Src family kinase phosphorylation of the motor domain of the human kinesin‐5, Eg5

Abstract

Spindle formation in mammalian cells requires precise spatial and temporal regulation of the kinesin-5, Eg5, which generates outward force to establish spindle bipolarity. Our results demonstrate that Eg5 is phosphorylated in cultured cells by Src family kinases (SFKs) at three sites in the motor head: Y125, Y211, and Y231. Mutation of these sites diminishes motor activity in vitro, and replacement of endogenous Eg5 with phosphomimetic Y211 in LLC-Pk1 cells results in monopolar spindles, consistent with loss of Eg5 activity. Cells treated with SFK inhibitors show defects in spindle formation, similar to those in cells expressing the non-phosphorylatable Y211 mutant, and distinct from inhibition of other mitotic kinases. We propose that this phosphoregulatory mechanism tunes Eg5 enzymatic activity for optimal spindle morphology.

A posttranslational modification of the mitotic kinesin Eg5 that enhances its mechanochemical coupling and alters its mitotic function

“…In kinesin 1, serine 175, at the amino-terminal end of the α3 helix, can be phosphorylated by the JNK3 kinase, which reduces stall force by 20% (54). A recent report has also described src-mediated phosphorylation of tyrosine residues in the motor domain of Eg5, including those located in the α3 helix and in Loop 5-in close proximity to residue K146 (12). However, these studies do not provide insight into how PTMs alter motor mechanochemistry.…”

“…However, in nearly all cases, the consequences of these PTMs on motor function remain unknown. Multiple PTMs in the motor domain of the mitotic kinesin Eg5 have been reported (12), including in Loop 5 and helices α2 and α3-all in the vicinity of the catalytic site ( Fig. 1A).…”

Non‐canonical functions of the mitotic kinesin Eg5

“…Recent studies have suggested acetylation of Eg5 at lysine 146, which is located in the α2 helix of the motor domain, enhances its mechanochemical coupling, and alters its mitotic function . In addition, Src is reported to phosphorylate three tyrosines in the motor domain of human Eg5 however, the molecular mechanisms regulating this modification and its functional significance remain unknown. It also remains to be determined whether the phosphorylation of Eg5 affects its role in plant growth and development.…”

Phospho‐regulation of mitotic spindle assembly

“…Cdk1 also phosphorylates Eg5 on Thr926 (Slangy et al, 1995), increasing the affinity of Eg5 for microtubules (Cahu et al, 2008).The PP2A-B55α complex dephosphorylates this residue to regulate Eg5 activity (Liu et al, 2017). SRC kinases phosphorylate Eg5 on Tyr125, Tyr211, and Tyr231 (Bickel et al, 2017). These modifications decrease Eg5 motor activity (Bickel et al, 2017).…”

“…SRC kinases phosphorylate Eg5 on Tyr125, Tyr211, and Tyr231 (Bickel et al, 2017). These modifications decrease Eg5 motor activity (Bickel et al, 2017). Of interest, Aurora A has been shown to phosphorylate Eg5 in X. laevis, but the site of phosphorylation and the consequence of this modification is unknown, and there are no reports of Aurora A phosphorylating Eg5 in human cells (Giet et al, 1999).…”