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Engineering Approaches to Biomolecular Motors: From in vitro to in vivo Poster Abstracts
61
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.