Approaches to Kinesin-1 Phosphorylation

  • Gerardo Morfini
  • Gustavo Pigino
  • Scott T. Brady
Part of the Methods in Molecular Biology™ book series (MIMB, volume 392)


Most mammalian proteins undergo reversible protein modification after or during synthesis. These modifications are associated, for the most part, with changes in protein functionality. Protein phosphorylation is the most common posttranslational modification in mammalian cells, regulating critical cellular processes that include cell division, differentiation, growth, and cell-cell signaling as well as fast axonal transport (FAT). Evidence has accumulated that kinesin-1 phosphorylation plays a key regulatory role in kinesin-based FAT. Multiple kinase and phosphatase activities with the ability to regulate kinesin-1 function and FAT have been identified. Moreover, additional pathways are likely to exist for regulating FAT through reversible phosphorylation/dephosphorylation of specific motor protein subunits. The present chapter describes specific biochemical assays to determine, or to perturb experimentally, the phosphorylation status of kinesin-1. These protocols provide assays for characterization of novel effectors (i.e., trophic factors, neurotransmitters, pharmacological inhibitors, pathogenic protein expression, etc.) that affect the phosphorylation status of kinesin-1. Finally, in vitro phosphorylation assays suitable for analyzing the direct effects of specific kinases on kinesin-1 are provided.

Key Words

Kinesin-1 fast axonal transport phosphorylation immunoprecipitation kinase phosphatase 


  1. 1.
    Brady, S.T. (1993) Axonal dynamics and regeneration. In: Neuroregeneration (Gorio, A., ed.), pp. 7–36. Raven Press, New York.Google Scholar
  2. 2.
    Morfini, G., Pigino, G., Beffert, U., Busciglio, J., and Brady, S.T. (2002) Fast axonal transport misregulation and Alzheimer’s disease. Neuromol. Med. 2(2), 89–99.CrossRefGoogle Scholar
  3. 3.
    Morfini, G., Pigino, G., and Brady, S.T. (2005) Polyglutamine expansion diseases: failing to deliver. Trends Mol. Med. 11, 64–70.CrossRefPubMedGoogle Scholar
  4. 4.
    Brady, S.T. and Sperry, A.O. (1995) Biochemical and functional diversity of microtubule motors in the nervous system. Curr. Opin. Neurobiol. 5, 551–558.CrossRefPubMedGoogle Scholar
  5. 5.
    Vale, R.D. (2003) The molecular motor toolbox for intracellular transport. Cell 112(4), 467–480.CrossRefPubMedGoogle Scholar
  6. 6.
    Cyr, J.L., Pfister, K.K., Bloom, G. S., Slaughter, C.A., and Brady, S.T. (1991) Molecular genetics of kinesin light chains: generation of isoforms by alternative splicing. Proc. Natl. Acad. Sci. USA 88, 10114–10118.CrossRefPubMedGoogle Scholar
  7. 7.
    Hirokawa, N., Pfister, K.K., Yorifuji, H., Wagner, M.C., Brady, S.T., and Bloom, G.S. (1989) Submolecular domains of bovine brain kinesin identified by electron microscopy and monoclonal antibody decoration. Cell 56, 867–878.CrossRefPubMedGoogle Scholar
  8. 8.
    Stenoien, D.S. and Brady, S.T. (1997) Immunochemical analysis of kinesin light chain function. Mol. Biol. Cell 8, 675–689.PubMedGoogle Scholar
  9. 9.
    Morfini, G., Szebenyi, G., Richards, B., and Brady, S.T. (2001) Regulation of kinesin: implications for neuronal development. Dev. Neurosci. 23, 364–376.CrossRefPubMedGoogle Scholar
  10. 10.
    Morfini, G., Pigino, G., Szebenyi, G., Zou, Y., Pollema, S., and Brady, S.T. (2006) JNK mediates pathogenic effects of polyglutamine-expanded androgen receptor on fast axonal transport. Nat. Neurosci. 9(7), 907–916.CrossRefPubMedGoogle Scholar
  11. 11.
    Morfini, G., Szebenyi, G., Brown, H., Pant, H.C., Pigino, G., DeBoer, S., Beffert, U., and Brady, S.T. (2004) A novel CDK5-dependent pathway for regulating GSK3 activity and kinesin-driven motility in neurons. EMBO J. 23, 2235–2245.CrossRefPubMedGoogle Scholar
  12. 12.
    Morfini, G., Szebenyi, G., Elluru, R., Ratner, N., and Brady, S.T. (2002) Glycogen synthase kinase 3 phosphorylates kinesin light chains and negatively regulates kinesin-based motility. EMBO J. 23, 281–293.CrossRefGoogle Scholar
  13. 13.
    Donelan, M.J., Morfini, G., Julyan, R., Sommers, S., Hays, L., Kajio, H., Briaud, I., Easom, R.A., Molkentin, J.D., Brady, S.T., and Rhodes, C.J. (2002) Ca2+-dependent dephosphorylation of kinesin heavy chain on beta-granules in pancreatic beta-cells. Implications for regulated beta-granule transport and insulin exocytosis. J. Biol. Chem. 277(27), 24232–24242.CrossRefPubMedGoogle Scholar
  14. 14.
    Hollenbeck, P.J. (1993) Phosphorylation of neuronal kinesin heavy and light chains in vivo. J. Neurochem. 60, 2265–2275.CrossRefPubMedGoogle Scholar
  15. 15.
    Ratner, N., Bloom, G.S., and Brady, S.T. (1998) A role for Cdk5 kinase in fast anterograde axonal transport: novel effects of olomoucine and the APC tumor suppressor protein. J. Neurosci. 18, 7717–7726.PubMedGoogle Scholar
  16. 16.
    Pigino, G., Morfini, G., Mattson, M.P., Brady, S.T., and Busciglio, J. (2003) Alzheimer’s presenilin 1 mutations impair kinesin-based axonal transport. J. Neurosci. 23, 4499–4508.PubMedGoogle Scholar
  17. 17.
    Szebenyi, G., Morfini, G.A., Babcock, A., Gould, M., Selkoe, K., Stenoien, D.L., Young, M., Faber, P.W., MacDonald, M.E., McPhaul, M.J., and Brady, S.T. (2003) Neuropathogenic forms of huntingtin and androgen receptor inhibit fast axonal transport. Neuron 40, 41–52.CrossRefPubMedGoogle Scholar
  18. 18.
    Elluru, R.G., Bloom, G.S., and Brady, S.T. (1990) Axonal transport of kinesin in the rat optic nerve/tract. J. Cell Biol. 111, 417a.Google Scholar
  19. 19.
    Pfister, K.K., Wagner, M.C., Stenoien, D., Bloom, G.S., and Brady, S.T. (1989) Monoclonal antibodies to kinesin heavy and light chains stain vesicle-like structures, but not microtubules, in cultured cells. J. Cell Biol. 108, 1453–1463.CrossRefPubMedGoogle Scholar
  20. 20.
    Encinas, M., Iglesias, M., Liu, Y., Wang, H., Muhaisen, A., Cena, V., Gallego, C., and Comella, J.X. (2000) Sequential treatment of SH-SY5Y cells with retinoic acid and brain-derived neurotrophic factor gives rise to fully differentiated, neurotrophic factordependent, human neuron-like cells. J. Neurochem. 75(3), 991–1003.CrossRefPubMedGoogle Scholar
  21. 21.
    Simeoni, S., Mancini, M.A., Stenoien, D.L., Marcelli, M., Weigel, N.L., Zanisi, M., Martini, L., and Poletti, A. Motoneuronal cell death is not correlated with aggregate formation of androgen receptors containing an elongated polyglutamine tract. Hum. Mol. Genet. 9(1), 133–144.Google Scholar
  22. 22.
    Trettel, F., Rigamonti, D., Hilditch-Maguire, P., Wheeler, V.C., Sharp, A.H., Persichetti, F., Cattaneo, E., and MacDonald, M.E. (2000) Dominant phenotypes produced by the HD mutation in STHdh(Q111) striatal cells. Hum. Mol. Genet. 9(19), 2799–2809.CrossRefPubMedGoogle Scholar
  23. 23.
    Anantharam, V., Kitazawa, M., Latchoumycandane, C., Kanthasamy, A., and Kanthasamy, A.G. (2004) Blockade of PKC delta proteolytic activation by loss of function mutants rescues mesencephalic dopaminergic neurons from methyl-cyclopentadienyl manganese tricarbonyl (MMT)-induced apoptotic cell death. Ann. N.Y. Acad. Sci. 1035, 271–289.CrossRefPubMedGoogle Scholar
  24. 24.
    Goslin, K., Asmussen, H., and Banker, G. (1998) Rat hippocampal neurons in low density culture. In: Culturing Nerve Cells (Goslin, K. and Banker, G., eds.), pp. 339–370. MIT Press, Cambridge.Google Scholar
  25. 25.
    Avila, D.M., Allman, D.R., Gallo, J.M., and McPhaul, M.J. (2003) Androgen receptors containing expanded polyglutamine tracts exhibit progressive toxicity when stably expressed in the neuroblastoma cell line, SH-SY 5Y. Exp. Biol. Med. (Maywood) 228(8), 982–990.Google Scholar
  26. 26.
    Greene, L.A., Farinelli, S.E., Cunningham, M.E., and Park, D.S. (1998) Culture and experimental use of PC12 rat pheochromocytoma cell line. In: Culturing Nerve Cells (Banker G. and Goslin, K., eds.), pp. 161–188. MIT Press, Cambridge.Google Scholar
  27. 27.
    Miki, H., Setou, M., Kaneshiro, K., and Hirokawa, N. (2001) All kinesin super-family protein, KIF, genes in mouse and human. Proc. Natl. Acad. Sci. USA 98(13), 7004–7011.CrossRefPubMedGoogle Scholar
  28. 28.
    Rahman, A., Friedman, D.S., and Goldstein, L.S. (1998) Two kinesin light chain genes in mice. J. Biol. Chem. 273, 15395–15403.CrossRefPubMedGoogle Scholar
  29. 29.
    Junco, A., Bhullar, B., Tarnasky, H.A., and van der Hoorn, F.A. (2001) Kinesin light-chain KLC3 expression in testis is restricted to spermatids. Biol. Reprod. 64(5), 1320–1330.CrossRefPubMedGoogle Scholar
  30. 30.
    Wagner, M.C., Pfister, K.K., Brady, S.T., and Bloom, G.S. (1991) Purification of kinesin from bovine brain and assay of micro tubule-stimulated ATPase activity. Methods Enzymol. 196, 157–175.CrossRefPubMedGoogle Scholar
  31. 31.
    Tsai, M.-Y., Morfini, G., Szebenyi, G., and Brady, S.T. (2000) Modulation of kinesin-vesicle interactions by Hsc70: implications for regulation of fast axonal transport. Mol. Biol. Cell 11, 2161–2173.PubMedGoogle Scholar
  32. 32.
    Bain, J., McLauchlan, H., Elliott, M., and Cohen, P. (2003) The specificities of protein kinase inhibitors: an update. Biochem. J. 371 (Pt. 1), 199–204.CrossRefPubMedGoogle Scholar
  33. 33.
    Davies, S.P., Reddy, H., Caivano, M., and Cohen, P. (2000) Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem. J. 351 (Pt. 1), 95–105.CrossRefPubMedGoogle Scholar
  34. 34.
    Lindesmith, L., McIlvain, J.M., Jr., Argon, Y., and Sheetz, M.P. (1997) Phosphotransferases associated with the regulation of kinesin motor activity. J. Biol. Chem. 272(36), 22929–22933.CrossRefPubMedGoogle Scholar
  35. 35.
    Sato-Yoshitake, R., Yorifuji, H., Inagaki, M., and Hirokawa, N. (1992) The phosphorylation of kinesin regulates its binding to synaptic vesicles. J. Biol. Chem. 267, 23930–23936.PubMedGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2007

Authors and Affiliations

  • Gerardo Morfini
    • 1
  • Gustavo Pigino
    • 1
  • Scott T. Brady
    • 1
  1. 1.Department of Anatomy and Cell BiologyUniversity of Illinois at ChicagoChicago

Personalised recommendations