Phosphorylation by Cdk1 Increases the Binding of Eg5 to Microtubules In Vitro and in Xenopus Egg Extract Spindles

Abstract

BackgroundMotor proteins from the kinesin-5 subfamily play an essential role in spindle assembly during cell division of most organisms. These motors crosslink and slide microtubules in the spindle. Kinesin-5 motors are phosphorylated at a conserved site by Cyclin-dependent kinase 1 (Cdk1) during mitosis. Xenopus laevis kinesin-5 has also been reported to be phosphorylated by Aurora A in vitro.Methodology/Principal FindingsWe investigate here the effect of these phosphorylations on kinesin-5 from Xenopus laevis, called Eg5. We find that phosphorylation at threonine 937 in the C-terminal tail of Eg5 by Cdk1 does not affect the velocity of Eg5, but strongly increases its binding to microtubules assembled in buffer. Likewise, this phosphorylation promotes binding of Eg5 to microtubules in Xenopus egg extract spindles. This enhancement of binding elevates the amount of Eg5 in spindles above a critical level required for bipolar spindle formation. We find furthermore that phosphorylation of Xenopus laevis Eg5 by Aurora A at serine 543 in the stalk is not required for spindle formation.Conclusions/SignificanceThese results show that phosphorylation of Eg5 by Cdk1 has a direct effect on the interaction of this motor with microtubules. In egg extract, phosphorylation of Eg5 by Cdk1 ensures that the amount of Eg5 in the spindle is above a level that is required for spindle formation. This enhanced targeting to the spindle appears therefore to be, at least in part, a direct consequence of the enhanced binding of Eg5 to microtubules upon phosphorylation by Cdk1. These findings advance our understanding of the regulation of this essential mitotic motor protein.

The NIMA-like kinase Nek2 is a key switch balancing cilia biogenesis and resorption in the development of left-right asymmetry

“…Xenopus nek2B was cloned from stage 15 Xenopus tropicalis cDNA and GATEWAY-cloned in a similar manner to nek2A. his-Eg5 plasmid was a gift from Thomas Surrey, EMBL (Cahu et al, 2008). eg5 was sub-cloned into pCS108 for RNA expression.…”

Ase1p phosphorylation by cyclin-dependent kinase promotes correct spindle assembly inS. cerevisiae

“…27 Similarly, CDK phosphorylation enhances recruitment of the kinesin-5 Eg5 onto MTs, thus promoting spindle assembly in vitro. 28 Here, we address the significance of CDK-mediated phosphorylation of Ase1p toward spindle assembly. Ase1p is a preferred substrate of S-phase CDK.…”

Kinesin-5 mitotic motors: Is loop5 the on/off switch?

“…Kinesin-5 primary structure with an N-terminal motor domain followed by a kinesin-5-specific neck-linker, the conformation of which responds to nucleotide and Mt binding. 17,18,21 the neck-linker connects the motor domain with the rest of the molecule, through a coiled-coil and a C-terminal tail domain, which may also bind Mts (left); 30,33 quarternary arrangement of the bipolar kinesin-5 tetramer (middle); 11 the tetramer is involved in cross-linking and sliding Mt arrays (in blue) in the bipolar mitotic spindle (right). (B) structure of the drug-bound conformation of kinesin-5 (PDB: 1QOB).…”

“…34 addition to the Wee1 sites, 26 cell cycledependent phosphorylation of kinesin-5 also occurs in the tail domain (including by Cdk1), which regulates localization to the spindle and enhances MT association. [28][29][30][31] Phosphorylation of a kinesin-13 MT depolymerising motor within its conserved motor domain has also been described. 32 The structural consequences of kinesin phosphorylation are not understood though they clearly have a critical role to play in multiple aspects of kinesin function, particularly during mitosis.…”