CRMP Family Protein: Novel Targets for Cdk5 That Regulates Axon Guidance, Synapse Maturation, and Cell Migration

  • Yoshio Goshima
  • Yukio Sasaki
  • Yutaka Uchida
  • Naoya Yamashita
  • Fumio Nakamura


In the developing nervous system, post-mitotic neurons migrate and extend their neurites and form precise patterns of connections that emerge through the interaction between the growth cone and a myriad of environmental cues such as attractive or repulsive axon guidance molecules. Semaphorin3A (Sema3A) is the prototypical repulsive axon guidance molecule that potently induces growth cone collapse stalling neurite extension. Neuropilin-1 (NRP-1) and Plexin-As are ligand-binding and signal-transducing receptor components for Sema3A, respectively. Collapsin response mediator protein (CRMP) was identified as a signaling molecule of Sema3A. However, its molecular mechanisms have been ill-defined. CRMPs are now known to be composed of five homologous cytosolic proteins CRMP1–5; all of the family proteins are highly phosphorylated in developing brains. By screening pharmaceutical reagents and utilizing gene-deficient mice and through biochemical analysis, we found that Fyn and cyclin-dependent kinase 5 (Cdk5) mediate Sema3A-induced response in dorsal root ganglion (DRG) neurons. Cdk5 was associated with PlexA2 through the active state of Fyn. This raised the possibility that Sema3A induced growth cone collapse response through phosphorylation of CRMPs by Cdk5. The 2-D gel analysis of brain lysate from Cdk5-deficient mice revealed that CRMP2 was a substrate for Cdk5 in vivo. In vitro kinase assay revealed that Ser522 was the major site of CRMP1 and CRMP2 phosphorylation by Cdk5. Cdk5 primarily phosphorylated CRMP2 at Ser522, and GSK3β secondarily phosphorylates at Thr509. The dual-phosphorylated CRMP2 was recognized by the antibody 3F4, which is highly reactive with the neurofibrillary tangles of Alzheimer’s disease. In DRG neurons, Sema3A stimulation enhanced the levels of the phosphorylated form of CRMP2 detected by 3F4. Overexpression of CRMP2 mutant substituting either Ser522 or Thr509 with Ala attenuated Sema3A-induced growth cone collapse. Knockdown of CRMP1 and CRMP2 inhibited Sema3A-induced growth cone collapse. The phosphorylation of CRMP1 and/or CRMP2 is therefore an essential step for Sema3A signaling. CRMP1 and CRMP2 were also good substrates for Fyn. The phosphorylation of CRMP1 by Cdk5 and Fyn also appears to be involved in Sema3A and Reelin signaling, contributing to spine maturation and the regulation of cell migration during the development of the cerebral cortex.


Dorsal Root Ganglion Dorsal Root Ganglion Neuron Growth Cone Collapsin Response Mediator Protein Retrograde Axonal Transport 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



I am grateful to many colleagues who have participated in developing these ideas, especially my Yokohama collaborators, T. Nakayama, K. Takei, and other laboratory members for instructive inputs. I am grateful also to T. Ohshima for our fruitful collaboration. This work was supported by Grants-in-aid for Scientific Research in a Priority Area and The Yokohama City University Center of Excellence Program form the Ministry of Education, Science, Sports and Culture, Yokohama Medical Foundation, CREST (Core Research for Evolutional Science and Technology) of JST (Japan Science and Technology Corporation), Uehara Memorial Foundation.


  1. Arimura N, Inagaki N, Chihara K, Menager C, Nakamura N, Amano M, Iwamatsu A, Goshima Y, Kaibuchi K (2000) Phosphorylation of collapsin response mediator protein-2 by Rho-kinase: evidence for two separate signaling pathways for growth cone collapse. J Biol Chem 275:23973–23980.PubMedCrossRefGoogle Scholar
  2. Arimura N, Kaibuchi K (2005) Key regulators in neuronal polarity. Neuron 48:881–884.PubMedCrossRefGoogle Scholar
  3. Byk T, Dobransky T, Cifuentes-Diaz C, Sobel A (1996) Identification and molecular characterization of Unc-33-like phosphoprotein (Ulip), a putative mammalian homolog of the axonal guidance-associated unc-33 gene product. J Neurosci 16:688–701.PubMedGoogle Scholar
  4. Chae T, Kwon YT, Bronson R, Dikkes P, Li E, Tsai LH (1997) Mice lacking p35, a neuronal specific activator of Cdk5, display cortical lamination defects, seizures, and adult lethality. Neuron 18:29–42.PubMedCrossRefGoogle Scholar
  5. Charrier E, Mosinger Jr B, Meissirel C, Aguera M, Rogemond V, Reibel S, Salin P, Chounlamountri N, Perrot V, Belin M, Goshima Y, Honnorat J, Thomasset N, Kolattukudy P (2006) Transient Alterations in granule cell proliferation, apoptosis and migration in post-natal developing cerebellum of CRMP1–/– mice. Genes Cells 10:165–179.Google Scholar
  6. Cheng Q, Sasaki Y, Shoji M, Sugiyama Y, Tanaka H, Nakayama T, Mizuki N, Nakamura F, Takei K, Goshima Y (2003) Cdk5/p35 and Rho-kinase mediate ephrin-A5-induced signaling in retinal ganglion cells. Mol Cell Neurosci 24:632–645.PubMedCrossRefGoogle Scholar
  7. Cho JH, Johnson GV (2003) Glycogen synthase kinase 3beta phosphorylates tau at both primed and unprimed sites: differential impact on microtubule binding. J Biol Chem 278:187–193.PubMedCrossRefGoogle Scholar
  8. Fournier AE, Nakamura F, Kawamoto S, Goshima Y, Kalb RG, Strittmatter SM (2000) Semaphorin3A enhances endocytosis at sites of receptor-F-actin colocalization during growth cone collapse. J Cell Biol 149:411–422.PubMedCrossRefGoogle Scholar
  9. Fukata Y, Itoh TJ, Kimura T, Menager C, Nishimura T, Shiromizu T, Watanabe H, Inagaki N, Iwamatsu A, Hotani H, Kaibuchi K (2002) CRMP-2 binds to tubulin heterodimers to promote microtubule assembly. Nat Cell Biol 4:583–591.PubMedGoogle Scholar
  10. Goshima Y, Nakamura F, Strittmatter P, Strittmatter SM (1995) Collapsin-induced growth cone collapse mediated by an intracellular protein related to UNC-33. Nature 376:509–514.PubMedCrossRefGoogle Scholar
  11. Goshima Y, Kawakami T, Hori H, Sugiyama Y, Takasawa S, Hashimoto Y, Kagoshima-Maezono M, Takenaka T, Misu Y, Strittmatter SM (1997) A novel action of collapsin: collapsin-1 increases antero- and retrograde axoplasmic transport independently of growth cone collapse. J Neurobiol 33:316–328.PubMedCrossRefGoogle Scholar
  12. Goshima Y, Hori H, Sasaki Y, Yang T, Kagoshima-Maezono M, Li C, Takenaka T, Nakamura F, Takahashi T, Strittmatter SM, Misu Y, Kawakami T (1999) Growth cone neuropilin-1 mediates collapsin-1/Sema III facilitation of antero- and retrograde axoplasmic transport. J Neurobiol 39:579–589.PubMedCrossRefGoogle Scholar
  13. Grimes CA, Jope RS (2001) The multifaceted roles of glycogen synthase kinase 3beta in cellular signaling. Prog Neurobiol 65:391–426.PubMedCrossRefGoogle Scholar
  14. Gu Y, Hamajima N, Ihara Y (2000) Neurofibrillary tangle-associated collapsin response mediator protein-2 (CRMP-2) is highly phosphorylated on Thr-509, Ser-518, and Ser-522. Biochemistry 39:426–4275.Google Scholar
  15. Helmke S, Pfenninger KH (1995) Growth cone enrichment and cytoskeletal association of non-receptor tyrosine kinases. Cell Motil Cytoskel 30:194–207.CrossRefGoogle Scholar
  16. Hosoi T, Uchiyama M, Okumura E, Saito T, Ishiguro K, Uchida T, Okuyama A, Kishimoto T, Hisanaga S (1995) Evidence for cdk5 as a major activity phosphorylating tau protein in porcine brain extract. J Biochem (Tokyo) 117:741–749.Google Scholar
  17. Huang EJ, Reichardt LF (2001) Neurotrophins: roles in neuronal development and function. Annu Rev Neurosci 24:677–736.PubMedCrossRefGoogle Scholar
  18. Kolodkin AL (1998) Semaphorin-mediated neuronal growth cone guidance. Prog Brain Res 117:115–132.PubMedCrossRefGoogle Scholar
  19. Lew J, Wang JH (1995) Neuronal cdc2-like kinase. Trends Biochem Sci 20:33–37.PubMedCrossRefGoogle Scholar
  20. Li C, Sasaki Y, Takei K, Yamamoto H, Shouji M, Sugiyama Y, Kawakami T, Nakamura F, Yagi T, Ohshima T, Goshima Y (2004) Correlation between semaphorin3A-induced facilitation of axonal transport and local activation of a translation initiation factor eukaryotic translation initiation factor 4E. J Neurosci 24:6161–6170.PubMedCrossRefGoogle Scholar
  21. Li W, Herman RK, Shaw JE (1992) Analysis of the Caenorhabditis elegans axonal guidance and outgrowth gene unc-33. Genetics 132:675–689.PubMedGoogle Scholar
  22. Liu G, Beggs H, Jurgensen C, Park HT, Tang H, Gorski J, Jones KR, Reichardt LF, Wu J, Rao Y (2004) Netrin requires focal adhesion kinase and Src family kinases for axon outgrowth and attraction. Nat Neurosci 7:1222–1232.PubMedCrossRefGoogle Scholar
  23. Meriane M, Tcherkezian J, Webber CA, Danek EI, Triki I, McFarlane S, Bloch-Gallego E, Lamarche-Vane N (2004) Phosphorylation of DCC by Fyn mediates Netrin-1 signaling in growth cone guidance. J Cell Biol 167:687–698.PubMedCrossRefGoogle Scholar
  24. Morita A, Yamashita N, Sasaki Y, Uchida Y, Nakajima O, Nakamura F, Yagi T, Taniguchi M, Usui H, Katoh-Semba R, Takei K, Goshima Y (2006) Regulation of dendritic branching and spine maturation by semaphorin3A-Fyn signaling. J Neurosci 26:2971–2980.PubMedCrossRefGoogle Scholar
  25. Morse WR, Whitesides III JG, LaMantia AS, Maness PF (1998) p59fyn and pp60c-src modulate axonal guidance in the developing mouse olfactory pathway. J Neurobiol 36:53–63.PubMedCrossRefGoogle Scholar
  26. Nakayama T, Goshima Y, Misu Y, Kato T (1999) Role of cdk5 and tau phosphorylation in heterotrimeric G protein-mediated retinal growth cone collapse. J Neurobiol 41:326–339.PubMedCrossRefGoogle Scholar
  27. Ohshima T, Ward JM, Huh CG, Longenecker G, Veeranna, Pant HC, Brady RO, Martin LJ, Kulkarni AB (1996) Targeted disruption of the cyclin-dependent kinase 5 gene results in abnormal corticogenesis, neuronal pathology and perinatal death. Proc Natl Acad Sci USA 93:11173–11178.Google Scholar
  28. Puschel AW (2007) GTPases in semaphorin signaling. Adv Exp Med Biol 600:12–23.PubMedCrossRefGoogle Scholar
  29. Raper JA (2000) Semaphorins and their receptors in vertebrates and invertebrates. Curr Opin Neurobiol 10:88–94.PubMedCrossRefGoogle Scholar
  30. Rosslenbroich V, Dai L, Franken S, Gehrke M, Junghans U, Gieselmann V, Kappler J (2003) Subcellular localization of collapsin response mediator proteins to lipid rafts. Biochem Biophys Res Commun 305:392–329.PubMedCrossRefGoogle Scholar
  31. Sasaki Y, Cheng C, Uchida Y, Nakajima O, Ohshima T, Yagi T, Taniguchi M, Nakayama T, Kishida R, Kudo Y, Ohno S, Nakamura F, Goshima Y (2002) Fyn and Cdk5 mediate semaphorin-3A signaling, which is involved in regulation of dendrite orientation in cerebral cortex. Neuron 35:907–920.PubMedCrossRefGoogle Scholar
  32. Segal RA (2003) Selectivity in neurotrophin signaling: theme and variations. Annu Rev Neurosci 26:299–330.PubMedCrossRefGoogle Scholar
  33. Songyang Z, Lu KP, Kwon YT, Tsai LH, Filhol O, Cochet C, Brickey DA, Soderling TR, Bartleson C, Graves DJ, DeMaggio AJ, Hoekstra MF, Blenis J, Hunter T, Cantley LC (1996) A structural basis for substrate specificities of protein Ser/Thr kinases: primary sequence preference of casein kinases I and II, NIMA, phosphorylase kinase, calmodulin-dependent kinase II, CDK5, and Erk1. Mol Cell Biol 16:6486–6493.PubMedGoogle Scholar
  34. Takahashi T, Fournier A, Nakamura F, Wang LH, Murakami Y, Kalb RG, Fujisawa H, Strittmatter SM (1999) Plexin-neuropilin-1 complexes form functional semaphorin-3A receptors. Cell 99:59–69.PubMedCrossRefGoogle Scholar
  35. Tamagnone L, Artigiani S, Chen H, He Z, Ming GI, Song H, Chedotal A, Winberg ML, Goodman CS, Poo M, Tessier-Lavigne M, Comoglio PM (1999) Plexins are a large family of receptors for transmembrane, secreted and GPI-anchored semaphorins in vertebrates. Cell 99:71–80.PubMedCrossRefGoogle Scholar
  36. Tanaka E, Sabry J (1995) Making the connection: cytoskeletal rearrangements during growth cone guidance. Cell 83:171–176.PubMedCrossRefGoogle Scholar
  37. Tessier-Lavigne M, Goodman CS (1996) The molecular biology of axon guidance. Science 274:1123–1133.PubMedCrossRefGoogle Scholar
  38. Uchida Y, Ohshima T, Sasaki Y, Suzuki H, Yanai S, Yamashita N, Nakamura F, Takei K, Ihara Y, Mikoshiba K, Kolattukudy P, Honnorat J, Goshima Y (2005) Semaphorin3A signalling is mediated via sequential Cdk5 and GSK3beta phosphorylation of CRMP2: implication of common phosphorylating mechanism underlying axon guidance and Alzheimer's disease. Genes Cells 10:165–179.PubMedCrossRefGoogle Scholar
  39. Wang LH, Strittmatter SM (1996) A family of rat CRMP genes is differentially expressed in the nervous system. J Neurosci 16:6197–6207.PubMedGoogle Scholar
  40. Wang LH, Strittmatter SM (1997) Brain CRMP forms heterotetramers similar to liver dihydropyrimidinase. J Neurochem 69:2261–2269.PubMedCrossRefGoogle Scholar
  41. Yagi T, Aizawa S, Tokunaga T, Shigetani Y, Takeda N, Ikawa Y (1993) A role for Fyn tyrosine kinase in the suckling behaviour of neonatal mice. Nature 366:742–745.PubMedCrossRefGoogle Scholar
  42. Yamashita N, Uchida Y, Ohshima T, Hirai S, Nakamura F, Taniguchi M, Mikoshiba K, Honnorat J, Kolattukudy P, Thomasset N, Takei K, Takahashi T, Goshima Y (2006) Collapsin response mediator protein 1 mediates reelin signaling in cortical neuronal migration. J Neurosci 26:13357–13362.PubMedCrossRefGoogle Scholar
  43. Yamashita N, Morita A, Uchida Y, Nakamura F, Usui H, Ohshima T, Taniguchi M, Honnorat J, Kolattukudy P, Thomasset N, Takei K, Takahashi T, Goshima Y (2007) Regulation of spine development by Semaphorin3A through cyclin-dependent kinase 5 phosphorylation of collapsin response mediator protein 1. J Neurosci 27:12546–12554.Google Scholar
  44. Yoshida H, Watanabe A, Ihara Y (1998) Collapsin response mediator protein-2 is associated with neurofibrillary tangles in Alzheimer's disease. J Biol Chem 273:9761–9768.PubMedCrossRefGoogle Scholar
  45. Yoshimura T, Kawano Y, Arimura N, Kawabata S, Kikuchi A, Kaibuchi K (2005) GSK-3beta regulates phosphorylation of CRMP-2 and neuronal polarity. Cell 120:137–149.PubMedCrossRefGoogle Scholar
  46. Zukerberg LR, Patrick GN, Nikolic M, Humbert S, Wu CL, Lanier LM, Gertler FB, Vidal M, Van Etten RA, Tsai LH (2000) Cables links Cdk5 and c-Abl and facilitates Cdk5 tyrosine phosphorylation, kinase upregulation, and neurite outgrowth. Neuron 26:633–646.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • Yoshio Goshima
    • 1
  • Yukio Sasaki
  • Yutaka Uchida
  • Naoya Yamashita
  • Fumio Nakamura
  1. 1.Department of Molecular Pharmacology & NeurobiologyYokohama City University Graduate School of MedicineJapan

Personalised recommendations