Cellular and Molecular Neurobiology

, Volume 26, Issue 2, pp 163–175 | Cite as

Phosphoproteomic Analysis of Neurotrophin Receptor TrkB Signaling Pathways in Mouse Brain

  • Artour Semenov
  • Gundars Goldsteins
  • Eero Castrén


1. The signaling pathways activated by trkB neurotrophin receptor have been studied in detail in cultured neurons, but little is known about the pathways activated by trkB in intact brain. TrkB is a tyrosine kinase and protein phosphorylation is a key regulatory process in the neuronal signal transduction pathways.

2. We have investigated trkB signaling in the transgenic mice overexpressing trkB in postnatal neurons (trkB.TK) using phosphoproteomics.

3. We found that several proteins are overphosphorylated on tyrosine residues in the brain of trkB.TK mice and identified some of these proteins.

4. We demonstrate that the well characterized signaling molecules mitogen-activated protein kinase (MAPK) and cyclic AMP responsive element binding protein (CREB) were phosphorylated at a higher level in the brain of trkB.TK mice when compared to the wild type littermates. Furthermore, we found that β-actin was tyrosine phosphorylated in the brain of the transgenic mice.

5. Our results demonstrate that phosphoproteomics is a sensitive approach to investigate signaling pathways activated in mouse brain.


BDNF phosphorylation MAPK CREB actin proteomics 



Mass spectrometric protein identifications were performed at the Protein Chemistry Research Group and Core Facility, Institute of Biotechnology,University of Helsinki. We would like to thank MSc. Saara Ihalainen, Dr. Nisse Kalkkinen and Dr. Gunilla Rönnholm for their help in the analysis and Dr. Moshe Finel for his help with electrophoresis.


  1. Aigner, L., Arber, S., Kapfhammer, J. P., Laux, T., Schneider, C., Botteri, F., Brenner, H. R., and Caroni, P. (1995). Overexpression of neural growth-associated protein GAP-43 induces nerve sprouting in the adult nervous system of transgenic mice. Cell 83:269–278.CrossRefPubMedGoogle Scholar
  2. Baba, T., Fusaki, N., Shinya, N., Iwamatsu, A., and Hozumi, N. (2003). Actin tyrosine dephosphorylation by the Src homology 1-containing protein tyrosine phosphatase is essential for actin depolymerization after membrane IgM cross-linking. J. Immunol. 170:3762–3768.Google Scholar
  3. Barbacid, M. (1994). The Trk family of neurotrophin receptors. J. Neurobiol. 25:1386–1403.CrossRefPubMedGoogle Scholar
  4. Castrén, E. (2004). Neurotrophic effects of antidepressant drugs. Curr. Opin. Pharmacol. 4:58–64.CrossRefPubMedGoogle Scholar
  5. Finkbeiner, S., Tavazoie, S. F., Maloratsky, A., Jacobs, K. M., Harris, K. M., and Greenberg, M. E. (1997). CREB: a major mediator of neuronal neurotrophin responses. Neuron 19:1031–1047.CrossRefPubMedGoogle Scholar
  6. Gorski, J. A., Balogh, S. A., Wehner, J. M., and Jones, K. R. (2003). Learning deficits in forebrain-restricted brain-derived neurotrophic factor mutant mice. Neuroscience 121:341–354.CrossRefPubMedGoogle Scholar
  7. Howard, P. K., Sefton, B. M., and Firtel, R. A. (1993). Tyrosine phosphorylation of actin in Dictyostelium associated with cell-shape changes. Science 259:241–244.PubMedCrossRefGoogle Scholar
  8. Huang, E. J. and Reichardt, L. F. (2001). Neurotrophins: roles in neuronal development and function. Annu. Rev. Neurosci. 24:677–736.CrossRefPubMedGoogle Scholar
  9. Huang, E. J., and Reichardt, L. F. (2003). Trk receptors: roles in neuronal signal transduction. Annu. Rev. Biochem. 72:609–642.CrossRefPubMedGoogle Scholar
  10. Ingraham, H. A., and Evans, G. A. (1986). Characterization of two atypical promoters and alternate mRNA processing in the mouse Thy-12 glycoprotein gene. Mol. Cell Biol. 6:2923–2931.PubMedGoogle Scholar
  11. Kameyama, K., Kishi, Y., Yoshimura, M., Kanzawa, N., Sameshima, M., and Tsuchiya, T. (2000). Tyrosine phosphorylation in plant bending. Nature 407:37.CrossRefPubMedGoogle Scholar
  12. Kaplan, D. R., and Miller, F. D. (2000). Neurotrophin signal transduction in the nervous system. Curr Opin Neurobiol. 10:381–391.CrossRefPubMedGoogle Scholar
  13. Klein, R., Nanduri, V., Jing, S., Lamballe, F., Tapely, P., Bryant, S., Cordon-Cardo, C., Jones, K. R., Reichardt, L. F., and Barbacid, M. (1991). The TrkB Tyrosine protein kinase is a receptor for brain-derived neurotrophic factor and neurotrophin-3. Cell 66:395–403.CrossRefPubMedGoogle Scholar
  14. Koponen, E., Lakso, M., and Castren, E. (2004a). Overexpression of the full-length neurotrophin receptor TrkB regulates the expression of plasticity-related genes in mouse brain. Brain Res. Mol. Brain Res. 130:81–94.CrossRefPubMedGoogle Scholar
  15. Koponen, E., Voikar, V., Riekki, R., Saarelainen, T., Rauramaa, T., Rauvala, H., Taira, T., and Castrén, E. (2004b). Transgenic mice overexpressing the full-length neurotrophin receptor TrkB exhibit increased activation of the TrkB-PLCgamma pathway, reduced anxiety, and facilitated learning. Mol. Cell Neurosci. 26:166–181.CrossRefPubMedGoogle Scholar
  16. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685.CrossRefPubMedGoogle Scholar
  17. Lim, Y. P., Wong, C. Y., Ooi, L. L., Druker, B. J., and Epstein, R. J. (2004). Selective tyrosine hyperphosphorylation of cytoskeletal and stress proteins in primary human breast cancers: implications for adjuvant use of kinase-inhibitory drugs. Clin. Cancer Res. 10:3980–3987.CrossRefPubMedGoogle Scholar
  18. Linnarsson, S., Bjorklund, A., and Ernfors, P. (1997). Learning deficit in BDNF mutant mice. Eur. J. Neurosci. 9:2581–2587.CrossRefPubMedGoogle Scholar
  19. Lu, B. (2003). BDNF and activity-dependent synaptic modulation. Learn Mem. 10:86–98.CrossRefPubMedGoogle Scholar
  20. Mann, M., Ong, S. E., Gronborg, M., Steen, H., Jensen, O. N., and Pandey, A. (2002). Analysis of protein phosphorylation using mass spectrometry: Deciphering the phosphoproteome. Trends Biotechnol. 20:261–268.CrossRefPubMedGoogle Scholar
  21. Minichiello, L., Calella, A. M., Medina, D. L., Bonhoeffer, T., Klein, R., and Korte, M. (2002). Mechanism of TrkB-mediated hippocampal long-term potentiation. Neuron 36:121–137.CrossRefPubMedGoogle Scholar
  22. Minichiello, L., Casagranda, F., Tatche, R. S., Stucky, C. L., Postigo, A., Lewin, G. R., Davies, A. M., and Klein, R. (1998). Point mutation in TrkB causes loss of NT4-dependent neurons without major effects on diverse BDNF responses. Neuron 21:335–345.CrossRefPubMedGoogle Scholar
  23. Minichiello, L., Korte, M., Wolfer, D., Kuhn, R., Unsicker, K., Cestari, V., Rossi-Arnaud, C., Lipp, H. P., Bonhoeffer, T., and Klein, R. (1999). Essential role for TrkB receptors in hippocampus-mediated learning. Neuron 24:401–414.CrossRefPubMedGoogle Scholar
  24. O’Connell, K. L., and Stults, J. T. (1997). Identification of mouse liver proteins on two-dimensional electrophoresis gels by matrix-assisted laser desorption/ionization mass spectrometry of in situ enzymatic digests. Electrophoresis 18:349–359.CrossRefPubMedGoogle Scholar
  25. Pandey, A., Andersen, J. S., and Mann, M. (2000). Use of mass spectrometry to study signaling pathways. Sci. STKE. 2000:L1.CrossRefGoogle Scholar
  26. Pandey, A., and Mann, M. (2000). Proteomics to study genes and genomes. Nature 405:837–846.CrossRefPubMedGoogle Scholar
  27. Saarelainen, T., Hendolin, P., Lucas, G., Koponen, E., Sairanen, M., MacDonald, E., Agerman, K., Haapasalo, A., Nawa, H., Aloyz, R., Ernfors, P., and Castrén, E. (2003). Activation of the TrkB neurotrophin receptor is induced by antidepressant drugs and is required for antidepressant-induced behavioral effects. J. Neurosci. 23:349–357.PubMedGoogle Scholar
  28. Saarelainen, T., Pussinen, R., Koponen, E., Alhonen, L., Wong, G., Sirviö, J., and Castrén, E. (2000). Transgenic mice overexpressing truncated TrkB neurotrophin receptors in neurons have impaired long-term spatial memory but normal hippocampal LTP. Synapse 38:102–104.CrossRefPubMedGoogle Scholar
  29. Shaywitz, A. J., and Greenberg, M. E. (1999). CREB: a stimulus-induced transcription factor activated by a diverse array of extracellular signals. Annu. Rev. Biochem. 68:821–861.CrossRefPubMedGoogle Scholar
  30. Tolwani, R. J., Buckmaster, P. S., Varma, S., Cosgaya, J. M., Wu, Y., Suri, C., and Shooter, E. M. (2002). BDNF overexpression increases dendrite complexity in hippocampal dentate gyrus. Neuroscience 114:795–805.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, Inc. 2006

Authors and Affiliations

  • Artour Semenov
    • 1
  • Gundars Goldsteins
    • 2
  • Eero Castrén
    • 1
  1. 1.Neuroscience CenterUniversity of HelsinkiHelsinkiFinland
  2. 2.Department of Neurobiology, A.I. Virtanen InstituteUniversity of KuopioKuopioFinland

Personalised recommendations