Mechanism of endogenous phosphorylation of microtubule proteins during GTP-induced microtubule assembly and implications for stability of the assembled structures

Rosario Palomares, Veena Prasad, Richard Froilan Luduena, Rafael Manso-Martínez

Research output: Contribution to journalArticle

1 Scopus citations

Abstract

Cycle-purified microtubule protein from mammalian brain incorporated [32P]Pi upon incubation with [γ-32P]GTP under the conditions used to promote assembly. This phosphorylation also occurred in the same proteins when phosphorylated with [γ-32P]ATP and was only slightly stimulated by cAMP. GTP was a much less effective substrate than ATP. The transfer of phosphoryl groups from [γ-32P]GTP to endogenous proteins followed a linear time-course and was stimulated by low concentrations of ATP and, more efficiently, by ADP. These data are in agreement with the predictions derived from a mechanism of phosphorylation by which [γ-32P]GTP does not act as a phosphoryl donor for the protein kinase activity but, instead, only as a repository of high group transfer potential phosphoryl groups used to make [γ-32P]ATP, from contaminating ADP, by means of the nucleoside diphosphate kinase activity. Using 100 mM fluoride, which suppressed protein phosphorylation without inhibiting the nucleoside diphosphate kinase activity, formation of [γ-32P]ATP was detected. Fluoride was also able to protect microtubules from a slow depolymerization which was found to occur during long-term incubation of microtubules. This indicates that the phosphorylation observed in the presence of GTP is sufficient to destabilize microtubules.

Original languageEnglish (US)
Pages (from-to)139-147
Number of pages9
JournalBBA - Molecular Cell Research
Volume927
Issue number1
DOIs
StatePublished - Jan 19 1987

Keywords

  • GTP
  • Microtubule protein
  • Microtubule stability
  • Phosphorylation

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

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