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Effects of ATP and cyclic AMP on the in vitro assembly and stability of mammalian brain microtubules

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Abstract

The relevance of protein phosphorylation, transphosphorylation and binding phenomena in the kinetics of the ATP-induced assembly of cycle-purified microtubule protein from mammalian brain were studied. ATP was able to induce the polymerization of microtubules of normal appearance. However, the assembled structures, were unstable and microtubules depolymerized after achievement of a transitory maximum. Cyclic AMP reduced the amplitude of the polymerization maximum in a concentration-dependent manner, correlating with the stimulation of the endogenous phosphorylation reaction. When microtubule assembly was induced by GTP, in the presence of various concentrations of ATP, the slope of the depolymerization phase was found to depend on the concentration of ATP. Fluoride ion inhibited the endogenous phosphorylation reaction and reduced the disassembly rate, in a concentration-dependent manner. Evidence is also presented indicating that ATP did not bind to phosphocellulose-purified tubulin. These results further contribute to indicate that ATP and cyclic AMP, acting coordinately to control the phosphorylation extent of microtubule proteins are important factors to determine microtubule stability within the cell. Some implications of this mechanism for the regulation by cAMP of the initiation of DNA synthesis and mitosis are considered.

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Abbreviations

MAPs:

microtubule-associated proteins

MAP2:

microtubule-associated protein 2, Mes-4-morpholinoethanesulfonic acid

EGTA:

ethylene-glycol bis (β-aminoethyl ether)N,N,N′,N′-tetraacetic acid

PMSF:

phenylmethylsulfonyl fluoride

PEI:

polyethyleneimine

PC:

phosphocellulose

DEAE:

Diethylaminoethyl

SDS-PAGE:

polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate

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Authors and Affiliations

  1. Centro de Biología Molecular (C.S.L.-U.A.M.), Universidad Autónoma de Madrid, 28049, Madrid, Spain

    Felix Pariente & Rafael Manso-Martínez

  2. Department of Biochemistry, The University of Texas Health Science Center, 78284, San Antonio, Texas, USA

    Veena Prasad & Richard Froilan Ludueña

Authors
  1. Felix Pariente
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  2. Veena Prasad
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  3. Richard Froilan Ludueña
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  4. Rafael Manso-Martínez
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Pariente, F., Prasad, V., Ludueña, R.F. et al. Effects of ATP and cyclic AMP on the in vitro assembly and stability of mammalian brain microtubules. Mol Cell Biochem 74, 43–54 (1987). https://doi.org/10.1007/BF00221911

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  • DOI: https://doi.org/10.1007/BF00221911

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