Motile properties of the bi-directional kinesin-5 Cin8 are affected by phosphorylation in its motor domain.

Ofer Shapira,Larisa Gheber

doi:10.1038/srep25597

Abstract

The Saccharomyces cerevisiae kinesin-5 Cin8 performs essential mitotic functions in spindle assembly and anaphase B spindle elongation. Recent work has shown that Cin8 is a bi-directional motor which moves towards the minus-end of microtubules (MTs) under high ionic strength (IS) conditions and changes directionality in low IS conditions and when bound between anti-parallel microtubules. Previous work from our laboratory has also indicated that Cin8 is differentially phosphorylated during late anaphase at cyclin-dependent kinase 1 (Cdk1)-specific sites located in its motor domain. In vivo, such phosphorylation causes Cin8 detachment from spindles and reduces the spindle elongation rate, while maintaining proper spindle morphology. To study the effect of phosphorylation on Cin8 motor function, we examined in vitro motile properties of wild type Cin8, as well as its phosphorylation using phospho-deficient and phospho-mimic variants, in a single molecule fluorescence motility assay. Analysis was performed on whole cell extracts and on purified Cin8 samples. We found that addition of negative charges in the phospho-mimic mutant weakened the MT-motor interaction, increased motor velocity and promoted minus-end-directed motility. These results indicate that phosphorylation in the catalytic domain of Cin8 regulates its motor function.

Highlights

The anti-parallel MT-sliding activity at the midzone can only be achieved by the plus-end-directed motility of kinesin-5 motors bound between the anti-parallel MTs13,22
Mediated phosphorylation on Cin[8] motility, we analyzed the motile properties of three Cin[8] variants, namely wt Cin[8], phospho-deficient Cin8-3A, in which the serines and threonine in Cdk1-specific sites within the Cin[8] catalytic domain were mutated to alanine (S277A, T285A, S493A), and the phospho-mimic, Cin8-3D mutant, in which the same amino acids were mutated to negatively-charged aspartic acids (S277D, T285D, S493D)
In vitro single molecule motility measurements were performed under two IS conditions, i.e. low IS (motility buffer (MB) supplemented with 30 mM NaCl, IS = 0.172) and intermediate IS (MB supplemented with 80 mM NaCl, IS = 0.222) on polarity-marked MTs

Summary

Introduction

The anti-parallel MT-sliding activity at the midzone can only be achieved by the plus-end-directed motility of kinesin-5 motors bound between the anti-parallel MTs13,22. Several kinesin-5 motors have been shown to be plus-end-directed in vitro[12,13]. It has been previously shown that Cin[8] is regulated by phosphorylation of at least one of the Cdk1-specific sites located in its catalytic domain, i.e. S277, T285 and S49330,41 (Fig. 1a). Residues S277 and T285 are located in the loop 8 of Cin[8], which is the longest loop 8 among kinesin family members and is not conserved among kinesin-5 motors of higher eukaryotes. Our results indicate that phosphorylation of Cdk1-specific sites within the Cin[8] catalytic domain affects motor-MT interactions and alters Cin[8] velocity and directionality. These results suggest that phospho-regulation of the intracellular functions of mitotic kinesins involves regulation of their motile properties

Methods

Results

Conclusion