Engineering Approaches to Biomolecular Motors

Engineering Approaches to Biomolecular Motors: From in vitro to in vivo Poster Abstracts

6-POS Board 6 Study of Phospho-regulation of a Mitotic Motor Protein by Systematic Mutagenesis Alina Goldstein 1 , Darya Goldman 1 , Ervin Valk 2 , Mart Loog 2 , Liam Holt 3 , Leah Gheber 1 . 1 Ben Gurion University of the Negev, Beer Sheva, Israel, 2 University of Tartu, Tartu, Estonia, 3 UC Berkeley, Berkeley, CA, USA. The S. cerevisiae Cin8 belongs to the kinsesin-5 family of mitotic motor proteins. During mitosis, Cin8 orchestrates the mitotic spindle assembly and its elongation. Recent work from our laboratory indicated that phosphorylation of Cin8 at cyclin-dependent kinase 1 (Cdk1) sites located in its catalytic domain governs its localization to the mitotic spindle during mitosis. Here we tested the flexibility of phosphoregulation of Cin8, and examined whether phosphorylation at newly created Cdk1 sites can mimic the known phospho-regulation or create new regulation. For this purpose, we first generated a phospho-deficient mutant of Cin8 and then introduced new Cdk1 sites by single amino acid replacement. We examined the mutants by viability test, live imaging and quantitation of phosphorylation by Cdk1 in vitro . We found that out of 32 novel sites, only one site at position 276, which is located in high proximity to a native Cdk1 phosphorylation site (S277), recapitulated the original phospho-regulation of Cin8. Although several sites were created nearby, and some of them were found to undergo phosphorylation in vitro , only this site exhibits localization and viability phenotypes similar to those of the wt Cin8. This result indicates that phospho-regulation of Cin8 by Cdk1 is rigid and highly dependent on the structural context. However, several additional mutants bearing novel Cdk1 sites which are not adjacent to native sites, exhibited new phenotypes, suggesting that phsopho-regulation by Cdk1 at additional sites of Cin8 can affect its activity. The mechanism and physiological significance of phospho-regulation at these new sites needs to be further investigated.

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