1. Mechanism of endogenous phosphorylation of microtubule proteins during GTP-induced microtubule assembly and implications for stability of the assembled structures.
- Author
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Palomares R, Prasad V, Luduena RF, and Manso-Martínez R
- Subjects
- Adenosine Diphosphate metabolism, Adenosine Diphosphate pharmacology, Adenosine Triphosphate metabolism, Adenosine Triphosphate pharmacology, Animals, Cattle, Fluorides pharmacology, Guanosine Triphosphate metabolism, Kinetics, Microtubules drug effects, Phosphates metabolism, Phosphorylation, Cerebral Cortex analysis, Guanosine Triphosphate pharmacology, Microtubule Proteins metabolism, Microtubules physiology
- Abstract
Cycle-purified microtubule protein from mammalian brain incorporated [32P]Pi upon incubation with [gamma-32P]GTP under the conditions used to promote assembly. This phosphorylation also occurred in the same proteins when phosphorylated with [gamma-32P]ATP and was only slightly stimulated by cAMP. GTP was a much less effective substrate than ATP. The transfer of phosphoryl groups from [gamma-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 [gamma-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 [gamma-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 [gamma-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.
- Published
- 1987
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