Complex phospho-regulation pathways of a cancer related mitotic motor protein

Introduction: Kinesin-5 motor proteins play pivotal roles in mitotic cell division since they are involved in essential functions of mitotic spindle dynamics. These motor proteins are attractive targets for anti-cancer treatment by arresting mitosis progression and since high expression of kinesin-5 motors is correlated with poor prognosis. Few such drugs were previously investigated and tested in clinical trials, however, attempts to finalize an effective anti-cancer drug were futile. In order to investigate regulation pathways of kinesin-5 motor proteins we use the S. cerevisiae yeast model which express two kinesin-5 homologues, Cin8 and Kip1. Recently it was found that Cin8 is phospho-regulated by Cdk1, which governs its localization to the mitotic spindle during mitosis. Although it was demonstrated that phospho-regulation of Cin8 at its motor domain by Cdk1 regulates its localization, the contribution of each of the three Cdk1 sites was never explored.

Materials and methods: We first established the role of each of the three Cdk1 sites in the motor domain by characterizing phospho-deficient and phospho-mimic mutants of Cin8 by several in vivo methods such as viability assay and live cell imaging. We then combined these results to analysis by in vitro methods such as quantitative phosphorylation assay and single molecule in vitro motility assay conducted on purified mutants of Cin8.

Results and discussion: We found that each of the sites serve different role in phospho-regulating Cin8; S277, in the motor domain of Cin8, is the major regulator of localization and functionality in vivo. However, the mostly conserved site at position S493 is responsible for regulating Cin8 motile properties. In addition, we tested the rigidity of phospho-regulation of Cin8, and examined whether phosphorylation at newly created Cdk1 sites can mimic the known phospho-regulation or create new phenotypes. For this purpose, we generated phospho-deficient mutant of Cin8 and introduced novel Cdk1 sites by single amino acid replacement. We found that out of 29 novel sites, only two sites resulted in phospho-regulation of Cin8, although they were not able to fully recapitulate the original phospho-regulation of Cin8. Position 276 which is in high proximity to the native site S277, and position 148, which was sampled in evolution in different homologues and is located in α-helix 1 which has unknown contribution to functionality to kinesin-5 motor proteins.

Conclusions: Our results indicate that phospho-regulation of Cin8 by Cdk1 is rigid and highly dependent on the structural context and that phospho-regulation pathways of kinesin-5 motor proteins are much more complex than initially anticipated. This complex regulation suggests that the activity of kinesin-5 motor proteins is highly susceptible to regulation by phosphorylation.