Skip to main content
SpringerLink
Log in

Calpain-mediated proteolysis of microtubule associated proteins MAP1B and MAP2 in developing brain

  • Original Articles
  • Published:
Neurochemical Research Aims and scope Submit manuscript

Abstract

Microtubule associated proteins MAP1B and MAP2 are important components of the neuronal cytoskeleton. During early development of the brain, MAP1B (340 kDa) is present as two isoforms that differ in their level of phosphorylation, while MAP2 is expressed as a single high molecular weight isoform (MAP2B, 280 kDa) and a low molecular weight form (MAP2C, 70 kDa). In this study we examined and compared the sensitivities of MAP1B and MAP2, obtained from MT preparations and brain homogenates of young rats, to degradation by calcium-activated neutral protease, calpain II. We found that in MAPs prepared from microtubules the two isoforms of MAP1B had comparable sensitivity to calpain-mediated proteolysis. Similarly, the high and low molecular weight forms of MAP2 were equally sensitive to digestion by calpain. However, although both MAPs were very susceptible to calpain-mediated proteolysis, MAP1B was more resistant to degradation by calpain than MAP2. Furthermore, the endogenous degradation of MAPs in neonate brain homogenates was calcium-dependent and inhibited by leupeptin, and the pattern of degradation products for MAP1B and MAP2 was similar to that of calpain-mediated proteolysis. These data suggest that calpain can play a role in the regulation of MAPs levels during brain development, in relation to normal neuronal differentiation and disorders associated with neurodegeneration.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Yamada, K. M., Spooner, B. S. and Wessells, N. K. 1970. Axon growth; roles of microfilaments and microtubules. Proc. Natl. Acad. Sci. USA. 66:1206–1212.

    Google Scholar 

  2. Bernhardt, R., and Matus, A. 1982. Initial phase of dendrite growth: evidence for the involvement of high molecular weight microtubule-associated proteins (HMWP) before the appearance of tubulin. J. Cell Biol. 92:589–593.

    Google Scholar 

  3. Burgoyne, R., and Cumming, R. 1984. Ontogeny of microtubule-associated protein 2 in rat cerebellum: Differential expression of the double polypeptides. Neuroscience 11:156–167.

    Google Scholar 

  4. Caceres, A., Banker, G., Steward, O., Binder, L., and Payne, M. 1984. MAP 2 is localized to the dendrites of hippocampal neurons which develop in culture. Dev. Brain Res. 13:314–318.

    Google Scholar 

  5. De Cammili, P., Miller, P. E., Navone, F., Theurkauf, W. E., and Vallee, R. B. 1984. Distribution of microtubule-associated protein 2 in the nervous system of the rat studied by immunofluorescence. Neuroscience 11:817–46.

    Google Scholar 

  6. Caceres, A., Banker, G. A., and Binder, L. 1986. Immunocytochemical localization of tubulin and microtubule associated protein 2 during the development of hippocampal neurons in culture. J. Neurosci. 6:714–722.

    Google Scholar 

  7. Fischer, I., Shea, T. B., Sapirstein, V. S., and Kosik, K. S. 1986. Expression and distribution of microtubule-associated protein 2 (MAP2) in neuroblastoma and primary neuronal cells. Dev. Brain Res. 25:99–109.

    Google Scholar 

  8. Binder, L. I., Frankfurter, A., Kim, H., Caceres, A., Payne, M. R., and Rebhun, L. I. 1984. Heterogeneity of microtubule-associated protein during rat brain development. Proc. Natl. Acad. Sci. USA 81:5613–17.

    Google Scholar 

  9. Fischer, I., Kosik, K. S., and Sapirstein, V. S. 1987. Heterogeneity of microtubule-associated protein (MAP2) in vertebrate brains. Brain Res. 436:39–48.

    Google Scholar 

  10. Garner, C. C., Brugg, B., and Matus, A. 1988. A 70 kDa microtubule-associated protein (MAP2c), related to MAP2. J. Neurochem. 50:609–615.

    Google Scholar 

  11. Crandall, J. E., and Fischer, I. 1989. Developmental regulation of MAP2 expression in regions of mouse brain. J. Neurochem. 53:1910–1917.

    Google Scholar 

  12. Kuznetsov, S. A., Rodinov, V. I., Gelfand, V. A., and Rosenblat, V. A. 1981. microtubule-associated protein MAP1 promotes microtubule assembly in vitro. FEBS Lett. 135:241–244.

    Google Scholar 

  13. Vallee, R. B., and Davis, S. D. 1983. Low molecular weight microtubule-associated proteins are light chains of microtubule-associated protein 1 (MAP 1). Proc. Natl. Acad. Sci. USA 80:1342–1346.

    Google Scholar 

  14. Bloom, G. C., Luca, F. C., and Vallee, R. B. 1984. Widespread distribution of a major component of MAP 1 (microtubule-associated protein 1) in nervous system. J. Cell Biol. 98:320–330.

    Google Scholar 

  15. Bloom, G. C., Luca, F. C., and Vallee, R. B. 1985. microtubule-associated protein 1B: identification of a major component of the neuronal cytoskeleton. Proc. Natl. Acad. Sci. USA 82:5404–5408.

    Google Scholar 

  16. Herrmann, H., Dalton, J. M., and Wiche, G. 1985. Microheterogeneity of microtubule-associated proteins, MAP 1 and MAP 2, and differential phosphorylation of individual subcomponents. J. Biol. Chem. 260:5797–803.

    Google Scholar 

  17. Safaei, R., and Fischer, I. 1989. Cloning of a cDNA encoding MAP1B in rat brain: regulation of mRNA levels during development. J. Neurochem. 52:1871–9.

    Google Scholar 

  18. Schoenfeld, T. A., McKerracher, L., Obar, R., and Vallee, R. B. 1989. MAP1A and MAP1B are structurally related microtubule associated proteins with distinct developmental pattern in the CNS. J. Neurosci. 9:1712–1730.

    Google Scholar 

  19. Calvert, R., and Anderton, B. H. 1985. A microtubule-associated protein MAP 1 which is expressed at elevated levels during development of rat cerebellum. EMBO J. 4:1171–76.

    Google Scholar 

  20. Riederer, B., and Matus, A. 1985. Differential expression of distinct microtubule-associated proteins during brain development. Proc. Natl. Acad. Sci. USA 82:6006–9.

    Google Scholar 

  21. Aletta, J. M., Lewis, S. A., Cowan, N. J., and Greene, L. A. 1988. Nerve growth factor regulates both the phosphorylation and steady-state levels of microtubule-associated protein 1.2 (MAP1.2). J. Cell Biol. 106:1573–81.

    Google Scholar 

  22. Paschal, B. M., Shpetner, H. S., and Vallee, R. B. 1987. MAP 1C is a microtubule-associated ATPase which translocates microtubules in vitro and has dynein-like properties. J. Cell Biol. 105:73–82.

    Google Scholar 

  23. Shiomura, N., and Hirokawa, Y. 1987. The molecular structure of microtubule-associated protein 1A (MAP1A) in vivo and in vitro. An immunoelectron microscopy and quick-freeze, deep-etch study. J. Neurosci. 7:1461–1469.

    Google Scholar 

  24. Sato-Yoshitake, R., Shiomura, Y., Miyasaka, H., and Hirokawa N. 1989. microtubule-associated protein 1B: molecular structure, localization, and phosphorylation-dependent expression in developing neurons. Neuron 3:229–38.

    Google Scholar 

  25. Vallee, R. B. 1986. Reversible assembly purification of microtubules without assembly-promoting agents and further purification of tubulin, microtubule-associated proteins, and MAP fragments. Meth. Enzymol. 134:89–104.

    Google Scholar 

  26. Noble, M., Lewis, S. A., and Cowan, N. J. 1989. The microtubule binding domain of microtubule-associated protein MAP1B contains a repeated sequence motif unrelated to that of MAP2 and tau. J. Cell Biol. 109:3367–76.

    Google Scholar 

  27. Luca, F. C., Bloom, G. S., and Vallee, R. B. 1986. A monoclonal antibody that cross-reacts with phosphorylated epitopes on two microtubule-associated proteins and two neurofilament polypeptides. Proc. Natl. Acad. Sci. USA 83:1006–1010.

    Google Scholar 

  28. Diaz-Nido, J., Serrano, L., Mendez, E., and Avila, J. 1988. A casein kinase II-related activity is involved in phosphorylation of microtubule-associated protein MAP1-B during neuroblastoma cell differentiation. J. Cell Biol. 106:2057–2065.

    Google Scholar 

  29. Viereck, C., Tucker, R. P., and Matus, A. 1989. The adult rat olfactory system expresses microtubule-associated proteins found in the developing brain. J Neurosci. 9:3547–57.

    Google Scholar 

  30. Fischer, I., and Romano-Clarke, G. 1990. Changes in microtubule-associated protein MAP1B phosphorylation during rat brain development. J Neurochem. 55:328–33.

    Google Scholar 

  31. Calvert, R. A., Woodhams, P. L., and Anderton, B. H. 1987. Localization of an epitope of a microtubule associated protein 1x in outgrowing axons of the developing central nervous system. Neurosci. 23:131–141.

    Google Scholar 

  32. Dotti, C. G., Sullivan, C. A., and Banker, G. A. 1988. The establishment of polarity by hippocampal neurons in culture. J. Neurosci. 8:1454–1468.

    Google Scholar 

  33. Black, M. M., and Smith, W. 1988. Regional differentiation of the neuronal cytoskeleton. in Intrinsic determinants of neuronal form and function, eds Lasek, R. J. and M. M. Black (Liss, New York), pp. 463–486.

    Google Scholar 

  34. Vallee, R. B. and Bloom, S. G. 1984. High molecular weight microtubule-associated proteins. Mod. Cell. Biol. 3:21–76.

    Google Scholar 

  35. Olmsted, J. B. 1986. microtubule-associated proteins. Annu. Rev. Cell Biol. 2:421–457.

    Google Scholar 

  36. Matus, A. 1988. microtubule-associated proteins: Their potential role in determining neuronal morphology. Ann. Rev. Neurosci. 11:29–44.

    Google Scholar 

  37. Sandoval, I. V., and Weber, K. 1979. Calcium-induced inactivation of microtubule formation in brain extracts: presence of a calcium-dependent protease acting on polymerization-stimulating microtubule-associated proteins. Eur. J. Biochem. 92:463–470.

    Google Scholar 

  38. Nixon, R. A. 1983. Proteolysis of neurofilaments. in Neurofilaments. (Morrota C. A., ed.), pp. 117–154. University of Minnesota Press, Minneapolis.

    Google Scholar 

  39. Nixon, R. A. 1989. Calcium-activated neutral proteinases as regulators of cellular function. Annals N.Y. Aca. Sci. 568:198–208.

    Google Scholar 

  40. Vitto, A., and Nixon, R. A. 1986. Calcium-activated neutral proteinase of human brain: subunit structure and enzymatic properties of multiple molecular forms. J. Neurochem. 47:1039–1051.

    Google Scholar 

  41. Billger, M., Wallin, M., and Karlsson, J.-O. 1988. Proteolysis of tubulin and microtubule-associated proteins 1 and 2 by calpain I and II. Differences in sensitivity of assembled and disassembled microtubules. Cell Calcium 9:33–44.

    Google Scholar 

  42. Siman, R., and Noszek, J. C. 1988. Excitatory amino acids activate calpain I and induce structural protein breakdown in vivo. Neuron 1:279–287.

    Google Scholar 

  43. Johnson, G. V. W., Jope, R. S., and Binder, L. I. 1989. Proteolysis of tau by calpain. Biochem. Biophy. Res. Comm. 163:1505–1511.

    Google Scholar 

  44. Hasegawa, M., Arai, T., and Ihara, Y. 1990. Immunocytochemical evidence that fragments of phosphorylated MAP5 (MAP1B) are bound to neurofibrillary tangles in alzheimer disease. Neuron 4:909–918.

    Google Scholar 

  45. Safaei, R., and Fischer, I. 1989. Regulation of microtubule-associated protein (MAP2) mRNA expression during rat brain development. J. Mol. Neurosci. 1:189–198.

    Google Scholar 

  46. Fischer, I., Shea, T. B. 1991. Differential appearance of extensively phosphorylated forms of the high molecular weight neurofilament protein in regions of mouse brain during postnatal development. J. Neuroimmun. 31:73–81.

    Google Scholar 

  47. Pant, H. C. 1988. Dephosphorylation of neurofilament proteins enhances their susceptibility to degradation by calpain. Biochem. J. 256:665–668.

    Google Scholar 

  48. Chen, M., and Stracher, A. 1989. In situ phosphorylation of platelet actin-binding protein by cAMP-dependent protein kinase stabilizes it against proteolysis by calpain. J. Biol. Chem. 264:1482–1489.

    Google Scholar 

  49. Safaei, R., and Fischer, I. 1990. Turnover of cytoskeletal proteins in vivo. Brain Research 533:83–90.

    Google Scholar 

  50. Chakrabarti, A. K., Dasgupta, S., Banik, N. L., and Hogan, L. 1990. Ganglioside-modulated proteolysis by Ca2+-activated neutral proteinase (CANP): a role of glycoconjugates in CANP regulation. J. Neurochem. 54:1816–1819.

    Google Scholar 

  51. Cooligan, S. H., and Hathaway, D. R. 1984. Effect of L-alphaphosphatidylinositol on a vascular smooth muscle Ca2+ requirement for autolysis. J. Biol. Chem. 259:11627–11630.

    Google Scholar 

  52. Etienne, P., and Baudry, M. 1987. Calcium dependent aspects of synaptic plasticity, excitatory amino acid neurotransmission, brain aging and schizophrenia: a unifying hypothesis. Neurobiol. Aging 8:362–366.

    Google Scholar 

  53. Gottlieb, R. A., and Murphy, D. B. 1985. Analysis of microtubule-binding domain of MAP2. J. Cell Biol. 101:1782–1789.

    Google Scholar 

  54. Siman, R., Ahdoot, M., and Lynch, G. 1987. Ontogeny, compartmentation and turnover of spectrin isoforms in rat central neurons. J. Neurosci. 7:55–64.

    Google Scholar 

  55. Peng, I., Binder, L. I., and Black, M. M. 1986. Biochemical and immunochemical analysis of cytoskeletal domains in neurons. J. Cell Biol. 102:252–262.

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Biochemistry, E. K. Shriver Center, 200 Trapelo Rd., 02254, Waltham, MA

    Itzhak Fischer & Giuseppina Romano-Clarke

  2. Department of Neurology (Neuroscience), Harvard Medical School, 02115, Boston, MA

    Itzhak Fischer

  3. Laboratories of Molecular Neuroscience and Aging, Mailman Research Center, McLean Hospital, 02178, Belmont, MA

    Frida Grynspan

Authors
  1. Itzhak Fischer
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Giuseppina Romano-Clarke
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Frida Grynspan
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Fischer, I., Romano-Clarke, G. & Grynspan, F. Calpain-mediated proteolysis of microtubule associated proteins MAP1B and MAP2 in developing brain. Neurochem Res 16, 891–898 (1991). https://doi.org/10.1007/BF00965538

Download citation

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00965538

Key Words

Search

Navigation