Modification of microtubule steady-state dynamics by phosphorylation of the microtubule-associated proteins.

Abstract

Phosphorylation of purified microtubule-associated proteins (MAP) inhibited the rate and extent of MAP-stimulated microtubule assembly. The extent of microtubule assembly was reduced as a result of a decrease in the fraction of tubulin polymerized, without a significant change in the critical protein concentration. The decreased rate of microtubule assembly using phosphorylated MAP reflected a reduction in microtubule nucleation resulting in fewer, but 2-fold longer, microtubules at steady state. Analysis of microtubule (MT) dynamics at steady state revealed that the rate of directional incorporation of subunits (flux) was 22 subunits .cntdot. MT-1 .cntdot. s-1 with phosphorylated MAPs, compared to 10 subunits .cntdot. MT-1 .cntdot. s-1 with unphosphorylated MAP. The initial rate of disassembly determined by isothermal dilution was 232 subunits .cntdot. MT-1 .cntdot. s-1 for microtubules assembled with phosphorylated MAP, compared to 102 subunits .cntdot. MT-1 .cntdot. s-1 for microtubules assembled with unphosphorylated MAP. The directionality (the number of successful subunit additions relative to the total number of association events per unit time) for subunit addition was 0.1 for microtubules assembled with either phosphorylated or unphosphorylated MAP. These observations are interpreted in terms of a mechanism in which phosphorylation of MAP increases the rate of steady-state subunit flux by an equivalent enhancement of the rates of subunit association and dissociation, such that the critical protein concentration and directionality remain unchanged.