In this study, we show that Eg2 phosphorylation of Eg5 is not important for its function in bipolar spindle formation in Xenopus egg extract. Conversely, Eg5 needs to be phosphorylated by Cdk1 to be targeted to spindle microtubules and hence assemble a bipolar spindle in Xenopus egg extract. These findings confirm previous studies and furthermore indicate that Cdk1 phosphorylation site is not only conserved among kinesin-5 members but also that its mechanism of regulation is conserved among this subfamily. Although further experiments are required to fully characterize Eg5 motor properties in by means of our microtubule gliding assay in Xenopus egg extract, Eg5 intrinsic motor properties are definitely crucial for bipolar spindle assembly as none of the Eg5 chimeras could rescue spindle formation in Xenopus egg extract. Moreover, these experiments provide the first experimental evidence that the classification of kinesins in different subfamilies, according to their conserved motor domain sequences, has also yielded to classify them according to their diverse function.
