- Micro report
- Open Access
Evolutionary analysis of proline-directed phosphorylation sites in the mammalian growth cone identified using phosphoproteomics
Molecular Brain volume 12, Article number: 53 (2019)
The growth cone is essential for nerve growth and axon regeneration, which directly form and rearrange the neural network. Recently, to clarify the molecular signaling pathways in the growth cone that utilize protein phosphorylation, we performed a phosphoproteomics study of mammalian growth cone membranes derived from the developing rodent brain and identified > 30,000 phosphopeptides from ~ 1200 proteins. We found that the phosphorylation sites were highly proline directed and primarily mitogen-activated protein kinase (MAPK) dependent, due to particular activation of c-jun N-terminal protein kinase (JNK), a member of the MAPK family. Because the MAPK/JNK pathway is also involved in axon regeneration of invertebrate model organisms such Caenorhabditis elegans and Drosophila, we performed evolutionary bioinformatics analysis of the mammalian growth cone phosphorylation sites. Although these sites were generally conserved within vertebrates, they were not necessarily conserved in these invertebrate model organisms. In particular, high-frequency phosphorylation sites (> 20 times) were less conserved than low-frequency sites. Taken together, the mammalian growth cones contain a large number of vertebrate-specific phosphorylation sites and stronger dependence upon MAPK/JNK than C. elegans or Drosophila. We conclude that axon growth/regeneration likely involves many vertebrate-specific phosphorylation sites.
The growth cone, a highly motile structure at the tip of extending axons in developing or regenerating neurons , is crucial for accurate synaptogenesis. Therefore, elucidating the molecular pathways for growth cone behavior is essential. At present, however, sufficient molecular information is not available regarding growth cones in the mammalian brain. We performed a proteomics analysis of mammalian growth cones and characterized approximately 1000 unique proteins ; the results revealed novel molecular mechanisms underlying nerve growth .
To further investigate molecular signaling in growth cones, we focused on protein phosphorylation, the most important regulatory mechanism in many cellular processes . Phosphoproteomics is a powerful technique for comprehensive and quantitative identification of in vivo phosphorylation sites . Thus, we performed phosphoproteomics analysis of the growth cone membrane (GCM). From among more than 30,000 phosphopeptides, this analysis identified ~ 4600 different phosphorylation sites from ~ 1200 proteins . Surprisingly, proline (P)-directed phosphorylation  was predominant, with more than 60% of serine (S) or threonine (T) phosphorylation sites predicted to depend on P-directed kinases . Bioinformatics analysis suggested that these frequent P-directed phosphorylation events were due to mitogen-activated protein kinase (MAPK) activation. In particular, we found that c-Jun N-terminal kinase (JNK)  was the major active member of the MAPK family and was responsible for several heavily phosphorylated sites .
The MAPK family includes extracellular signal–regulated kinase, p38, and JNK, among which JNK appeared to be the most likely kinase candidate for mammalian GCM phosphorylation. First, several recent reports showed that JNK is involved in multiple steps of mammalian brain development [7,8,9,10,11]. Second, JNK signaling is activated during axon regeneration, even in Caenorhabditis elegans . Together, these observations suggest the importance of JNK signaling in a wide range of organisms.
Thus, to understand and characterize MAPK signaling in the GCM, we used bioinformatics to examine whether the phosphorylation sites of the mammalian GCM proteins that were identified using phosphoproteomics were conserved within a wide range of animals. If so, the signaling pathways involving those phosphosites were expected to be widely conserved from mammals to nematodes or insects. We first made an evolutionary comparison between vertebrates and invertebrates such as C. elegans and Drosophila, using comparative genomics data in “Ensembl” . Surprisingly, we found that MAPK-dependent substrates with very frequently phosphorylated sites (detected ≥20 times) were conserved in vertebrates, but were less abundant in invertebrates; more than 70% of the very frequent sites were vertebrate specific (Fig. 1a; also see Additional file 1: Figure S1), suggesting the importance of JNK signaling in a wide range of animals. In addition, we classified these MAPK-dependent phosphoprotein-coding genes using KinasePhos 2.0 , a kinase prediction site, into three groups. We found that the vertebrate-specific phosphoproteins had more high-frequency sites compared to the evolutionarily older ones (Fig. 1b). Namely, highly MAPK-dependent sites were conserved within vertebrates, as were the genes encoding these sites, which newly emerged in vertebrates (Fig. 1b). Taken together, the data revealed that the substrates of MAPK signaling in rodent GCM included many vertebrate-specific proteins and vertebrate-specific phosphorylation sites, suggesting that axonal growth may be controlled by considerably distinct signaling pathways in vertebrates and invertebrates.
In the case of downstream genes of DLK-JNK signaling in C. elegans axon regeneration , few P-directed substrates were identified in our phosphoproteomics study , suggesting that different molecular mechanisms involving JNK play a role in mammalian axon growth/regeneration compared to C. elegans, although JNK is activated in neurons of both organisms. We conclude that the molecular signaling in mammalian growth cones for axon growth/regeneration may more frequently use evolutionarily newer phosphoproteins or phosphorylated sites that depend on MAPK/JNK, in addition to older ones that are also present in invertebrate phosphoproteins. These newly identified phosphorylated sites may have allowed more sophisticated signaling pathways that are more suitable for neural network formation in vertebrate brain, where the neuronal number is much larger than in invertebrates.
Growth cone membrane
c-jun N-terminal protein kinase
Mitogen-activated protein kinase
Igarashi M. Proteomic identification of the molecular basis of mammalian CNS growth cones. Neurosci Res. 2014;88C:1–15.
Nozumi M, Togano T, Takahashi-Niki K, Lu J, Honda A, Taoka M, Shinkawa T, Koga H, Takeuchi K, Isobe T, Igarashi M. Identification of functional marker proteins in the mammalian growth cone. Proc Natl Acad Sci U S A. 2009;106:17211–6.
Needham EJ, Parker BL, Burykin T, James DE, Humphrey SJ. Illuminating the dark phosphoproteome. Sci Signal. 2019;12:eaau8645.
Kawasaki A, Okada M, Tamada A, Okuda S, Nozumi M, Ito Y, Kobayashi D, Yamasaki T, Yokoyama R, Shibata T, Nishina H, Yoshida Y, Fujii Y, Takeuchi K, Igarashi M. Growth cone Phosphoproteomics reveals that GAP-43 phosphorylated by JNK is a marker of axon growth and regeneration. iScience. 2018;4:190–203.
Villén J, Beausoleil SA, Gerber SA, Gygi SP. Large-scale phosphorylation analysis of mouse liver. Proc Natl Acad Sci U S A. 2007;104:1488–93.
Bogoyevitch MA, Ngoei KR, Zhao TT, Yeap Y, Ng DC. C-Jun N-terminal kinase (JNK) signaling: recent advances and challenges. Biochim Biophys Acta. 2010;1804:463–75.
Oliva AA Jr, Atkins CM, Copenagle L, Banker GA. Activated c-Jun N-terminal kinase is required for axon formation. J Neurosci. 2006;26:9462–70.
Barnat M, Enslen H, Propst F, Davis RJ, Soares S, Nothias F. Distinct roles of c-Jun N-terminal kinase isoforms in neurite initiation and elongation during axonal regeneration. J Neurosci. 2010;30:7804–16.
Hirai S, Banba Y, Satake T, Ohno S. Axon formation in neocortical neurons depends on stage-specific regulation of microtubule stability by the dual leucine zipper kinase-c-Jun N-terminal kinase pathway. J Neurosci. 2011;31:6468–80.
Yamasaki T, Kawasaki H, Arakawa S, Shimizu K, Shimizu S, Reiner O, Okano H, Nishina S, Azuma N, Penninger JM, Katada T, Nishina H. Stress-activated protein kinase MKK7 regulates axon elongation in the developing cerebral cortex. J Neurosci. 2011;31:16872–83.
Coffey ET. Nuclear and cytosolic JNK signalling in neurons. Nat Rev Neurosci. 2014;15:285–99.
Chen L, Wang Z, Ghosh-Roy A, Hubert T, Yan D, O'Rourke S, Bowerman B, Wu Z, Jin Y, Chisholm AD. Axon regeneration pathways identified by systematic genetic screening in C. elegans. Neuron. 2011;71:1043–57.
Cunningham F, Amode MR, Barrell D, Beal K, Billis K, Brent S, Carvalho-Silva D, Clapham P, Coates G, Fitzgerald S, Gil L, Girón CG, Gordon L, Hourlier T, Hunt SE, Janacek SH, Johnson N, Juettemann T, Kähäri AK, Keenan S, Martin FJ, Maurel T, McLaren W, Murphy DN, Nag R, Overduin B, Parker A, Patricio M, Perry E, Pignatelli M, Riat HS, Sheppard D, Taylor K, Thormann A, Vullo A, Wilder SP, Zadissa A, Aken BL, Birney E, Harrow J, Kinsella R, Muffato M, Ruffier M, Searle SM, Spudich G, Trevanion SJ, Yates A, Zerbino DR, Flicek P. Ensembl 2015. Nucleic Acids Res. 2015;43(Database issue):D662–9.
Wong YH, Lee TY, Liang HK, Huang CM, Yang YH, Chu CH, Huang HD, Ko MT, Hwang JK. KinasePhos 2.0: a web server for identifying protein kinase-specific phosphorylation sites based on sequences and coupling patterns. Nucleic Acids Res. 2007;35:W588–94.
Nix P, Hisamoto N, Matsumoto K, Bastiani M. Axon regeneration requires coordinate activation of p38 and JNK MAPK pathways. Proc Natl Acad Sci U S A. 2011;108:10738–43.
We thank Asami Kawasaki, PhD for help with figures.
This work was supported in part by KAKENHI from JSPS and MEXT of Japan (#221S0003, #22240040, #24111515, #18H04670, and #18H04013 to M.I.; and #18H04123 to S.O.), and Uehara Memorial Foundation for Life Sciences (to M.I.).
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.