scholar, 26Jørgensen C. Linding R. Curr. Opin. Genet. Dev. 2010; 20: 15-22Crossref PubMed Scopus (0) Google Scholar, 27Tan C.S. Linding R. Proteomics. 2009; 9: 5233-5242Crossref PubMed Scopus (0) Google Scholar, 28Linding R. Jensen L.J. Pasculescu A. Olhovsky M. Colwill K. Bork P. Yaffe M.B. Pawson T. Nucleic Acids Res. 2008; 36: D695-D699Crossref PubMed Scopus (0) Google Scholar). When combined, in silico programs have been shown to predict kinase activity with significant accuracy. This approach has been applied to large phosphoproteomic data sets consisting of thousands of phosphorylation sites revealing important kinase signaling networks that modulate cell migration, DNA damage, and yeast biology (13Jørgensen C. Sherman A. Chen G.I. Pasculescu A. Poliakov A. Hsiung M. Larsen B. Wilkinson D.G. Linding R. Pawson T. Science. 2009; 326: 1502-1509Crossref PubMed Scopus (176) Google Scholar, 25Linding R. Jensen L.J. Ostheimer G.J. van Vugt M.A. Jørgensen C. Miron I.M. Diella F. Colwill K. Taylor L. Elder K. Metalnikov P. Nguyen V. Pasculescu A. Jin J. Park J.G. Samson L.D. Woodgett J.R. Russell R.B. Bork P. Yaffe M.B. Pawson T. Cell. 2007; 129: 1415-1426Abstract Full Text Full Text PDF PubMed Scopus (553) Google Scholar, 29Van Hoof D. Muñoz J. Braam S.R. Pinkse M.W. Linding R. Heck A.J. Mummery C.L. Krijgsveld J. Cell Stem Cell. 2009; 5: 214-226Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar, 30van Vugt M.A. Gardino A.K. Linding R. Ostheimer G.J. Reinhardt H.C. Ong S.E. Tan C.S. Miao H. Keezer S.M. Li J. Pawson T. Lewis T.A. Carr S.A. Smerdon S.J. Brummelkamp T.R. Yaffe M.B. PLoS Biol. 2010; 8: e1000287Crossref PubMed Scopus (145) Google Scholar).Although significant progress has been made in identifying specific signals regulating neuritogenesis, a system-wide analysis of the phosphoprotein signals that control this process has not been investigated. Consequently, our understanding of the complex kinase/substrate signaling networks that regulate neurite formation and guidance is not defined. Here we describe a microporous filter method that facilitates selective purification of neurites undergoing growth or retraction. This fractionation method combined with immobilized metal-ion affinity chromatography and LC-MS protein identification technologies facilitated the identification of thousands of protein phosphorylation signatures that mediate neurite extension or retraction events induced by growth promoting or collapsing stimuli, respectively. Functional annotation and kinase activity predications in silico revealed phosphoprotein networks controlling cytoskeletal remodeling, axon/dendrite specification, and cell adhesion. Kinase signaling analysis and cell-based assays revealed that an integrin/MEK/ERK signaling pathway operates as a critical switch controlling neurite extension and retraction dynamics.DISCUSSIONOur ability to differentially isolate neurites in a state of growth or retraction for proteomic analyses provided a simple and robust system to globally profile thousands of protein phosphorylation and kinase changes that mediate neuritogenesis. An important aspect of this model system is the ability to specifically isolate the neurite from the soma for proteomic analyses. This unique strategy allowed us to mine deeper into the neurite phosphoproteome, uncovering many more low abundant phosphoproteins. Because of this, we were able to obtain a more precise picture of the complex spatial kinase/substrate networks that control neurite growth/retraction kinetics. Also, the neurite purification methods described here will likely transfer to other neuronal cell types, guidance factors, and collapsing agents. For example, we have previously reported that neurites can be readily purified from PC12 cells responding to laminin and fibronectin guidance cues (35Pertz O.C. Wang Y. Yang F. Wang W. Gay L.J. Gristenko M.A. Clauss T.R. Anderson D.J. Liu T. Auberry K.J. Camp 2nd, D.G. Smith R.D. Klemke R.L. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 1931-1936Crossref PubMed Scopus (61) Google Scholar). It may also be possible to selectively purify axons and dendrites from primary neurons for detailed proteomic analyses using specific differentiation and/or guidance factors. In any case, the utility of this model system combined with the rapid progress being made in quantitative mass spectrometry will help provide a more comprehensive understanding of the neurite proteome and its spatial organization, which in turn will provide valuable insight into the various disease pathologies caused by deregulation of this process.Using NetworKIN, we find that a large panel of kinases is active during neurite growth, whereas a relatively reduced number of kinases are active during retraction. Many of these kinases have been previously implicated in the regulation of different aspects of neurite outgrowth. PKA (PKACA) can trigger neurite outgrowth by phosphorylating synapsins (89Kao H.T. Song H.J. Porton B. Ming G.L. Hoh J. Abraham M. Czernik A.J. Pieribone V.A. Poo M.M. Greengard P. Nat. Neurosci. 2002; 5: 431-437Crossref PubMed Scopus (125) Google Scholar) and CREB (cAMP-response element-binding protein) (90Vaudry D. Stork P.J. Lazarovici P. Eiden L.E. Science. 2002; 296: 1648-1649Crossref PubMed Scopus (669) Google Scholar). The MET receptor (MET) enhances axonal outgrowth of cultured dorsal root ganglions by potentiating NGF signaling (91Maina F. Hilton M.C. Ponzetto C. Davies A.M. Klein R. Genes Dev. 1997; 11: 3341-3350Crossref PubMed Google Scholar). The tyrosine kinase BTK (BTK) phosphorylates the actin regulator N-WASP, which is essential for neurite outgrowth (92Suetsugu S. Hattori M. Miki H. Tezuka T. Yamamoto T. Mikoshiba K. Takenawa T. Dev. Cell. 2002; 3: 645-658Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). The cyclin kinase Cdk5 (CDK5) regulates neurite and axon outgrowth by phosphorylating the kinase PAK1 and the microtubule-associated protein MAP1B (93Paglini G. Cáceres A. Eur. J. Biochem. 2001; 268: 1528-1533Crossref PubMed Scopus (63) Google Scholar). GSK3 (GSK3A) phosphorylates the collapsin response mediator protein-2 (Crmp-2) to promote neurite elongation via microtubule assembly (94Yoshimura T. Kawano Y. Arimura N. Kawabata S. Kikuchi A. Kaibuchi K. Cell. 2005; 120: 137-149Abstract Full Text Full Text PDF PubMed Scopus (696) Google Scholar). The MAP kinase kinase MKK4 (MAP2K4) and its substrate JNK (MAPK9) have also been involved in the regulation of neurite outgrowth through the phosphorylation of the microtubule binding proteins MAP, MAP1, doublecortin, and SGC10 (95Haeusgen W. Boehm R. Zhao Y. Herdegen T. Waetzig V. Neuroscience. 2009; 161: 951-959Crossref PubMed Scopus (70) Google Scholar). Although these findings point to a wide array of kinase classes that regulate neuritogenesis, it is likely that they work together in time and space to fine-tune changes in the actin and microtubule cytoskeletons that drive this process. Using similar proteomics and bioinformatic methods, we recently revealed a Rho GTPase signaling network that regulates neurite outgrowth (35Pertz O.C. Wang Y. Yang F. Wang W. Gay L.J. Gristenko M.A. Clauss T.R. Anderson D.J. Liu T. Auberry K.J. Camp 2nd, D.G. Smith R.D. Klemke R.L. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 1931-1936Crossref PubMed Scopus (61) Google Scholar). This allowed us to uncover an unexpected signaling complexity, with different Rho GTPase signaling modules operating in time and space to regulate distinct neurite functions including neurite initiation, elongation, pathfinding, and filopodial stabilization. Similarly, the wide kinase activation and signal annotation profiles we observes hint to a high degree of modularity in fine tuning neuritogenesis. On the other hand, the precise functions of the protein kinases that modulate LPA-induced neurite collapse were less clear from our phosphoproteomic datasets. However, one of these kinases, serum- and glucocorticoid-inducible kinase 1, regulates microtubule depolymerization and the shortening of neuronal processes (96Yang Y.C. Lin C.H. Lee E.H. Mol. Cell. Biol. 2006; 26: 8357-8370Crossref PubMed Scopus (41) Google Scholar). It will be important in the future to systematically probe these different kinase-substrate networks to reveal their specialized contribution to the dynamic processes of neurite growth and retraction.Our combined informatics and functional studies revealed that integrin to MEK/ERK signaling plays a prominent role in controlling neuritogenesis. Based on our findings and the work of others, we have constructed a working model for how MEK/ERK activation and deactivation by LPA control neuritogenesis (Fig. 6C). The signaling components that were used to construct the schematic and their relative abundance in the soma and neurite as well as their observed phosphorylation sites are listed in Fig. 6C and supplemental Table 3. Fig. 6C panel a shows the proposed pathway for how integrins mediate ERK activation leading to neurite extension. ECM proteins like laminin and fibronectin are present as adhesive gradients in the extracellular environment that can initiate and guide neurite protrusion from the cell body through activation of integrin receptors and the recruitment of their downstream effectors including FAK/CAS/Crk (8Dent E.W. Gertler F.B. Neuron. 2003; 40: 209-227Abstract Full Text Full Text PDF PubMed Scopus (668) Google Scholar, 83Chodniewicz D. Klemke R.L. Exp. Cell Res. 2004; 301: 31-37Crossref PubMed Scopus (53) Google Scholar, 84Chodniewicz D. Klemke R.L. Biochim. Biophys. Acta. 2004; 1692: 63-76Crossref PubMed Scopus (143) Google Scholar). This canonical pathway operates spatially in the neurite to drive localized Rac activation of PAK. PAK then phosphorylates MEK Ser-298, leading to enhanced MEK and ERK activity as described in non-neuronal cells (31Park E.R. Eblen S.T. Catling A.D. Cell. Signal. 2007; 19: 1488-1496Crossref PubMed Scopus (60) Google Scholar, 32Eblen S.T. Slack-Davis J.K. Tarcsafalvi A. Parsons J.T. Weber M.J. Catling A.D. Mol. Cell. Biol. 2004; 24: 2308-2317Crossref PubMed Scopus (105) Google Scholar, 33Slack-Davis J.K. Eblen S.T. Zecevic M. Boerner S.A. Tarcsafalvi A. Diaz H.B. Marshall M.S. Weber M.J. Parsons J.T. Catling A.D. J. Cell Biol. 2003; 162: 281-291Crossref PubMed Scopus (196) Google Scholar, 34Eblen S.T. Slack J.K. Weber M.J. Catling A.D. Mol. Cell. Biol. 2002; 22: 6023-6033Crossref PubMed Scopus (190) Google Scholar). The integrin/Rac/PAK/MEK-Ser-298 signaling cascade promotes strong sustained ERK activation, which serves as a switch to mediate neurite outgrowth while suppressing basal RhoA/ROCK/MLC-mediated membrane contractility, possibly through PKA activation (panel b).Although the spatial mechanisms that promote localized ERK activation in the neurite are not clear, the integrin/Rac/PAK activation pathway most likely works in close conjunction with common upstream activators of MEK including Rap-1, Ras, and Raf (22Yee K.L. Weaver V.M. Hammer D.A. IET Syst. Biol. 2008; 2: 8-15Crossref PubMed Scopus (80) Google Scholar, 97Pouysségur J. Volmat V. Lenormand P. Biochem. Pharmacol. 2002; 64: 755-763Crossref PubMed Scopus (346) Google Scholar, 98Howe A.K. Aplin A.E. Juliano R.L. Curr. Opin. Genet. Dev. 2002; 12: 30-35Crossref PubMed Scopus (238) Google Scholar, 99McLeod S.J. Shum A.J. Lee R.L. Takei F. Gold M.R. J. Biol. 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It is notable that although MEK activation may occur through either B-Raf and/or Raf-1 activation, our phosphoproteomics work suggests that B-Raf is more likely involved as Raf-1 is highly phosphorylated on Ser-43 (growth specific) in extending neurites (supplemental Table 1). Ser-43 phosphorylation has previously been associated with PKA activation and is inhibitory to Raf-1 kinase activity (105Gerits N. Kostenko S. Shiryaev A. Johannessen M. Moens U. Cell. Signal. 2008; 20: 1592-1607Crossref PubMed Scopus (123) Google Scholar, 106Dhillon A.S. Meikle S. Peyssonnaux C. Grindlay J. Kaiser C. Steen H. Shaw P.E. Mischak H. Eychène A. Kolch W. Mol. Cell. Biol. 2003; 23: 1983-1993Crossref PubMed Scopus (41) Google Scholar). How then is B-Raf activity modulated to increase MEK/ERK signaling? Integrin activation of Rap-1 can modulate B-Raf activity, as can cAMP/PKA signaling (102Retta S.F. Balzac F. Avolio M. Eur J. Cell Biol. 2006; 85: 283-293Crossref PubMed Scopus (61) Google Scholar). Rap1A and -B isoforms are present in the neurite, and several Rap1 GEF (guanine nucleotide exchange factor) and GAP (GTPase-activating protein) effector proteins are differentially phosphorylated during neurite growth and retraction (supplemental Table 1). Rap activity and its effectors could be modulated directly or indirectly by integrin activation and/or cell spreading on laminin, possibly through PKA activation (102Retta S.F. Balzac F. Avolio M. Eur J. Cell Biol. 2006; 85: 283-293Crossref PubMed Scopus (61) Google Scholar). Alternatively, integrin activation of FAK/Src can facilitate activation of the canonical Grb2 (growth factor rector-bound protein 2)/SOS (son of sevenless)/Ras/Raf/MEK/ERK pathway (22Yee K.L. Weaver V.M. Hammer D.A. IET Syst. Biol. 2008; 2: 8-15Crossref PubMed Scopus (80) Google Scholar). In the end, ERK activity self-regulates by modulation of its upstream activators through negative feedback phosphorylation of MEK-S292, which suppresses PAK phosphorylation of 298 and phosphorylation of B-Raf-Ser-151, which inhibits B-Raf activity (102Retta S.F. Balzac F. Avolio M. Eur J. Cell Biol. 2006; 85: 283-293Crossref PubMed Scopus (61) Google Scholar). Fig. 6C panel c shows a list of cytoskeletal effector proteins that are either known and/or predicted to be phosphorylated by ERK and, thus, may modulate neurite growth. Of particular interest are α-parvin, which modulates focal adhesion and actin cytoskeletal dynamics and the microtubule modulating proteins Crmp-1, MARK2 (microtubule-associated regulatory kinase 2), MAP1B, and MAP2 (10Dehmelt L. Halpain S. Curr. Biol. 2007; 17: R611-R614Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 107Halpain S. Dehmelt L. Genome Biol. 2006; 7: 224Crossref PubMed Scopus (173) Google Scholar, 108Arimura N. Ménager C. 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Cell Biol. 2002; 34: 731-745Crossref PubMed Scopus (117) Google Scholar). Interestingly, the RhoA/ROCK/MLC pathway has been reported to mediate neurite collapse by modulating Crmp family proteins and microtubule dynamics (5Arimura N. Kaibuchi K. Neuron. 2005; 48: 881-884Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 108Arimura N. Ménager C. Kawano Y. Yoshimura T. Kawabata S. Hattori A. Fukata Y. Amano M. Goshima Y. Inagaki M. Morone N. Usukura J. Kaibuchi K. Mol. Cell. Biol. 2005; 25: 9973-9984Crossref PubMed Scopus (197) Google Scholar, 109Inagaki N. Chihara K. Arimura N. Ménager C. Kawano Y. Matsuo N. Nishimura T. Amano M. Kaibuchi K. Nat. Neurosci. 2001; 4: 781-782Crossref PubMed Scopus (428) Google Scholar, 110Arimura N. Inagaki N. Chihara K. Ménager C. Nakamura N. Amano M. Iwamatsu A. Goshima Y. Kaibuchi K. J. Biol. Chem. 2000; 275: 23973-23980Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar). In our work here, Crmp-1 was observed to be enriched by 7-fold in the neurite and to be differentially phosphorylated on multiple sites in neurites undergoing growth or retraction (supplemental Table 1). Although the mechanisms that block LPA-induced integrin signaling in the neurite are not defined, Abl family kinases (Abl and Arg) are likely to play a central role through their ability to modulate CAS/Crk coupling and Rac activity at adhesion sites as described (8Dent E.W. Gertler F.B. Neuron. 2003; 40: 209-227Abstract Full Text Full Text PDF PubMed Scopus (668) Google Scholar, 83Chodniewicz D. Klemke R.L. Exp. Cell Res. 2004; 301: 31-37Crossref PubMed Scopus (53) Google Scholar, 84Chodniewicz D. Klemke R.L. Biochim. Biophys. Acta. 2004; 1692: 63-76Crossref PubMed Scopus (143) Google Scholar, 85Kain K.H. Klemke R.L. J. Biol. Chem. 2001; 276: 16185-16192Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 111Lanier L.M. Gertler F.B. Curr. Opin. 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It is also notable that LPA did not inhibit MEK-Ser-298 phosphorylation by PAK in cells expressing constitutively activated MEK, which was associated with decreased neurite retraction (Fig. 5, A and D). This suggests that MEK/ERK signaling provides a positive feedback to maintain integrin/CAS/Crk/Rac/PAK signaling and, thus, prevents neurite retraction.Taken together our work and the work of others suggest a model whereby the net effect of high Rac/PAK/MEK/ERK activation and RhoA inhibition is increased membrane protrusion and decreased neurite contraction events leading to net neurite growth. Increased Rac activity has been shown to suppress RhoA signaling, which leads to neurite growth in neurons (7Nikolic M. Int. J. Biochem. Cell Biol. 2002; 34: 731-745Crossref PubMed Scopus (117) Google Scholar, 114Ehler E. van Leeuwen F. Collard J.G. Salinas P.C. Mol. Cell. Neurosci. 1997; 9: 1-12Crossref PubMed Scopus (75) Google Scholar). 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Our findings provide a plausible mechanism for how neuronal growth cones spatially interpret extracellular cues and navigate through complex tissues, which is critical for proper brain development and spinal cord regeneration. IntroductionNeuritogenesis is a dynamic process involving the extension of long, thin protrusions called neurites that will subsequently differentiate into long axons or an elaborate dendritic arbor (1Giannone G. Mège R.M. Thoumine O. Trends Cell Biol. 2009; 19: 475-486Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 2Drees F. Gertler F.B. Curr. Opin. Neurobiol. 2008; 18: 53-59Crossref PubMed Scopus (81) Google Scholar, 3Koh C.G. Neurosignals. 2006; 15: 228-237Crossref PubMed Scopus (76) Google Scholar, 4Allen J. Chilton J.K. Dev. Biol. 2009; 327: 4-11Crossref PubMed Scopus (21) Google Scholar). 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This is further enriched by computational models that predict higher order contextual features of kinases such as protein scaffolding, subcellular localization, and expression patterns (13Jørgensen C. Sherman A. Chen G.I. Pasculescu A. Poliakov A. Hsiung M. Larsen B. Wilkinson D.G. Linding R. Pawson T. Science. 2009; 326: 1502-1509Crossref PubMed Scopus (176) Google Scholar, 25Linding R. Jensen L.J. Ostheimer G.J. van Vugt M.A. Jørgensen C. Miron I.M. Diella F. Colwill K. Taylor L. Elder K. Metalnikov P. Nguyen V. Pasculescu A. Jin J. Park J.G. Samson L.D. Woodgett J.R. Russell R.B. Bork P. Yaffe M.B. Pawson T. Cell. 2007; 129: 1415-1426Abstract Full Text Full Text PDF PubMed Scopus (553) Google Scholar, 26Jørgensen C. Linding R. Curr. Opin. Genet. Dev. 2010; 20: 15-22Crossref PubMed Scopus (0) Google Scholar, 27Tan C.S. Linding R. Proteomics. 2009; 9: 5233-5242Crossref PubMed Scopus (0) Google Scholar, 28Linding R. Jensen L.J. Pasculescu A. Olhovsky M. Colwill K. Bork P. Yaffe M.B. Pawson T. Nucleic Acids Res. 2008; 36: D695-D699Crossref PubMed Scopus (0) Google Scholar). When combined, in silico programs have been shown to predict kinase activity with significant accuracy. This approach has been applied to large phosphoproteomic data sets consisting of thousands of phosphorylation sites revealing important kinase signaling networks that modulate cell migration, DNA damage, and yeast biology (13Jørgensen C. Sherman A. Chen G.I. Pasculescu A. Poliakov A. Hsiung M. Larsen B. Wilkinson D.G. Linding R. Pawson T. Science. 2009; 326: 1502-1509Crossref PubMed Scopus (176) Google Scholar, 25Linding R. Jensen L.J. Ostheimer G.J. van Vugt M.A. Jørgensen C. Miron I.M. Diella F. Colwill K. Taylor L. Elder K. Metalnikov P. Nguyen V. Pasculescu A. Jin J. Park J.G. Samson L.D. Woodgett J.R. Russell R.B. Bork P. Yaffe M.B. Pawson T. Cell. 2007; 129: 1415-1426Abstract Full Text Full Text PDF PubMed Scopus (553) Google Scholar, 29Van Hoof D. Muñoz J. Braam S.R. Pinkse M.W. Linding R. Heck A.J. Mummery C.L. Krijgsveld J. Cell Stem Cell. 2009; 5: 214-226Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar, 30van Vugt M.A. Gardino A.K. Linding R. Ostheimer G.J. Reinhardt H.C. Ong S.E. Tan C.S. Miao H. Keezer S.M. Li J. Pawson T. Lewis T.A. Carr S.A. Smerdon S.J. Brummelkamp T.R. Yaffe M.B. PLoS Biol. 2010; 8: e1000287Crossref PubMed Scopus (145) Google Scholar).Although significant progress has been made in identifying specific signals regulating neuritogenesis, a system-wide analysis of the phosphoprotein signals that control this process has not been investigated. Consequently, our understanding of the complex kinase/substrate signaling networks that regulate neurite formation and guidance is not defined. Here we describe a microporous filter method that facilitates selective purification of neurites undergoing growth or retraction. This fractionation method combined with immobilized metal-ion affinity chromatography and LC-MS protein identification technologies facilitated the identification of thousands of protein phosphorylation signatures that mediate neurite extension or retraction events induced by growth promoting or collapsing stimuli, respectively. Functional annotation and kinase activity predications in silico revealed phosphoprotein networks controlling cytoskeletal remodeling, axon/dendrite specification, and cell adhesion. Kinase signaling analysis and cell-based assays revealed that an integrin/MEK/ERK signaling pathway operates as a critical switch controlling neurite extension and retraction dynamics." @default.
W2023488851 created "2016-06-24" @default.
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W2023488851 date "2011-05-01" @default.
W2023488851 modified "2023-10-18" @default.
W2023488851 title "Spatial Phosphoprotein Profiling Reveals a Compartmentalized Extracellular Signal-regulated Kinase Switch Governing Neurite Growth and Retraction" @default.
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